NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless...

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NMR studies of the amyloid β-peptide Jens Danielsson Stockholm University

Transcript of NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless...

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NMR studies of the

amyloid ββββ-peptide

Jens Danielsson

Stockholm University

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© Jens Danielsson, Stockholm 2007

ISBN (91-7155-349-5 pp 1-86)

Printed in Sweden by Printers name, Stockholm 2007

Distributor: Department of Biochemistry and Biophysics

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Dedicated to my Family

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List of Papers

I. Jens Danielsson, Jüri Jarvet, Peter Damberg and Astrid Gräs-lund, “Translational diffusion measured by PFG-NMR on full length and fragments of the Alzheimer Aβ(1-40) peptide. De-termination of hydrodynamic radii of random coil peptides of varying length”, Magn Res Chem., 2002; 40:S89-S97

II. Jüri Jarvet, Peter Damberg, Jens Danielsson, Ingrid Johans-

son, Göran Eriksson and Astrid Gräslund, “A left-handed 31 helical conformation in the Alzheimer Aβ(12-28) peptide”, FEBS letters , 2003, 555(2), 371-374

III. Jens Danielsson, Jüri Jarvet, Peter Damberg, and Astrid

Gräslund, “Two-Site Binding of β-Cyclodextrin to the Alz-heimer Aβ(1-40) Peptide Measured with Combined PFG-NMR Diffusion and Induced Chemical Shifts”, Biochemistry, 2004, 43, 6261-6269

IV. Jens Danielsson, Jüri Jarvet, Peter Damberg and Astrid Gräs-

lund, “The Alzheimer β-peptide shows temperature-dependent transitions between left-handed 31-helix, β-strand and random coil secondary structures”, FEBS J., 2005, 272, 3938-3949.

V. Jens Danielsson, August Andersson, Jüri Jarvet and Astrid

Gräslund. “15N-relaxation study of the Alzheimer β-peptide: structural propensities and persistence length”, 2006, Magn

Res Chem. 2006, 44, S114-S121 VI. Jens Danielsson, Roberta Pierattelli, Lucia Banci and Astrid

Gräslund, “High Resolution NMR Studies of the Zinc Bind-ing Site of the Alzheimer’s Aβ-peptide”, 2006, FEBS J., doi: 10.1111/j.1742-4658.2006.05563.x

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VII. Jüri Jarvet, Jens Danielsson, Peter Damberg, Marta Oleszczuk and Astrid Gräslund, “Structure and positioning of the Alzheimer Aβ(1-40) peptide in SDS micelles using NMR and paramagnetic probes.”, Manuscript in preparation

VIII. Martin Dahlberg, Jens Danielsson, Astrid Gräslund and Atto Laksonen, “Structure of the Amyloid β-peptide Fragment 1-9, A combined molecular dynamics and NMR study”, Manu-

script in preparation

Paper I and V is reproduced by permission of John Wiley & Sons Limited.

Paper II, IV and VI is reprinted by permission of Federation of the Euro-

pean Biochemical Societies

Paper III is reproduced with permission from American Chemical Society

Papers not included in this thesis Amina S. Woods, Rafal Kaminski, Murat Oz, Yun Wang, Kurt Hauser, Robin Goody,Hay-Yan J. Wang, Shelley N. Jackson, Peter Zeitz, Karla P. Zeitz, Dorota Zolkowska, Raf Schepers, Michael Nold, Jens Danielson, Astrid Gräslund, Vladana Vukojevic, Georgy Bakalkin, Allan Basbaum and Toni Shippenberg, “Decoy Peptides that Bind Dynorphin Noncovalently Prevent NMDA Receptor-Mediated Neurotoxicity”, 2006, J. Proteome Res. 3, 478-484. August Andersson, Henrik Biverståhl, Jon Nordin, Jens Danielsson, Emma Lindahl, Lena Mäler, “The membrane-induced structure of melittin is corre-lated with the fluidity of the lipids”, 2006, Biochim Biophys Acta, doi:10.1016/j.bbamem.2006.07.009. August Andersson, Jens Danielsson, Astrid Gräslund and Lena Mäler, “Ki-netic models for peptide-induced membrane leakage in vesicles and cells”, Submitted Manuscript

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Contents

Introduction ...................................................................................................11

Biological background...................................................................................13 Misfolding diseases.....................................................................................................13 Alzheimer’s disease is related to the amyloid β-peptide ..............................................15 Amyloid precursor protein ...........................................................................................15 Proteolytic degradation of APP and the formation of soluble Aβ .................................17 Oligomerization of the Aβ-peptide and neurotoxic mechanisms ..................................19 Fibrilization and structure of amyloid fibrils..................................................................21 Metal interaction of the soluble Aβ-peptide .................................................................22 The soluble Aβ-peptide’s membrane interactions........................................................23 Ligand binding to Aβ, and other strategies to prevent Aβ-toxicity ................................25

Theory of hydrodynamic dimensions and structural conversions of peptides

......................................................................................................................28 Dimensions of polymers and polypeptides ..................................................................28 Translational Diffusion of peptides ..............................................................................31 Structural transitions ...................................................................................................32

Spectroscopic methods.................................................................................35 NMR............................................................................................................................37 J-couplings and the structural interpretation thereof....................................................37 Nuclear spin relaxation and dynamics.........................................................................38 Linebroadening in NMR ..............................................................................................39 Diffusion measurements with NMR .............................................................................42 Circular Dichroism and secondary structure of peptides .............................................45 Estimation of secondary structure fraction with CD .....................................................46 Fluorescence spectroscopy.........................................................................................47

Results and discussion .................................................................................50 The soluble Aβ occurs as a mainly unfolded monomer ...............................................51 The monomeric Aβ has some residual structural propensities ....................................53 The Aβ peptide shows a temperature dependent structural behavior..........................55 The structural propensities of Aβ corresponds to structure both in membrane mimicking

media and in fibrils ......................................................................................................58

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Metals such as copper and zinc interact with and alter the aggregation properties of Aβ

....................................................................................................................................62 Aβ binding of β-cyclodextrin involves aromatic residues and reduces aggregation .....65

Outlook..........................................................................................................68

Sammanfattning på svenska.........................................................................70

Acknowledgements .......................................................................................72

References....................................................................................................74

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Abbreviations

β-cd β-cyclodextrin Aβ Amyloid β peptide AD Alzheimer’s disease AFM Atomic force microscopy ALS Amyotrophic lateral sclerosis APP Amyloid Precursor Protein CD Circular dichroism COSY Correlation spectroscopy CSA Chemical shift anisotropy CSF Cerebrospinal fluid FTIR Fourier transformed infrared spectroscopy HSQC Heteronuclear single quantum coherence NMR Nuclear magnetic resonance NOE Nuclear Overhauser effect PFG-NMR Pulsed field gradient NMR (see above) PII Polyproline type II TFE Trifluoroethanol ThT Thioflavin T TMS Transmembrane segment

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Introduction

Life is a delicate matter; the molecular machinery has to be tuned to function in an extremely complex environment. The central dogma of biochemistry describes the storage (DNA) and flow (RNA) of genetic information and the transcription of the information into functional molecular units, proteins. In order for life to be maintained, all three parts of the central dogma must work, and the direct functional work is, with certain exceptions, done by the proteins. Proteins are polypeptide chains constructed from a pool of twenty different amino acids, each of which has its own specific characteristic prop-erties. The function of a protein is closely related to the three dimensional structure. This relationship may look different for different proteins; a pro-tein may adopt its structure directly after the production and after that have a rather rigid structure. Other proteins are unstructured, partly or totally, and a well defined structure is induced when the protein exhibits its function. Yet another possibility is the protein structure has to change for the protein to function, maybe due to binding to a target.

The three dimensional structure of a protein is determined by the primary structure, the amino acid sequence of the polypeptide chain. The relationship between the primary structure and the secondary and tertiary structure is not straightforward, and the relationship between sequence and structure is not entirely understood. The folding process of the protein is quite fast, millisec-onds to seconds and occurs spontaneously, at least for small and medium size soluble proteins. Protein folding is a cooperative process where locally folded regions fold together and stabilize the overall structure. This, in turn, implies that the folding energy landscape is not flat, but can rather be seen as a rough funnel with the native structure having the lowest energy and one or more transition states manifested as local minima in the wall of the funnel.

This very complicated process is performed in a “biomolecular soup” with proteins, RNA, DNA and membrane lipids (and much more) as ingredi-ents. The polypeptide concentration in the cell is typically 10-20 mg/ml which is very high. In order to make this complex machinery work, an intri-cate control system has evolved with chaperon proteins that keep the un-folded proteins from aggregation during the folding process and with degra-dation systems in the cases when folding fails.

In some cases when proteins are not folded correctly, either the protein is not folded at all or folded in a way that inhibits the normal function of the protein. In some cases the new structure, or lack of structure gives the pro-

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tein a new, possibly pathogenic function. One could also imagine this as an evolutionary process where the new fold somehow is favored while the old one is not. In the case of prion diseases the misfolded protein induces mis-folding of other proteins of the same kind. Considering the complexity of folding it is surprising that diseases arising from misfolded proteins are not more common than they are. Nevertheless misfolding diseases are an impor-tant field in medicine and pathophysiology and also in biophysics. Under-standing the events that lead to misfolding of a protein may lead to the un-derstanding of the mechanisms of normal protein folding. In addition, knowledge about the molecular mechanisms underlying any disease provides a possibility to design prevention or a cure for that specific disease. Impor-tant misfolding diseases are e.g. type II diabetes, Parkinson disease and Alz-heimer’s disease.

This thesis is about the peptide which, misfolded and oligomerized, is be-lieved to be the molecular basis of Alzheimer’s disease. I have made bio-physical studies of this peptide in order to better understand the misfolding and aggregation process of this peptide. The peptide is called the amyloid β peptide and is the cleavage product of a ubiquitous membrane bound protein called amyloid precursor protein or APP.

There is no established unambiguous definition of a peptide, but usually a polypeptide chain with less than about 50 amino acids is considered a pep-tide. Peptides may or may not have a well defined structure, but short poly-peptides usually have a high order of flexibility and no ordered structure. Unstructured peptides and proteins or protein domains play an important part in biology. One way to understand the underlying mechanisms for misfold-ing, and consequently also folding, is to understand the physical interactions stabilizing and inducing structure, either the native or the misfolded.

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Biological background

“Digerdöden drabbar alla, även den som inte vill”

Anonymous graffiti

This thesis concerns biophysical studies of Alzheimer’s amyloid β-peptide (Aβ) in order to elucidate the molecular properties of the peptide that cause it to form oligomers, aggregate into insoluble aggregates, form fibrils rich in cross β secondary structure and finally accumulate in the brain and form amyloid plaques. The Aβ-peptide arises as a cleavage product of a precursor protein that is anchored to the neuronal cell membrane. In the membrane bound state the peptide is assumed to be in an α-helical structure, but upon cleavage the peptide leaves the membrane and goes into solution. The neuro-toxic effect of Aβ seems to be linked to the presence of soluble aggregates of the peptide and not directly to fibrils and plaques (Hsia et al., 1999; Lue et

al., 1999; McLean et al., 1999; Wang et al., 1999; Klein et al., 2001). Upon aggregation and oligomerization and upon membrane interaction

the peptide undergoes a conformational change towards a β-rich structure which builds up the fibrils that are the typical histopathological landmark of Alzheimer’s disease. The life of the Aβ-peptide thus involves a change in the secondary structure from the generic α-helical structure to the pathological β sheet structure, with an important mainly unstructured solvent intermediate. This means that Alzheimer’s disease may be classified as a protein misfold-ing disease. In this section, the life of the Aβ-peptide will be discussed, from the membrane bound APP-form to the final aggregated state of the peptide.

Misfolding diseases

The molecular basis of misfolding diseases that are associated with fibrillar amyloid aggregates is not yet fully understood. The family of these diseases may be divided into two major groups, one which concerns neurological

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degeneration and one that does not. The neurodegenerative misfolding dis-eases, in turn, consist of a wide variety of syndromes, including well-known diseases as Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), Hunt-ington’s and Parkinson’s diseases. These diseases are so called neurodegen-erative amyloid-related disorders (Chiti and Dobson, 2006). Among the non-neurodegenerative diseases one may mention the systemic amyloidosis and the more localized type II diabetes.

The prion diseases are other important examples, causing spongiform neuropathies. Among the most well known prion diseases are Creutzfeldt-Jacobs disease, bovine spongiform encephalopathy (mad cow disease) and scrapie. The macroscopic appearance of the tissues affected by these mis-folding disorders is similar. The term spongiform gives a hint of the appear-ance of the amyloid affected tissues: The accumulation of amyloid in some cases kills the surrounding cells and thus creates “holes” in the tissue. This makes the tissue appear as a sponge. These features were described already in the 17th century as waxy liver and spongy spleens. In the mid 19th cen-tury, staining experiments showed that affected tissue stained similar to cel-lulose, and the scientist Rudolph Virchow drew the conclusion that the caus-ing substance was cellulose and named it amyloid from Latin amylum and Greek amylon for cellulose (Sipe and Cohen, 2000) .

The stained structures, i.e. the amyloid plaques, are aggregates of fibrils composed of one or more types of molecules, specific to the disease. In some cases the amyloid deposition in the tissues can be huge, up to kilograms, as in systemic amyloidosis (Bucciantini et al., 2002). In other cases as in neu-rodegenerative amyloid diseases the pathological impact has a much earlier onset and the depositions are microscopic even when the disease has caused the death of the patient. The fibrils are long and thin, 60-130Å, and are com-posed of otherwise soluble proteins (Sipe and Cohen, 2000; Ghiso and Frangione, 2002).

Independent of the molecular origin of the fibrils they have some com-mon properties, e.g. amyloid fibrils consist of aggregates of molecules in β-sheet conformation, regardless of the native peptide structure (Ghiso and Frangione, 2002; Murphy, 2002; Dobson, 2003). The cell dysfunction or death caused by the amyloid depositions may have different origins. The deposition itself can mechanically disturb its surroundings or the prefibrillar aggregates can be toxic or in other ways disturb or inhibit normal cell func-tions (Selkoe, 2003; Quist et al., 2005; Chiti and Dobson, 2006).

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Alzheimer’s disease is related to the amyloid β-peptide

One of the most important of the misfolding diseases is Alzheimer’s disease (AD), where the amyloid deposition is in the brain and causes severe demen-tia and eventually death. This disease affects approximately 10 % of all hu-mans at 65 years of age and every second of all those who have reached 85 years (Gaggelli et al., 2006).

AD is named after the psychiatrist and neuro-pathologist Alois Alz-heimer. He described the histological features of AD in a famous paper 1907 called “Über eine eigenartige Erkrankung der Hirnrinde” (About a peculiar disease of the brain cortex) (Alzheimer, 1907).

Alzheimer’s disease shows accumulation of amyloid both intra- and ex-tra-cellularly. Intracellularly the tau-protein forms amyloid fibrils and ex-tracellularly the fibrils are formed mainly by the amyloid β-peptide (Hardy and Selkoe, 2002; Selkoe, 2003) These extracellular depositions are called neuritic (or senile) plaques. Deposition of tau-protein alone gives a dementia other than AD, and this is called fronto-temporal dementia, while deposition of Aβ alone induces tau-deposition and results in AD (Evin and Weidemann, 2002; Hardy and Selkoe, 2002).

Aβ is a 39-42 residue peptide, most commonly 40 or 42 residues, that is cleaved from a transmembrane protein, the amyloid precursor protein (APP). In vivo, the Aβ concentration is nanomolar. In vitro the cleaved Aβ occurs a monomer at low concentrations, ≤ 30 µM, and at physiological pH and room temperature. Mainly, the Aβ-peptide is in random coil conformation at these conditions and there is no significant difference in structure between the 40 and 42 residue long fragments (Riek et al., 2001). However, the 42 residue fragment Aβ(1-42) is more prone to assemble into fibrils already at low con-centrations (Harper and Lansbury, 1997). Other fragments of the Aβ-peptide also form fibrils, and the central hydrophobic cluster Aβ(16-22) is the short-est among the fibril-forming fragments (Balbach et al., 2000).

Amyloid precursor protein

The Aβ-peptide is an enzymatic cleavage product of the amyloid precursor protein (APP), a ubiquitous protein found in different tissues, but exists in higher concentrations in the brain. APP, discovered as late as 1987, is a transmembrane protein that is anchored to the neuron cell membrane with one transmembrane segment (TMS). There are different isoforms of the pro-tein with different sizes, ranging from 695 to 770 residues. The most abun-

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dant isoform in the neuron is the 695-residue APP. The difference between the shorter and the longer variants is a central protease inhibitor segment that is present in the longer fragments. The longer isoforms are present in the blood platelets and are involved in the coagulation cascade regulation. All isoforms of APP contain the Aβ-segment (Selkoe, 1998).

Figure 1. Schematic picture of APP inserted into a biological membrane. The trans-membrane segment is represented as a cylinder and the Aβ fragment is highlighted in a box, with the sequence of the peptide shown. In the N-terminal extracellular part of APP two specific binding sites for divalent metal ions are located. The proteolytic cleavage sites are also shown.

APP has a hydrophobic segment that is assumed to be the TMS, and this hydrophobic TMS is located in the C-terminal region of the protein ranging from position G625 to L648 (Figure 1). The Aβ-segment ranges from residue D596 to V636 or A638 depending on Aβ length. Thus, the Aβ-segment is par-tially located in the membrane. The TMS is assumed to be an α-helix, even though no structure of the membrane bound APP in the membrane has been reported. Several studies of the peptide in membrane mimicking systems suggest that the Aβ-segment located in the membrane adopts an α-helical secondary structure (Coles et al., 1998; Shao et al., 1999). This, and the fact that the rest of the putative membrane spanning region of APP consists of residues that are prone to adopt α-helical secondary structure suggest that the whole TMS is helical.

The biological function of neuronal APP is not entirely known. The to-pology of the protein with an extracellular large part anchored to the mem-

D596 A638

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brane with one TMS resembles that of a receptor, and APP has been sug-gested to be involved in G-coupled cell signalling as well as being a regulat-ing protein of cell trafficking (Okamoto et al., 1995; Sabo et al., 2001).

APP has also been suggested to be involved in metal homeostasis of the cell. APP has selective metal binding sites and it participates in the regula-tion of the copper levels and is affected by the copper level. Lowering the copper levels down-regulates the gene expression of APP and APP gene knockout increases copper levels (Storey and Cappai, 1999; Phinney et al., 2003; Bellingham et al., 2004a-b; Maynard et al., 2005). Depletion of metals in drinking water gave lower Aβ fibril formation among rabbits in vivo (Sparks and Schreurs, 2003). In the extracellular part of APP one can find specific binding sites for copper and zinc, close to the N-terminus of the protein. There are also other specific binding sites on APP that bind heparin and collagen (Frederickson et al., 2005).

Not only the membrane bound APP may exhibit specific functions but also a soluble fragment of APP, that is the degradation product of α-secretase cleavage, is responsible for potential functions of APP. The struc-ture of this cleaved fragment has only been reported as a course grain model (Gralle et al., 2006). This soluble fragment is responsible for APP effects in coagulation (Selkoe DJ, 1998). APP may have multiple functions in the membrane-bound native state or in the soluble cleaved form. The neurotoxic peptide fragments Aβ(1-40) and Aβ(1-42) are however the results of double proteolytic cleavage of APP by two secretases, other than α-secretase.

Proteolytic degradation of APP and the formation of soluble Aβ

Normal APP is anchored to the membrane with one transmembrane segment as described above. In the normal degradation of APP two proteases are involved, which produce three APP fragments. First, α-secretase cleaves the protein at position 625 on the extracellular side and produces the large frag-ment that is suggested to have some biological function. The rest of the pro-tein is still attached to the membrane and is cleaved by γ-secretase at posi-tion 636-638 and the two produced fragments leave the membrane. This pathway of degradation of APP is called the non-amyloidogenic pathway because the released fragments form neither toxic oligomers nor fibrils (Maccioni et al., 2001; Hardy and Selkoe, 2002; Blennow et al., 2006). The pathologic, amyloidogenic pathway also involves the γ-secretase but the extracellular cleavage is performed by another protease, a β-secretase. The β-secretase is a membrane bound protease enzyme and the full name is β-site

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APP-cleaving enzyme 1 (BASE1) (Vassar et al., 1999). BASE1 cleaves the APP at position 596 and upon subsequent cleavage by γ-secretase the 40-42 residue Aβ-peptide is the produced (Figure 1).

This sequence of events results in that the peptide is being partly inserted into the membrane when the rest of the APP is enzymaticly removed by the secretases. Generally the Aβ peptide is assumed to immediately leave the membrane and go into solution, but this hypothesis has recently been chal-lenged and the peptide has been suggested to stay in the membrane and di-rectly exhibit its toxic effect (Marchesi, 2005). In solution the peptide ap-pears as a monomer at sufficiently low concentrations. The cleaved Aβ-peptide has the sequence:

DAEFR5HDSGY10EVHHQ15KLVFF20AEDVG25SNKGA30IIGLM35VGGVV40IA

The peptide has some amphipathic properties with a hydrophobic C-terminal region and a hydrophilic N-terminal region. This is a property that the Aβ-peptide shares with other amyloidogenic peptides and proteins such as those derived from Huntingtin and the 106-126 fragment of the prion peptide. The much longer amyloidogenic protein α-synuclein shows a similar pattern, but this protein has periodic alternating regions of hydrophobic and hydrophilic regions (Murphy, 2002; Chiti and Dobson, 2006).

Escaping the membrane involves a structural transition of the Aβ-peptide from the membrane-bound α-helical secondary structure to the mainly un-structured solution state peptide. Solution state studies of Aβ reveal that there are some non-random regions of the peptide, in the central parts, but that no well-defined secondary structure is present in aqueous solution (Riek

et al., 2001). Molecular dynamics simulations of the Aβ(1-42) peptide showed that also the N-terminal region exhibited some order (Flöck et al., 2006). MD studies of the monomeric soluble Aβ-peptide have mainly con-cerned the α-helix to β-sheet structural conformational transition and the peptide was forced into a helical conformation as an initial condition (Borreguero et al., 2005; Xu et al., 2005).

The physiological concentration of Aβ in the cerebrospinal fluid (CSF) is nanomolar, as in plasma. This is much below the critical concentration for spontaneous aggregation to initiate (Harper and Lansbury, 1997). The criti-cal concentrations for aggregation of Aβ(1-40) and Aβ(1-42) differ. The concentration is slightly higher for Aβ(1-40), meaning that monomeric Aβ(1-40) is more stable than the longer fragment. This implies that in order to aggregate, Aβ has to be enriched in specific regions in the brain to a con-centration above the critical concentration. This can be achieved in different ways, where one is obtained by binding of Aβ to a membrane which would increase the effective concentration (Terzi et al., 1997).

After escaping the membrane the Aβ-peptide is removed from the tissue by peptide degradation, performed by the enzymes neprilysin and endo-

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thelin-converting enzyme. A flux of Aβ across the blood-brain barrier is also present, assisted by co-proteins (Carson and Turner, 2002; Tanzi et al., 2004). An imbalance between production and removal of the peptide in-creases the amount of peptide available for toxic action.

Oligomerization of the Aβ-peptide and neurotoxic mechanisms

Despite the fact that there is strong evidence to support the hypothesis that Aβ is responsible for the dementia of AD (Chen et al., 2000; Janus et al., 2000; Selkoe and Podlisny, 2002; Westerman et al., 2002), the soluble monomeric form of Aβ does not seem to exhibit any direct neurotoxicity. The concentration of free monomeric Aβ does not directly relate to the se-verity of the memory impairment (Lesné et al., 2006), but soluble oligomeric forms of Aβ seem to exhibit that correlation (Hartley et al., 1999; Hsia et al., 1999; Ward et al., 2000; Klein et al., 2001). The discovery of the soluble oligomers and the correlation between their presence and dementia have led to an amyloid cascade hypothesis that describes the cause of AD on a mo-lecular level (Figure 2) (Hardy and Selkoe, 2002),.

Figure 2. Two pathways for aggregation of the amyloid β peptide. In the case of protofibril formation and subsequent assembly into fibrils the peptide is caught into the stable fibrils and may be kept from toxic effects. The neurotoxic effect brought about by Aβ may be caused by a dodecamer (n = 12) of the peptide.

The Aβ-peptide first undergoes oligomerization and then further aggrega-tion into protofibrils and fibrils. The oligomers are soluble and are suggested to play an important role in the pathogenic cascade of AD by being toxic to neurons (Bucciantini et al., 2002; Gong et al., 2003; Dobson, 2004). The

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oligomers share their topological features with the oligomers of other amy-loidogenic peptides. This suggests that there is a common cell-toxic mecha-nism exhibited by these peptides (Kayed et al., 2003).

The structure of the rather newly discovered oligomeric species is not known in detail. Recently a specific Aβ oligomer has been suggested to have specific toxic effects on neurons. This is a dodecamer that binds specifically to the dendritic processes of the neuron and blocks the membrane potentiation (Barghorn et al., 2005; Muresan and Muresan, 2006). This do-decamer also impairs memory function in mice, the memory impairment is directly linked to the prescence of dodecameric Aβ (Lesné et al., 2006). In molecular terms the oligomer can be characterized as a micelle of Aβ-peptides with the hydrophobic C-terminus buried in the micellar center and a critical micelle concentration of 17.6 µM (Kayed et al., 2003; Sabaté and Estelrich, 2005). The peptides in the oligomer are mainly unstructured (Chiti and Dobson, 2006).

The oligomeric Aβ may aggregate further and build up protofibrils and fibrils. However, the oligomers are not necessary for fibril formation and oligomerization and fibrillation are suggested to be different pathways in Aβ metabolism (Figure 2) (Bitan et al., 2003; Barghorn et al., 2005; Chen and Glabe, 2006). Formation of protofibrils and fibrils may be a protective event in order to lower the oligomeric concentration (Carrotta et al., 2005).

The mechanism by which the Aβ oligomer exhibits neurotoxicity is not clear, but production of reactive oxygen species has been suggested. Another suggested mechanism is that the oligomers change cell membrane function and thereby disturb calcium homeostasis and/or membrane dynamic proper-ties. Yet another mechanism suggested is alteration of metal homeostasis (Bush et al., 2003b; Walsh and Selkoe, 2004). A common feature for all these proposed mechanisms is that they lead to destruction of synapses and consequently cell death. The basic requisite for Aβ to become toxic is the structural conversion from the non-toxic soluble form to the toxic oligomeric form. The toxicity is induced by a misfolding event (Chiti and Dobson, 2006).

The oligomerization may expose certain reactive residues of the peptide and the number of reactive residues should increase upon aggregation into the dodecamer. More and more evidence suggests that the oligomeric forms of the peptide alter the membrane integrity of the cell. In vitro selective cation channels are formed by Aβ-peptide. This is also supported by the fact that Aβ changes the Ca2+ homeostasis giving rise to increased intracellular Ca2+ -levels. These channels do not show a single morphology, but an AFM study suggests that the channels have well-defined structures and similar topology as seen in channels formed by other amyloidogenic peptides (Arispe et al., 1993; Lin et al., 2001; Quist et al., 2005).

At this stage in the life of Aβ several structural transitions have occured, from the membrane-anchored APP-bound largely α-helical peptide to the

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soluble non-toxic monomeric peptide and further on to the oligomeric toxic state of the peptide. This is however not all, since the peptide can undergo yet another transition and accumulate to fibrils with a very well-defined structure.

Fibrilization and structure of amyloid fibrils

The assembly of Aβ peptides into fibrils may occur either on the so called activated monomer (Taylor et al., 2003) to fibril-end basis or by assembling of oligomers, involving a bead on a string model. The kinetics of fibriliza-tion of supersaturated Aβ includes a lag phase where the monomeric peptide is in fast exchange with oligomeric species. After some time a seed is formed, a nucleus of aggregated peptides in fibril formation, and this seed promotes rapid aggregation of peptides into the fibrillar structure. The time gap before the seed is formed and rapid aggregation begins is called the lag time and is very dependent on sample conditions and may range from min-utes to days (Murphy, 2002; Sabaté and Estelrich, 2005). When the aggrega-tion process is initiated it is a single non-cooperative process (Carrotta et al., 2005). The aggregated Aβ, earlier assumed to be a toxic species, has been suggested to be a protective escape route for the peptide. The peptide is kept out of the equilibrium between monomers and toxic oligomers (Carrotta et

al., 2005; Barnham et al., 2006). In the aggregated form Aβ forms amyloid fibrils, a structure that shows

features that are similar for all amyloidogenic proteins and peptides. The general topological properties of the fibrils are those of an elongated fiber, up to a µm long and approximately 20 nm in diameter. The fibril consists of two filaments, twisted around each other in a left-handed helix (Sachse et al., 2006).

On a molecular level the structure of Aβ in the fibrils has recently been reported using solid state NMR, site-specific mutagenesis and X-ray diffrac-tion. In the fibrils Aβ adopts a β-sheet secondary conformation. The N-terminus of the peptide is mainly unstructured and there are two β segments in the central region and the C-terminal regions. The residues that constitute the β-segments are 17-24/25 and 31-40 and these segments are separated by a turn (Figure 4). The segments are kept together by hydrophobic interac-tions and the peptide is hydrogen-bonded to the adjacent peptide along the fibril axis (Shivaprasad and Wetzel, 2004, 2006; Lührs et al., 2005; Nelson

et al., 2005; Chimon and Ishii, 2005; Sachse et al., 2006; Petkova et al., 2006).

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Figure 3. The molecular arrangement in Aβ fibrils. Each monomer has two regions with β sheets that are held together by hydrophobic interactions. The monomers are kept together by hydrogen bonds between the molecules. The hydrogen bonds are here represented by thick black lines.

The fibril is most likely the final stage of the Aβ-peptide and in this su-perstructure the peptide adopts a β-strand secondary structure. Thus, in its life-span Aβ undergoes three structural transition where the transition from mainly unordered monomeric peptide to the fibril-bound cross-β structure is the final one. This structural transition seems to remove the peptide from the toxic pool and fix it in an immobile state.

Metal interaction of the soluble Aβ-peptide

An increased copper, iron and zinc concentration has been found in the brains of Alzheimer’s disease patients, enriched in the core of the amyloid plaques but also generally in the cortical tissue (mainly zinc) (Lovell et al., 1998; Religa et al., 2006). As described above, APP has specific metal bind-ing sites for copper and zinc and is believed to participate in the regulation of metal homeostasis. The soluble monomeric Aβ-peptide also binds metals, mainly copper and zinc at specific binding site(s). These site(s) is/are not identical with the binding sites of APP which are located in the extracellular domain of APP. Binding of metal to the peptide alters the solubility proper-ties of the peptide in a non-trivial manner.

High concentrations of copper and zinc induce aggregation of Aβ, and high concentration in this case corresponds to a metal:peptide ratio >1 (Bush

et al., 1994b; Brown et al., 1997; Huang et al., 1997; Raman et al., 2005). The aggregate formed is suggested to be amorphous and unspecific and it does not contain any well-defined structure. High metal concentrations may therefore prevent the formation of the cross-β rich fibrils discussed above (Brown et al., 1997; Yoshiike et al., 2001; House et al., 2004; Raman et al.,

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2005). The metal induced aggregation of Aβ has been suggested to be the result of intermolecular His-His bridging after which an amorphous aggrega-tion occurs (Smith et al., 2006; Stellato et al., 2006; Syme and Viles, 2006).

Low concentrations of copper and zinc, on the other hand, reduce aggre-gation of Aβ and help in keeping it as a soluble monomer. Metal interaction is also able to destabilize Aβ oligomers and shift the monomer-oligomer equilibrium towards monomers (Cardoso et al., 2005; Garai et al., 2006). The normal zinc and copper concentrations in the cerebrospinal fluid (CSF) are 3 µM and 1 µM respectively, but during synaptic transmission the con-centration of Zn2+ locally increases up to 0.3 mM (Molina et al., 1998; Bush, 2003a). In the normal case (when the Aβ concentration in CSF is ~ nM) the low metal concentration in the CSF helps to keep the peptide monomeric and soluble but an altered metal homeostasis may, directly or indirectly, induce toxic oligomers.

Metal ions like copper and zinc bind with high affinity and specificity to the monomeric Aβ. The binding sites for copper and zinc have been shown to be located in the N-terminal part of the peptide and for copper the coordi-nation has been suggested to be planar. Increasing evidence show that the three histidines, His6, 13 and 14 are involved as ligands. The fourth ligand has been suggested to be Tyr10 but N-terminal mutations and acetylation suggest that the N-terminal amide nitrogen acts as the fourth ligand (Kowalik-Jankowska et al., 2003; Syme et al., 2004; Karr et al., 2005; Tickler et al., 2005). Also in the case of zinc the histidines have been shown to be necessary for high affinity binding to Aβ (Liu et al., 1999; Miura et al., 2000; Curtain et al., 2001; Syme and Viles, 2006). In the case of the N-terminal fragment Aβ(1-28) and Aβ(1-16) site specific mutations where one or two of the ligands were replaced by alanines show that His13 and His 14 are absolutely crucial for zinc binding while His6 increases the affinity sig-nificantly, in (Yang et al., 2000; Kowalik-Jankowska et al., 2003).

The identity of the fourth ligand, in addition to the three histidines, neces-sary for zinc coordination has been under some debate, and experiments have provided contradictory results. In most studies mainly shorter frag-ments of Aβ have been used. In some of these studies Tyr10 has been sug-gested to be a ligand, but also Glu11, Arg5 or the N-terminus (Zirah et al., 2004, 2006; Mekmouche et al., 2005; Syme and Viles, 2006). When the full length peptide was used to study metal binding the results suggest that the N-terminus is the fourth ligand (Hou and Zagorski, 2006).

The soluble Aβ-peptide’s membrane interactions

As described earlier Aβ is produced by the cleavage of APP inside the mem-brane and thus the peptide is initially located in the membrane. Immediately

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after cleavage the peptide leaves the membrane and ends up in monomeric, oligomeric and subsequently aggregated amyloid forms. The most likely toxic entity is the oligomeric form of the peptide but the exact toxic mecha-nism is still not entirely understood.

The toxic mechanism of the oligomers is thought to be either a direct or indirect mechanism, mediated through oxidative stress or by inducing in-flammatory processes. More and more evidence also suggests that the oli-gomeric forms of the peptide alter the membrane integrity of the cell. In vitro, selective cation channels are formed by the Aβ-peptide This is also supported by the fact that Aβ changes the Ca2+ homeostasis with increased intracellular Ca2+-levels (Arispe et al., 1993; Lin et al., 2001). These chan-nels do not show a single morphology, but an AFM study suggests the chan-nels to have a well-defined structure and consist of 4-6 peptides. The pore structures are not known in detail, but similarities with β-barrel pore forming toxins has led to the suggestion that the peptide, which is prone to form β structures, forms β-barrel pores (Lin et al., 2001; Lashuel et al., 2002).

Aβ induces leakage of sodium, potassium and calcium into lipid vesicles, but only in vesicles with negatively (partially or completely) charged headgroups (Kourie et al., 2001; Alarcón et al., 2006). The peptide does not insert itself into neutral membranes and this is suggested to explain the lack of peptide-induced influx of ions. In negatively charged model membranes the peptide both inserts into the membrane and induces leakage (Bokvist et

al., 2004). Interestingly, in the presence of metal ions (copper and zinc) the peptide, when interacting with the negatively charged vesicle, exhibits a structural transition from a dominating β structure to a high α-helical con-tent. This suggests that, in presence of copper and zinc the oligomeric aggre-gate in the membrane is formed by a small number of transmembrane heli-ces, and this may build up the channel, disrupting the membrane integrity. A particular property of the Aβ channel is that zinc inhibits the channel perme-ability, suggesting that zinc either blocks the channel or alters the channels properties, such as structure, in such way that ion leakage stops (Arispe et

al., 1996). The metal binding site of Aβ is not located in the assumed membrane

spanning region but still seems to influence the membrane interaction of the peptide. The shorter fragment Aβ(1-28) and the reversed sequence Aβ(40-1) neither insert into the vesicles, nor cause any leakage (Curtain et al., 2001, 2003; Alarcón et al., 2006). The hydrophobic region in the shorter fragment Aβ(1-28) is too short to penetrate the membrane. The hydrophobic region in the membrane corresponds to 20-23 amino acids in a α-helical conformation. It should be pointed out that the putative membrane spanning residues, in the Aβ peptide, are not the same residues that form the transmembrane segment of APP.

The structure of the membrane bound Aβ has been studied in various membrane mimicking media, such as SDS micelles or TFE/water mixtures.

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The results reveal two regions that adopt α-helices, i.e. the C-terminal region including residues 29-36/38 and a central region including residues 15/16-24. These regions could correspond to the transmembrane segment of the membrane bound soluble Aβ. The α-helical regions are separated by a kink corresponding to residues 25-29 (Coles et al., 1998; Watson et al., 1998; Shao et al., 1999; Crescenzi et al., 2002; Lau et al., 2003).

Ligand binding to Aβ, and other strategies to prevent Aβ-toxicity

The pathology of AD includes a series of stages starting with increased lev-els of soluble Aβ, possibly due to mutations in APP close to the cleavage sites of the proteases or a decreased clearance of produced Aβ. Following the increased level of Aβ-peptide, oligomerization occurs and oligomeric and protofibrilic forms of the peptide appear. The oligomers/protofibrils then either induce inflammatory processes in microglia and astrocytes or cause direct synaptic and neuritic injuries. Membrane integrity changes may cause changed ionic homeostasis, and thus oxidative stress and injury. This causes widespread neuritic death and consequently dementia and death (Ghiso and Frangione, 2002; Hardy and Selkoe, 2002; Chiti and Dobson, 2006).

Several families of strategies to prevent AD can be identified (Hardy and Selkoe, 2002; Dobson, 2004). First, the action of the proteases could be in-hibited or altered, which thereby lowers the level of Aβ-peptide. Inhibition of γ- or β-secretase would stop Aβ production totally. However, this strategy would also inhibit other, potentially important functions of the secretases (Masters et al., 2006). Related to this approach is the administration of a molecule that targets APP and inhibits the proteolytic effects of the proteases (Espeseth et al., 2005). Second, the oligomerization of the peptide could be a target for inhibition as well as the degradation of already formed oligomers. This may be done by changing the properties of the monomer to inhibit the aggregation process or by immuno-neutralization of soluble Aβ-oligomers (Klein et al., 2001; Brendza et al., 2005; Garai et al., 2006; Ali et al., 2006). Third, the inflammatory process induced in the disease can be a target for treatment. Another strategy is chelation of metal ions such as Cu2+ and Zn2+, by chelators such as Clioquinol. This is closely connected to the strategy that targets oligomerization of the peptide (Raman et al., 2005; White et al., 2006). In addition to these strategies selective Aβ-channel blocking and ad-dition of membrane stabilizing agents could be ways to in prevent the toxic events of AD (Kagan, 2005).

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One of the treatment/prevention strategies presented above is very suit-able for studies with biophysical methods; namely ligand binding to the pep-tide. The ligand should be constructed such that the properties of the com-plex differ from those of the Aβ-peptide alone, and thus the ligand prevents the toxic effects. Ligands are also a good strategy because it may be possible to administer them orally. A number of different ligands have been pro-posed. Some have an aggregation reducing effect and these ligands may also reduce toxicity. Among other substances, nicotine is reported to bind to and inhibit aggregation of the Aβ-peptide. The interaction is suggested to be non-specific and involves the N-terminal histidines, either direct or indirect by a chelating effect and thus inhibit normal metal interactions with the His residues (Salomon et al., 1996; Dickerson and Janda, 2003; Moore et al., 2004). As described above, metal binding to the Aβ-peptide alters the aggre-gation properties of the peptide. Curcumin from the Turmeric root has also been shown to interact with Aβ and reduce peptide aggregation. The interac-tion seems also to destabilize formed fibrils, possible by pushing the equilib-rium in the monomer-oligomer-fibril system towards the monomeric form (Ono et al., 2004).

Another strategy is to use peptide ligands to induce peptide-peptide inter-actions. Several short peptides interact specifically with the soluble Aβ-peptide and particularly sequences of the peptide itself have been studied (Santhoshkumar and Sharma, 2004; Schwarzman et al., 2005). Different fragments of the peptide, mainly including the hydrophobic central sequence 16-21, reduce fibril formation and neurotoxicity (Tjernberg et al., 1996; Hetényi et al., 2002; Matsunaga et al., 2004).

The cyclic oligosaccharide, β-cyclodextrin, interacts with the Aβ-peptide and the interaction is suggested to be between the inside of the cyclodextrin ring and Phe19 or Phe20 (Qin et al., 2002). The inhibition of aggregation has been determined with scintillation proximity assay to a 50 % inhibition by a 5 mM concentration of β-cyclodextrin and the interaction inhibits the forma-tion of the soluble oligomers (Yu et al., 2002). Mass spectroscopy has shown that the stoichiometry of the Aβ-peptide and β-cyclodextrin is one-to-one (Camilleri et al., 1994).

Cyclodextrins form a family of cyclic oligosaccharides that are e.g. used in pharmaceutical preparations when slow release of a drug is of interest. The cyclodextrins are torus-shaped rings built up by different numbers of glucose residues. There are three major types of cyclodextrins, the α-, β- and γ-cyclodextrins, in which the rings consist of six, seven and eight glu-copyranose units, respectively. The cyclodextrins have different characteris-tics due to differences in size, e.g. the solubility. The cyclodextrin mole-cules are amphiphilic molecules. The cavity of the torus is hydrophobic while the rest is hydrophilic, making the cavities favourable places for

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Figure 4. The structure of β-cyclodextrin. Light regions represent hydrophilic re-gions and dark regions represent hydrophobic regions. The two pictures reflect the two different sides of the oligosaccharide.

hydrophobic interactions (Figure 4). The differences in size of the hydro-phobic cavity in the different cyclodextrins give possibilities of specificity in interaction (Szejtli, 1998; Aachmann, 2003).

The main interaction with amino acid residues seems to be between the aromatic rings of phenylalanines and the hydrophobic cavity of cyclodextrin. The dissociation constant of a single phenylalanine amino acid and the dif-ferent cyclodextrins is 23 mM, 56 mM and 7 mM for the α, β, and γ- cyclo-dextrin, respectively (Matsuyama et al., 1987; Castronouvo et al., 1995; Aachmann, 2001). In this case, with phenylalanine alone, the interaction is strongest with the cyclodextrin with the largest hydrophopic cavity and gen-erally the interactions involving cyclodextrins are one-to-one (Szejtli, 1998). However, there is no direct correlation between cavity size and affinity for phenylalanine.

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Theory of hydrodynamic dimensions and structural conversions of peptides

“Whenever a theory appears to you as the only possible one,

take this as a sign that you have neither understood the theory

nor the problem which it was intended to solve”

Karl Popper

In addition to structural and dynamic properties of a peptide/protein it is important to characterize the hydrodynamic properties, such as hydrody-namic radius and diffusion coefficient that are related to structural and dy-namic properties. Stoke-Einstein’s equation relates the diffusion coefficient to the hydrodynamic radius, RH. RH may provide information on the folding and structural state as well as interaction with other molecules or self-aggregation (Cameron and Fielding, 2001; Dehner and Kessler, 2005). The dynamic hydration of a peptide or protein is also reflected in the hydrody-namic radius (Halle and Davidovic, 2003). In this section the framework for the studies of RH presented in this thesis are outlined. Some polypeptide the-ory used to describe general peptides, such as Aβ is discussed.

The Aβ peptide has some structural propensities that include undergoing a structural transition when raising the temperature. Studying the thermody-namics of this structural transition provides information on the stability and energetics of the structure. The method used to calculate enthalpies and co-operativity of the transition is also outlined in this section.

Dimensions of polymers and polypeptides

A polypeptide chain can in its simplest form be approximated with a random walk with stepsize l0. The radius of gyration for this simple model is given by:

(1)

∑ ∑ ∑

+

−+−

+=

= +=i

i

j

N

ij

jjg rN

jNr

N

j

NR

2

1 1

2

11

11

||

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Here N is the number of residues, ri is the vector pointing at residue i and < > represents the average. In this model the radius of gyration is the average distance of a residue to the center of mass.

In a simple random walk model the typical extension of a random walk with N steps and step size l0 is given by:

(2)

This gives the end-to-end distance, which is directly proportional to the ra-dius of gyration. Thus, the radius of gyration increases with the square root of the number of segments in a freely jointed chain. For such a freely jointed chain with no interactions between the segments the characteristic length, the step size, is equal to the length of the segment. For a random walk with con-strained angles the step size is bigger than the length of the segment but the overall scaling is the same, if the number of steps (segments) is large enough. This simple model of a polypeptide chain as a random walk is un-fortunately not very consistent with reality. First, all directions are not equally probable between subsequent segments. Second, a true polypeptide chain must be self-avoiding. It is not possible for two segments to exist at the same place at the same time.

A model with segments of finite volume with a repulsive interaction be-tween the segment and the rest of the chain was proposed by Flory and de Gennes (Flory, 1988). A simple argument where the free energy, F, of a chain with N segments is evaluated below. Assume that the radius of the volume occupied of the chain is R. The concentration of segments is then:

(3)

Where k1 is a proportionality constant. The repulsive interaction between the segments will give an interaction energy that is proportional to the number of pairs of segments and thus to the square of the concentration, and propor-tional to the volume.

The entropy, S, for a freely jointed chain is:

(4)

20

2

,

2 |||| NllllRN

i

i

ji

ji ==⋅= ∑∑

31R

Nkc =

20

2

23Nl

RkS B−=

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The free energy is given by F = E-TS, where E is the energy, and using equa-tions 3 and 4 an expression for the energy is obtained:

(5)

The radius that minimizes the free energy in equation 5 gives:

(6)

This result is remarkably close to experimental data despite the simplicity

and many approximations in the model (Flory, 1988). A more rigorous treatment of the free energy gives a more complex ex-

pression. If only the terms depending on R are kept it is:

(7)

In equation 7 three new parameters are introduced: d is the dimensionality, v is the volume of one segment in the chain and ε describes the monomer-monomer interaction, and is an attractive interaction if it is positive and a repulsive when it is negative.

When introducing the parameter δ = 1-ε / (vkBT), we see from examining equation 7 that there are three possibilities, δ > 0, δ = 0 and δ < 0.

When for δ > 0 differentiation of equation 7 gives the radius of gyration which scales with N as:

(8)

For the 3D case this is very close to experimental and simulation data and exactly as predicted by the simple approach above. This is called the Flory scaling and the chain is said to be in a swollen state (de Gennes, 1979).

A special case is obtained when δ = 0, the so called θ-point. Here the ra-dius scales as the random walk of the freely jointed chain, R ∝ N½. In this case the interactions between the monomers are equal to the interactions between the solvent and the segments.

20

2

3

2

2 23Nl

RTk

R

NkF B+=

5

3

320

25

NconstR

NTk

lkR

B

⋅=

=

06

32

20

22

ln262

3)1(

21

l

R

R

Nv

Nl

R

R

Nv

TvkTk

Fd

BB

−++−=ε

dNR +∝ 23

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In both of the above described cases the scaling depends on N if δ is small but positive. For short chains where N < 1 / δ

2 the random walk scaling dominates, and for longer chains the Flory scaling is valid. For δ < 0 the chain tends to collapse and the radius scales as R ∝ N1/3.

One can conclude that the radius of gyration and thus the hydrodynamic radius is related to the number of segments in the chain by a simple scaling law (Brochard and de Gennes, 1977; Fitzkee and Rose, 2004; Kohn et al., 2004):

(9)

All these expressions are derived and valid for long chains, such as polypep-tides. But equation 9 is of course valid for all types of macromolecules. Light scattering experiments on highly denaturated proteins and peptides shows a scaling factor of ν = 0.598 and Monte Carlo simulations yield the same result (Miller and Goebel, 1968; Lifshitz et al., 1978; Fitzkee and Rose, 2004; Kohn et al., 2004).

Translational Diffusion of peptides

The radius of gyration is related to the hydrodynamic radius, and is directly proportional, Rg = dRH. Where d is a proportionality factor. The upper limit of the proportionality factor, d = 0.775, is valid for spherical non-interacting particles. For polypeptides this value is always lower. However, the scaling is the same for the end-to-end distance, the radius of gyration and hydrody-namic radius.

One method to measure the hydrodynamic radius is to measure the trans-lational diffusion coefficient. Translational diffusion is caused by the ran-dom collisions with solvent molecules; this causes gain of momentum for the diffusing agent and thus random movement. The random movement is called Brownian motion and may occur in any number of spatial dimensions. The hydrodynamic dimension of the polypeptide chain reflects directly in the translational diffusion coefficient, Dt, through Stokes-Einsteins equation:

(10)

Here Boltzmann’s constant kB is introduced, T is the absolute temperature and η is the dynamic viscosity. The hydrodynamic radii of peptides and pro-teins can be determined via the measurements of their diffusion coefficients (Miller and Goebel, 1968; Lifshitz et al., 1978; Fitzkee and Rose, 2004;

νNR ∝

H

Bt

R

TkD

πη6=

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Kohn et al., 2004). The hydrodynamic properties yield information on the conformational state of the polypeptide, and if the chain follows the ex-pected Flory scaling or not. If peptides or proteins of various sizes have hy-drodynamic radii that scales with 1/3 one can draw the conclusion that they are in a folded state (Wilkins et al., 1999). Most reports on determining the hydrodynamic radii have been using small angle scattering or through the diffusion coefficient measured by dynamic light scattering.

The hydrodynamic radius of a peptide chain is, as shown above, depend-ent on the number of segments in the chain. It is also valid that a general volume can be written in terms of the hydrodynamic radius:

(11)

Where Θ is a proportionality constant. For a sphere it is 4π/3 and ϑ is a gen-eral exponent with the value 3 in the spherical case. The mass is related to the volume through the density and thus the diffusion coefficient via the hydrodynamic radius can be related to the mass of the peptide and the scal-ing behaviour of the peptide can be studied.

So far the diffusion is assumed to occur in an infinite dilute solution with no or very small interactions between the diffusing agents. In solutions with high concentrations the diffusion is not entirely free and is affected by ob-struction. The concentration dependence of the measured diffusion Dt is approximately given by:

Dobs = D0 (1-3.2λΦ) (12)

Here D0 is the diffusion coefficient at infinite dilution, λ is a shape and inter-action dependent parameter (λ = 1 for a hard, non-interacting sphere) and Φ is the dry volume fraction (Tokuyama and Oppenheim, 1994)

Structural transitions

Structural transitions in peptides and proteins can occur on different levels, on the tertiary structure level or the secondary structure level. For peptides the structural transitions mainly occur on the secondary structure level. In peptides an important structural state is the left-handed 31-helix, also called polyproline type II helical structure (PII) or the 32-helix. It has been discov-ered that PII is an important secondary structure in seemingly unstructured peptides, such as Aβ in solution. Not only the polyproline peptides adopt this

ϑHRV Θ=

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structure but many other peptides (Wilkins et al., 1999). Structural transi-tions can be induced by adding energy that exceeds the energy that stabilizes the structure. A typical example is adding heat to an α-helix until the stabi-lizing hydrogen bonds break and a transition towards random coil occurs. The structural transitions can be modelled using statistical physics. PII heli-ces do not have any inter-residue hydrogen bonds so a slightly modified Zimm-Bragg analyzis can be used (Zimm and Bragg, 1959). The modifica-tion is simply that the requirement to have at least three successive segments in helix conformation is removed and that only the first segment is assumed to be in a random coil conformation.

Assuming that a two state transition is studied, and every amino acid resi-due can adopt either PII-helix or random coil, then the partition function for a molecule is given by the sum of all possible states. All states are however not equally possible so a statistical weight is used. Each state’s statistical weight is the product of statistical weight factors from the different combina-torial possibilities. The model is presented in more detail in paper IV and by Zimm and Bragg (Zimm and Bragg, 1959).

Performing the calculations results in a surprisingly simple expression for the partition function, Q, and the fraction of residues in PII structural state, θ, can be calculated from Q.

(13)

The parameters s and σ can be interpreted in physical terms. s can be thought of as a equilibrium constant for the PII-helix to coil transition and thus re-lated to the enthalpy change of the system due to conversion of one segment from unordered to PII-helix. The relation is the well-known thermodynamic relation:

(14)

The parameter σ can be interpreted as the cooperativity of the conversion, a value close to one for low or no cooperativity and a low value for high coop-erativity. The temperature dependence of the transition can be studied and if the populations of the structural entities can be determined the parameters s

2

lnRT

H

dT

sd ∆=

))(1()(

))()()(1(

)(1

)(1

10

10

1100

011

1

1

100

0

0

0

0

λλ

γγ

λλλλ

λλλ

γ

λ

γλλ

λ

γ

λ

γ

θ−−

−−

−+−−

−++−

−+

=N

s

ssN

ss

Ns

s

Ns

NN

NN

( ){ }

s

sss

∂=

++±+=

1,01,0

21,0 411

21

λγ

σλ

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and σ can be determined from equation 13. s should be approximately linear close to the transition temperature Tm.

(15)

At s = 1 the two states are equally populated and σ determines the slope of the transition.

The outcome of this is that by measuring the PII-helical fraction at differ-ent temperatures, preferably close to the transition temperature, it is possible to calculate the enthalpy change of the system due the structural transition, the transition temperature and the cooperativity. This of course holds for all similar structural transitions, not only the PII-helix to random coil transition.

( )m

m

TTRT

Hs −

∆−= 21

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Spectroscopic methods

“It is a mistake to think you can solve any major problems

just with potatoes”

Douglas Adams

Different dynamical features occur on different time-scales. Time-scales in biomolecules can be defined in terms of correlation times, τc. Mathemati-cally the correlation time is defined by the correlation function. It is the char-acteristic time of the exponential decay of the time correlation function:

(16)

For the peptide studied in this thesis, the Alzheimer’s amyloid β-peptide, several time scales are of interest in characterizing the peptide’s properties. Local motions and vibrations of bonds occur on a femto- to picoseconds timescale. This ultra fast time scale is also that for the change in electronic configuration when a molecule is excited by the absorption of light, as seen in absorption- or CD-spectroscopy.

Molecular rotation is a slower process but still very fast. A typical rota-tional correlation time for a peptide is on the order of a few nanoseconds. For the Aβ-peptide the rotational correlation time can be calculated from the hydrodynamic radius.

(17)

The symbols were defined in the previous chapter. Assuming the hydrody-namic radius of Aβ to be 17Å and the temperature to be room temperature (T = 298K) in aqueous solution the rotational correlation time, τrot, of Aβ is 4.3 ns. A few nanoseconds are also the typical lifetime of the excited state of a fluorescent molecule.

Translational diffusion has no obvious correlation time but the expected time for the molecule to diffuse one molecular radius is for an Aβ-peptide 3 ns and thus occurs roughly on a similar time-scale as rotation.

Structural transitions and folding of proteins occur on longer time scales where induction of secondary structure occurs on pico- to microseconds and

c

t

eCtCτ

= )0()(

Tk

R

B

Hrot 3

4 3ηπτ =

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folding and induction of tertiary structure occur on micro- to seconds. The Aβ-peptide does not have any well defined structure, but has some secon-dary structure propensities. On the other hand, the Aβ-peptide undergoes other structural transitions. One important feature is the oligomerization of

Figure 5. Time-scales for proteins and NMR spectroscopy

the peptide into soluble toxic oligomers as described above and the subse-quent formation of fibrils and amyloid plaques.

The formation of soluble oligomers occurs on a much longer timescale and is seen after minutes to hours (Barghorn et al., 2005). Fibril formation is an even slower process and there is a lag time between the occurrence of a supersaturated monomer solution and fibril formation. This lag time is con-centration dependent but at physiological concentrations of Aβ the fibril formation occurs on a time-scale of hours to days (Harper and Lansbury, 1997).

Thus, the biomolecular processes that define the properties of Aβ occur on a wide variety of time-scales. These processes are however not independ-ent: the sub-molecular and molecular properties that occur on a very fast timescale underlie the macroscopic properties and pathogenic effects that occur on a much slower time-scale.

Many of these processes are within reach of spectroscopic methods (Fig-ure 5), as already mentioned above. NMR relaxation reflects both the very fast internal motion of the molecule, less than ns, and slow internal motions in micro- to milliseconds. NMR relaxation also carries information on mo-lecular rotation (nanosecond time scale) (Ishima and Torchia, 2000). The chemical shift in NMR can reflect very slow processes on second and longer time scales as well as fast processes on the µs time-scale. Measuring transla-tional diffusion with NMR does not reflect the time-scale of the correlation time of diffusion presented above, but instead reflects the timescale of diffu-sion time studied, typically 100 ms. The different time-scales within reach for NMR is one of the major advantages of this spectroscopic method.

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NMR

Nuclear magnetic resonance (NMR) is the main spectroscopic method used in the present thesis. NMR is a widely used spectroscopic method today and for purposes ranging from geological measurements via medical imaging to biomolecular dynamics. NMR has a long history and already 1936 Görter described an apparatus that, in theory, could detect the magnetic resonance of protons (Görter, 1936). In 1946 two groups independent of each other performed both solid-state and liquid-state NMR experiments (Bloch et al., 1946a-b; Purcell et al., 1946). Already early in NMR history biological stud-ies were performed: as early as in 1954 DNA was studied and 1957 a 40 MHz 1H-spectrum of ribonuclease was obtained (Jacobson et al., 1954; Saunders et al., 1957). Today the magnetic fields are significantly higher and 800-900 MHz magnets are not uncommon. The methods used to study bio-logical molecules have an increasing complexity and have developed from continuous wave 1D methods to Fourier transformed pulsed heteronuclear multidimensional experiments.

One important application of biological NMR is structure determination, but also dynamical properties, such as local mobility and hydrodynamics are within reach of NMR. In this thesis NMR has been used mainly to investi-gate dynamic properties of the Aβ-peptide using diffusion measurements, relaxation measurements but also to investigate ligand interactions with in-duced chemical shift changes and line-broadening. Some structural proper-ties of the peptide are also studied using measurements of J-coupling. For an introduction in NMR theory and basic principles of NMR spectroscopy I recommend the NMR textbook by Malcolm Levitt (Levitt, 2001).

J-couplings and the structural interpretation thereof

J-couplings, or indirect couplings, arise through the coupling of two

neighboring spins through covalent bonds. The indirect spin-spin coupling give rises to a splitting of the signal due to polarization of the spins and al-tered orbital motion of the valence electrons. It is almost exactly the same possibility for each spin to find its neighbor in an α or a β-state and therefore there will be a splitting in two peaks of equal intensity for each spin, in a two spin system. For larger spin systems the splitting gets more complicated and follows the statistics of a Pascal triangle. The splitting is typically 1-200 Hz.

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The J-coupling is most often measurable up to 3-bonds separation, and is not dependent on the magnetic field.

The 3-bond, 3J, couplings carry information on the structure of the mole-cule and are dependent on the dihedral angle between the spins. This relation can be parametrised and this was done by Karplus (Karplus, 1959, 1963) using the empirical relation J = A + Bcosφ +Ccos2φ. Here A, B and C are constants that are dependent on the molecular system and φ is the dihedral angle. For example the J-coupling between the amide proton and the α-proton is dependent on the φ - angle and thus carries information on the sec-ondary structure in the backbone.

The J-coupling can be measured directly from a 1D NMR spectrum if the studied peak is well resolved. If there is overlap in peaks 2D or 3D-spectra are necessary. Typically a COSY experiment yields the J-couplings in a pro-tein.

Different secondary structures show different J-couplings because the di-hedral φ-angle differs between different structures. However, as seen in the Karplus equation, the relation between J and φ is not one-to-one, and the interpretation of J-coupling data should be done with care.

Nuclear spin relaxation and dynamics

In order to obtain an NMR signal the spins have to be disturbed relative to

the thermal equilibrium state and the magnetic moments have to precess coherently. The loss of coherence and the process of returning the magneti-zation to thermal equilibrium are termed relaxation and carry information on molecular dynamics and motion on a fast time-scale. This time scale is in the nano- to pico-second range and involves local motion as well as molecular tumbling.

Relaxation of spins in a simple spin ½ system is caused by the stochastic fluctuating magnetic field that is created by dipole-dipole couplings and chemical shielding anisotropy (CSA) in a tumbling molecular system. The stochastic fluctuations in the magnetic field can be represented as a random perturbation term in the Hamiltonian of the system and can induce a spin flip, from β to α-state. Using this formalism relaxation can be described in terms of the spectral density function of the molecule (Wangsness and Bloch, 1953; Redfield, 1957, 1965).

Relaxation of the spin from a perturbed state back towards thermal equi-librium is called longitudinal relaxation because it involves the reconstitution of the macroscopic magnetization vector along the magnetic field-direction. This relaxation occurs with a characteristic time constant called T1 or the reciprocal rate R1. Loss of coherence of the precessing spins leads to loss of

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the detectable NMR signal, and this has another characteristic time constant called the tranverse relaxation time T2.

A third relaxation phenomenon is the steady state Nuclear Overhauser Ef-fect, NOE, that is due to dipolar-dipolar cross-correlation relaxation. This phenomenon is possible in a system with two dipole-dipole coupled spins. One way to measure the NOE effect, is to apply a radio frequency field to one of the coupled spins that equalizes the populations, i.e. removes the dif-ference in number of spins in the α- and β-state for that spin. This is called saturation. In the saturated system cross-relaxation rates cause an increase in the population difference in the other of the coupled spins. This effect is dependent on the rotational correlation time of the molecule studied and thus reflects both dynamic and structural properties of the molecule. (For a more complete description of relaxation phenomena in NMR, the text book of Kowalewski and Mäler is recommended (Kowalewski and Mäler, 2006)).

Longitudinal relaxation and NOE are both caused by spin flip between α- and β-state. NOE actually involves two simultaneous spin flips. These re-laxation parameters are mainly dependent on fast dynamics of the molecule. Transverse relaxation on the other hand is due to loss of spin coherence and also contains information on the molecular motion on a slower timescale. All three relaxation parameters can, as mentioned above, be described in terms of linear combinations of the spectral density function of the system studied and the magnetic field strength. The spectral density function is the Fourier transform of the correlation function of the perturbing magnetic field caused by the dipole-dipole-couplings and molecular tumbling. The dipole-dipole is just one of many possible interactions responsible for relaxation of spin = ½ systems. Relaxation through the chemical shift anisotropy interaction should also be considered.

To obtain dynamical parameters, relaxation data can be used to map the correlation function of the spectral density function and thereby obtain rota-tional correlation times both for local reorientation of the spin-spin vector and reorientation of the molecule studied. This approach is usually called the model-free approach and a generalized order parameter is obtained (Wennerström et al., 1974; Lipari and Szabo, 1982). The generalized order parameter reflects the local rigidity.

Linebroadening in NMR

Linebroadening of the NMR signal may be caused by a variety of mecha-nisms, mainly related to T2 relaxation. The NMR signal is represented as the Fourier transform of the induced sinusoidal signal. The loss of coherence of the individual spins reduces the magnitude of the signal and thus the sinu-

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soidal signal decays exponentially with the characteristic time T2 as de-scribed above. In the simplest case, a single spin, the obtained time-dependent signal is:

(18)

Here Meq is the magnetization at thermal equilibrium and ω is the larmor frequency. In a system of i spins the obtained signal is given by a sum of damped sinusoidal functions:

(19)

The Fourier transform of the function described by equations 18 and 19 is a Lorenzian function in the frequency domain. The Fourier transform is a lin-ear operation, and thus, a sum of signals yields a sum of Lorenzians. The frequency function is dependent on the Larmor frequency, ω0, and the re-laxation rate R2=1/T2:

(20)

This is a function with a maximum at the Larmor frequency and a line-width, in Hertz, that is R2/π. Thus the line-width of the signal in the Fourier transformed NMR spectrum is directly proportional to the relaxation rate. A high relaxation rate corresponds to broad lines. This is ultimately a conse-quence of Heisenberg’s uncertainty principle, where ∆E∆t ≥ ħ/2 → ∆ν∆t ≥ 1/4π, thus a relaxation time of 10 ms corresponds to an 8 Hz wide line which is a broad line in NMR.

This means that factors that influence the relaxation rate of a resonance are reflected in the line width of the signal. Typically this could be due to changed conformation of the molecule, oligomerization or ligand binding. If the relaxation rate is high enough the signal can be broadened beyond detec-tion. Another important relaxation mechanism is through the presence of paramagnetic species. If the studied molecule has unpaired electrons or if a paramagnetic agent is added to the sample an increased relaxation rate will be observed.

Paramagnetic relaxation is due to dipole-dipole couplings between the unpaired electron spin and the nuclear spin. The dipole-dipole-couplings are of course strongly distance dependent and decreases with the 6th power of the distance. The relaxation enhancement of the paramagnetic agent causes the NMR signal to broaden significantly if it is close enough. This can be used both to study interactions between paramagnetic molecules and dia-

teMMT

t

eqxy ωsin2

=

∑−

=i

i

T

t

i

eqxy teMMi

ωsin2

20

22

2

)()(

ωωω

−+=

R

RS

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magnetic ones, and to refine structures by either using intrinsic paramagnetic centres or by introducing paramagnetic markers (Figure 6).

Figure 6. Different strategies in using paramagnetic probes. Attaching a paramag-netic probe to the molecule (left) or addition of a probe into the solution. The dis-tance dependence of the PRE then helps to refine structure of the protein or peptide.

If the NMR signal disappears totally the spin is very close to the para-magnetic probe, but if the signal remains the change in relaxation rate can be used to measure the distance to the paramagnetic site (Bertini and Luchinat, 1996; Jacob et al., 1999).

Another property that may affect the NMR linewidth appearance is chemical exchange between states that exhibit different chemical shifts. The different states may be related to different conformations, bound and free states or different oligomeric states. Consider two states A and B, with their respective chemical shifts, δA and δB in Hertz [Hz]. If the exchange is slow on the NMR time scale there will be two lines in the spectra with intensities corresponding to the respective populations of states A and B. Slow ex-change on the NMR time scale corresponds to an exchange rate, k, that is orders of magnitude smaller than the difference in chemical shift, k << ∆δAB, (Figure 7). On the other hand, in the case of fast exchange, k >> ∆δAB, the two peaks will collapse into one, this peak will be centered at the frequency that corresponds to the weighted mean of the chemical shifts of state A and B. The weight is the equilibrium concentrations of state A and B:

(21)

Here K is the equilibrium constant. If the chemical shifts of state A and B are known, chemical shift studies may provide information on exchange rates and equilibrium constants of the system. In the intermediate exchange rate region, where k ≈ ∆δAB, the lineshapes are somewhat distorted and close

K

K BAobs

+

+=

1δδ

δ

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Figure 7. NMR line shapes for two spins in chemical exchange at different exchange rates.

to the transition point, k = ∆δAB, the lines are significantly broadened and often not visible in the NMR spectra (Figure 7).

To be able to resolve spectra that exhibit intermediate exchange the sys-tem may be studied at a different static magnetic field strength, or at a differ-ent temperature in order to change the chemical exchange rate or the NMR chemical shift time scale (Millet et al., 2000).

Diffusion measurements with NMR

Relaxation measurements yield information on local motion and molecu-

lar reorientation. Hydrodynamics reflects another scale of dynamics and may be characterized by its diffusion coefficient as described above. The transla-tional diffusion coefficient, Dt, can be obtained from NMR experiments by the use of gradient fields in addition to the strong static magnetic field (Hahn, 1950; Carr and Purcell, 1954; Stejskal and Tanner, 1965). Dt is calcu-lated from the mean square displacement of spins during a diffusion time and thus reflects both an ensemble and a time average and cannot be used to study diffusion on fast timescales nor on too slow timescales. In this section the basics of Pulsed Field Gradient (PFG)-NMR diffusion is described.

PFG-NMR is easiest described by representing the net number of spins in the α state as a magnetization vector M. However, the net magnetization does not produce any signal. An NMR signal is proportional to the magneti-zation vector along an orientation perpendicular to the field, B0. This mag-

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netization rotates with the Larmor frequency, induces an alternating current if the magnetization precesses in a coil.

The magnetization in the xy-plane after a 90°-pulse has a phase shift after a time τ according to the precession. If all spins experience the same mag-netic field the phase shift for spins with different location in the sample will be the same φ(τ) = γB0τ. If a field gradient along the z-axis over the sample, g(t,z), is added to B0, the field experienced by the spin will depend on the localization along the z-axis. The phase shift for a spin is then given by:

(22)

A gradient pulse with length τ induces a z-dependent phase shift that reduces the signal. A second gradient with reversed sign can rephase the signal, as-suming that they are at the same z-coordinate as they were at the first pulse. This is called a gradient echo. Small static inhomogenities in the magnetic field that cause dephasing and consequently line broadening can be rephased by a spin-echo where the spin precession direction is changed with a 180°-pulse and the precession times in both directions are equal, thus causing the total phase shift to be zero.

Figure 8. The NMR spin echo pulse sequence with gradient pulses. The first gradi-ent pulse dephases the signal and the second gradient pulse rephases them again. If the spins undergo Brownian motion during the delay ∆ the rephasing will be incom-plete and this causes a signal attenuation

The basic experiment for measuring diffusion by NMR is a spin-echo ex-periment with gradient pulses, where the spins undergo Brownian motion between the de- and refocusing gradients. The spin-echo experiment is initi-ated with a 90°-pulse followed by a delay τ. During this delay a gradient pulse with length δ and strength g is applied. A 180°-pulse is applied fol-lowed by a delay τ and an identical gradient pulse, figure 8. Small static

∫ ⋅+=τ

γτγφ0

0 )()( dttzgBt

)3

()2(

ln 222

0

δδγ

τ−∆−= Dg

S

S

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inhomogenities are refocused by the spin-echo. The gradient echo is fully effective only if the spins do not move in the time that separates the gradient pulses. In the presence of diffusion the refocusing will not be complete and the signal will be attenuated. Equation 23 give the signal attenuation known as the Stejskal-Tanner equation (Stejskal and Tanner, 1965; Price, 1997):

(23)

Measuring the signal intensity at different gradient strengths or different diffusion times makes it possible to calculate the diffusion coefficient (Fig-ure 9).

Figure 9. The signal attenuation due to incomplete rephasing of water proton spins that undergo Brownian motion (A). In B the attenuation is fitted to Stejskal-Tanners equation, equation 23.

Measurements of accurate diffusion coefficients require temperature time-stability and constant gradients over the whole sample. The diffusion coeffi-cient is highly temperature dependent in both the Boltzmann factor and the viscosity. Also temperature gradients over the sample cause convection and a flow term has to be added to equation 23. This can be compensated for by special pulse sequences (Momot and Kuchel, 2004); however, a homogenous temperature is preferable.

A constant field gradient over the sample is necessary, otherwise equation 23 will have a space dependent gradient term i.e. g(z). Measuring the diffu-sion coefficient using non-constant gradients gives an attenuation that devi-ates from the Stejskal-Tanner equation. This non-linearity of the gradient field can be dealt with using either a distribution function of gradient strengths (Damberg et al., 2001) or by replacing the slope in equation 23 with a power series (Nilsson and Morris, 2006). Then equation 23 can be used to measure the diffusion coefficient with high accuracy for any given substance with a measurable NMR signal.

)3

()2(

ln 222

0

δδγ

τ−∆−= Dg

S

S

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Circular Dichroism and secondary structure of peptides

CD (circular dichroism) is a low resolution spectroscopic technique from which structural information can be obtained. The phenomenon of chiropti-cal activity has a long history and was observed already in the early 19th century. In 1951 the structural motifs of biomolecules were proposed and the correlation of secondary structure and chiroptical activity was proposed just a couple of years later (Pauling et al., 1951; Yang and Foster, 1954). Nowa-days CD is a standard tool for estimating secondary structure content and to follow structural transitions.

In circular dichroism (CD) one takes advantage of the chirality of a mole-cule and the helicity of photons. The photon, is a massless boson with spin one. These massless bosons have two possible spin projections, -1 and +1, and these projections corresponds to left and right circular polarized light. Ordinary linearly polarized light is one-to-one superposition of -1 and +1 spin photons.

Biological molecules such as peptides and proteins have chiral properties and thus absorb circularly polarized light differently, dependent on if the light is left- or right-handed polarized. The difference results in elliptic po-larized light, and the ratio of the two semiaxises is called the ellipticity, θ (Figure 10).

Figure 10. The CD signal arises from that left and right circularly polarized light being absorbed differently. This gives elliptic polarized light and the ratio between the semiaxises is the ellipticity. If the absorption of the circularly polarized light also introduces a phase shift, α in the figure, the elliptic polarized light is tilted with an angle θ. This is called the ORD (optical rotatory dispersion)

This ellipticity is related to the difference in absorption of left- and right-handed circularly polarized light AL and AR according to:

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(24)

The ellipticity is usually normalised so that it is written in terms of molar ellipticity [degrees per mole residue and length2].

Different secondary structures have different chiroptical activity that gives characteristic CD spectra. Peptides and proteins that have well defined structures such as α-helices and β-sheets, are easy to recognize in CD spec-tra.

Estimation of secondary structure fraction with CD

The Aβ peptide has mainly disordered or random coil properties. Disordered peptides have some characteristics in their CD spectra (Woody, 1992). A true random coil spectrum shows no positive bands and has a distinct nega-tive band at 195 nm. A left-handed 31-helix (PII-helix) has a positive band at 225 nm and a negative band close to 200 nm (Woody, 1992; Shi et al., 2002). The positive band close to 225 nm is at 229 nm in a pure polyproline chain and is shifted towards shorter wavelengths when other residues are involved. This band corresponds to an n-π* transition (Ma et al., 1999). Using these characteristics the fraction of PII-helix in a peptide can be esti-mated from CD spectra. An empirical relation bteween the intensity at the obseved band at 225 nm and the amount of PII structure (assuming random coil to be the only other structure present) has been derived (Rucker et al., 2003):

(25)

Where [θ]max is the maximum CD signal at the local maximum close to 225 nm. This approximation is rather crude and gives only an estimate of the population. It is valid only if a two-state system is considered, such as a sys-tem with PII and random coil contributions.

The α-helix CD-spectrum shows two minima at 222 nm and 208 nm and one maximum at 190 nm. This spectra is the superposition of several exciton contributions: the 222 nm minimum is mainly due to the nπ* excitation, while the 208 nm minimum and the maximum mainly arise from the ππ* excitation. Estimation of α-helical content is possible in analogy with the PII case, usually from the ellipticity at 208 nm (Greenfield and Fasman, 1969).

[ ]13700

6100max +=

θpopPII

πθ 4180)(303.2 ⋅−⋅

= RL AA

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β-sheets show two different classes of CD-spectra, depending on if it is a parallel or anti-parallel structure. Both show one clear negative and one posi-tive band where the negative band corresponds to nπ* excitation and the positive ππ* excitation. The parallel β-sheet CD spectrum is red-shifted relative to that of the anti-parallel β-sheet.

Fluorescence spectroscopy

In fluorescence, the spontaneous emission of light due to de-excitation of a singlet state to the ground state of the molecule is studied. In the work pre-sented in this thesis fluorescence has been used to study metal interaction with Aβ.

When a molecule interacts with a photon the molecule may change its electronic configuration to an excited state, Si, from the ground state Sa. Si is a singlet state and from this state the molecule can deexcite via different processes. Sa may convert into a triplet state and subsequent phosphores-cence or the molecule can relax back to Sa under photon emission, fluores-cence. The excited singlet state can also relax due to collisions with the sol-vent and by dissipation into vibrational states. Another important mechanism is quenching where the molecule collides with solute molecules that are ca-pable of quenching the excited Si state, see figure 11. The fraction of excited singlets that deexcite through fluorescence is called the quantum yield.

In polypeptides the aromatic side chains of tryptophan, phenylalanine and tyrosine are intrinsic fluorophores. The Aβ-peptide contains three phenyla-lanine residues and one tyrosine but the phenylalanine quantum yield is only 20% of the quantum yield of tyrosine (Chen, 1967). Thus, tyrosine fluores-cence is the choice when intrinsic Aβ fluorescence is to be measured.

As mentioned above an excited molecule in state Si may deexcite non-radiatively due to collision with quenchers. One type of quenching mole-

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Figure 11. Energy diagram of a fluorescent molecule. After excitation the electron is in an excited state S1 and from S1 a spontaneous emission may occur back to ground state by emitting a photon. The fluorophore may also transfer into a triplet state with a substantially longer lifetime. The emission of light when relaxing from a triplet state to a ground state is called phosphorescence.

cules are the paramagnetic molecules. One of the most effective of these is molecular oxygen that quenches most fluorophores, but also copper ions give rise to a high degree of quenching. The mechanism of copper quenching is suggested to be that the paramagnetic molecule induces a triplet state of the flourophore which is not fluorescent. If the quenching is to be effective the quencher has to collide with the fluorophore while it is in the Si state, during the fluorescence lifetime,τ. Thus, the quenching rate is diffusion lim-ited. Consider the paramagnetic copper ion as a quencher and the tyrosine of Aβ as the fluorophore. The collision rate constant per molar is given by the solution of the Smoluchowski equation (Ware, 1962; Lakowicz and Weber, 1973):

(26)

where N*A is the number of molecules per millimole, D is the diffusion coef-ficient of Aβ and Cu respectively and r is the radii of the contributing mole-cules. The diffusion coefficient of the copper ion in water solution is 7.2⋅10-6 cm2/s and of Aβ is 1.5 ⋅10-6 cm2/s, and their radii is 1.5 Å and 17 Å. P(Q) is the probability that a collision leads to quenching of tyrosine fluorescence. Assuming that all collisions will lead to quenching, P(Q) ≈ 1 (which is un-reasonable since only collision with the tyrosine residue leads to quenching). This gives a collision rate k = 1010 s-1M-1. Tyrosine has a fluorescence life-time, τ ≈ 3.3⋅10-9 s (Ferreira et al., 1994) If the quenching concentration is millimolar to molar the collision rate is 107-1010 and the probability that a copper ion collides during the lifetime of the fluorophor is significant. This

( )( ) )(4 * QPrrDDNk CuACuAA ⋅++= ββπ

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means that at millimolar to molar concentrations of copper direct diffusion-controlled quenching is possible but at submillimolar concentrations specific binding of the quenching copper close to or at the fluorescent tyrosine is required.

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Results and discussion

“There is a theory which states that if ever for any reason

anyone discovers what exactly the Universe is for and why it

is here it will instantly disappear and be replaced by some-

thing even more bizarre and inexplicable”

Douglas Adams

It is a challenging task to characterize the biophysical properties of intrinsi-cally unstructured proteins and peptides. However, this task has attracted increasing interest in the recent years, because local residual structure may be important in determining the folding (and thus the misfolding) properties of peptides (Wirmer et al., 2006). In a true random coil structure no residual local structure and no preferred global structural propensity is present. In such case no specific parameters would be measurable along the peptide chain; all measurable properties would be coupled only to the specific details of each amino acid residue. However, for a number of intrinsically unfolded peptides, such as α-synuclein and tau, local residual structures have been found and their structure deviate significantly from the so called true random coil (Smet et al., 2004; Mukrasch et al., 2005; Bernadó et al., 2005; Lippens

et al., 2006). Also the amyloid β peptide has been shown to have some re-sidual structure that deviates from random coil (Riek et al., 2001).

Studies of unfolded peptides can include the studies of local dynamics and local residual structure, as well as the properties of domains of the pep-tide and the global molecular dimensions of the peptide. These different properties can be measured using a variety of different spectroscopic meth-ods described earlier in this text. The local residual structure and dynamics may be measured using NMR relaxation and chemical shifts. The global structural propensities and the molecular dimensions are readily measured using low resolution methods such as CD or fluorescence and also by PFG-NMR diffusion measurements.

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The soluble Aβ occurs as a mainly unfolded monomer

After degradation of the membrane-bound APP the Aβ peptide is released into solution. In vitro, the peptide exists as a soluble entity until it aggregates into soluble oligomers (the toxic species?) and finally into insoluble fibrils and amyloid. The global molecular dimension of the peptide is directly re-flected in the hydrodynamic properties.

The expected scaling of the hydrodynamic radius for a long polypeptide is ν = 3/5, in agreement with the self-avoiding random coil in the swollen state. This means that the diffusion coefficient of sufficiently long peptides scales with -ν. Peptides belonging to the same structural family should scale in the same way and measurement for fragments of a peptide with different lengths gives information on whether there are structural changes or aggregation processes that are chain-length dependent. Fragments that differ in the ag-gregation or structural state would fall outside the scaling law.

In Paper I the diffusion coefficients of fragments as well as the full length Aβ-peptide were measured using the NMR diffusometry described above. All measurements were performed at physiological pH and room temperature (25 °C) and in H2O/D2O or pure D2O. Viscosity effects arising from the heavy water were accounted for using a linear dependence on the fraction D2O used. The validity of this approximation was tested by meas-urements of a molecule with a known diffusion coefficient at different H2O/D2O ratios, assuming that the overall structure of that molecule did not change when changing the heavy water content.

In the diffusion measurements non-constant gradient profiles were ac-counted for with the method developed by Damberg et al (Damberg et al., 2001). The studied fragments ranged from the hydrophobic central pentamer Aβ(16-20) up to the full-length Aβ peptide. The fragment variant Aβ(12-28)G19G20 was included since it is known not to aggregate and could there-fore serve as a control.

The measured diffusion coefficients were fitted to a simple scaling law for the diffusion coefficient:

21

ξξ −= MDT (27)

Data for all fragments fitted well to equation 27 and no systematic deviation was noticed. Thus we could conclude that all peptides exhibit the same ag-gregation state, and in this case they are monomeric. If any of the fragments would exist as a dimer the diffusion constant would deviate from eq. 27 with a factor of 1/2ξ2. No points deviate by more than 0.02 << 1/2ξ2, when using the evaluated parameters: ξ1 = 6.06 10-9 m2 s-1 and ξ2 = 0.44. Equation 27 is a scaling law for the diffusion coefficient, which may easily be changed to a

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relation between the hydrodynamic radius and the mass using Stoke’s-Einstein’s equation.

The hydrodynamic radii of single amino acids and small di- and tri-peptides as well as denatured proteins, up to a molecular mass of 12 kDa were compared to the results obtained in Paper I. All these additional data fit well to a simple power law, RH = 0.27M0.50 Å, over all peptide sizes, from 70 Da to 12kDa. Using literature data obtained from native folded proteins

Figure 12. Hydrodynamic radius versus the molecular mass of Aβ and literature values for small di- and tripeptides and large denatured proteins. In A the scaling law is fitted to Aβ results only and here ν = 0.44. In B the scaling law is fitted to the shorter Aβ fragments and the di- and tripeptides, here the fitted parameter ν = 0.50. Using only the longer fragments of Aβ and the denatured proteins as in C, ν = 0.57, close to the expected Flory-scaling.

(Wilkins et al., 1999) an exponent of 0.30 was obtained, in good agreement with a spherical model.

The results are however not in agreement with the predicted Flory scaling of ν = 3/5 for an excluded volume chain but rather in agreement with a chain in θ-solvent, ν = 1/2. The chain interactions with itself are equal to the inter-actions with the solvent. Other studies of unfolded proteins and peptides using X-ray scattering, coil libraries or PFG-NMR diffusion to measure the radius of gyration give the predicted value of the exponent, close to 0.6, but these measurements were performed in high concentrations of denaturant, which may give a different interaction pattern with the solvent (Wilkins et

al., 1999; Kohn et al., 2004; Fitzkee and Rose, 2004). The theoretical scaling laws are calculated to be valid for long polypep-

tide chains. Using only the mono-, di- and tripeptides hydrodynamic radii from the literature (Longsworth, 1953) and the penta-, hexa- and septameric fragment’s hydrodynamic radii presented in Paper I a scaling of ν = 0.50 is obtained. Using data from the the longer fragments together with the long peptide chain diffusion constants published by Wilkins et al. (Wilkins et al., 1999) yields a scaling closer to the theoretically expected value, ν = 0.57.

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This suggests that including too short fragments may bias the calculation of the scaling of the hydrodynamic radius (Figure 12). Using only the frag-ments from the Aβ peptide studied here a scaling of ν = 0.44 is found and this deviates both from the θ-solvent (ν = 0.5) and from the Flory scaling (ν = 0.6). A scaling below 0.5 suggests a more compact overall structure and this may be interpreted in terms of some residual structure in the peptide which reflects in the hydrodynamic scaling. Only the data for really short fragments scale as true random coil structures in a θ-solvent.

We conclude that the Alzheimer Aβ-peptide is in monomeric form at low concentrations and physiological pH. The peptide chain behaves as more compact than a Gaussian chain in a θ-solvent and this is in agreement with a residual secondary structure propensity along the peptide chain.

The monomeric Aβ has some residual structural propensities

In solution the monomeric Aβ peptide secondary structure is not entirely random, as shown earlier by Riek et al. (Riek et al., 2001) and also sug-gested by the hydrodynamic studies in Paper I. Structural studies were per-formed on the fragment Aβ(12-28), a plaque competent fragment with higher solubility than the full length peptide, in paper II. CD data and high resolution NMR experiments reveal that Aβ(12-28) at low temperature has a high propensity to adopt a left-handed 31-helix, (polyproline type II helix (PII helix) or 32-helix).

The amyloid Aβ-peptide as well as a number of other peptides partly adopt PII-helix, and this holds not only for proline-rich chains (Rucker and Creamer, 2002; Ding et al., 2003; Eker et al., 2004a, c).

The PII-helix is defined by its dihedral backbone angles, [φ, ψ] = [-75°, 145°]. In this helix a full turn is completed by only three residues. There are no interresidual hydrogen bonds that stabilise the structure or any salt bridges (Whittington SJ and Creamer TP, 2003). The stabilisation is instead proposed to be mediated by backbone-solvent interactions. This is supported by the fact that heavy water, D2O, stabilises the PII-helix in favour of normal water. This could mean that the hydrogen bonds between solvent water molecules and the backbone are stronger with D2O, making the structure more stable than in H2O (Shi et al., 2002; Schweitzer-Stenner et al., 2004; Chellgren and Creamer, 2004). The high resolution NMR studies of Aβ(12-28) in Paper II showed large variations in amide proton exchange times which suggest that some of the amides are more protected than others. How-ever, the experimental amide proton exchange rates of most residues in

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Aβ(12-28) are close to the theoretical values for unstructured peptides, and this suggests that no stable hydrogen bonds involving the amide protons are present in this structure.

The amount of PII-helix is strongly temperature dependent with a higher population of the PII-state at low temperatures. This temperature dependence is shown for the shorter fragment studied in Paper II and is further studied for the full length peptide in Paper IV and discussed below.

The backbone φ,ψ-angles for the PII-helix in a Ramachandran plot is very close to those of a β-strand, so the geometric differences between β-strand and PII-helix are small. By various methods, e.g. FTIR and Raman spectros-copy, the propensity for different amino acids to adopt PII-helix structure has been estimated. Using these estimations for the Aβ-peptide reveals that the N-terminal half of the peptide seems to have a higher propensity for forming PII helix than the C-terminal part. In total, about 42% of the resi-dues in Aβ have a high PII propensity and all these are found between resi-dues 1 and 28 (Eker et al., 2004b). In paper IV the PII-helix content of the full length peptide is studied and at low temperature the amount of 31-helix corresponds well with the predicted values. The fraction PII-helix calculated from CD data using equation 25 described earlier in this text, is 45% com-pared to the predicted 42%. Shorter fragments of the Aβ-peptide also show CD spectra that well correspond to the predicted fraction of PII-helix. This suggests that the propensity to adopt PII-helix may be an intrinsic amino acid residue property rather than a polypeptide chain property.

To further elucidate what regions in the peptide that adopt PII-helices 1H-NMR studies were performed. In Paper II NMR spectra show higher amide proton dispersion at low temperatures corresponding to a higher degree of general order in the structure. Measurements of the 3JHNHα-couplings suggest that the N-terminal part of the peptide adopts a PII-helix while some of the central residues, corresponding to the central hydrophobic cluster, mainly adopt a β-strand conformation, as discussed further in Paper IV. 15N-relaxation measurements, presented in Paper V, reveal that there are two regions with dynamics that differ from the rest of the peptide. Relaxation data, i.e. R1 and R2 rates were used to calculate effective local correlation times for each residue that reflect the local mobility. The effective correla-tion time in these two regions was found to be shorter than the average value, suggesting an increased mobility in these regions. The mobile regions may correspond to highly unordered segments surrounded by regions with higher propensities to adopt an extended structure. Secondary chemical shifts together with results from Paper II and IV suggest that the ordered regions mainly consist of PII-helix and β-strand. All these data taken to-gether suggest a model consisting of six regions with different structural propensities. The peptide has two regions with high propensity to adopt PII-helix, corresponding to residues 1-4 and 11-15 separated by a flexible region and two regions with a high β-strand propensity, residues 16-24 and 31-40.

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Also these two structured regions are separated by a flexible region (25-30). The highest fraction of structure propensities is seen at low temperature indi-cating that at higher temperatures the thermal motions reach an amplitude that shadows the structural propensities.

The Aβ peptide shows a temperature dependent structural behavior

In Paper II it is shown that the amount of PII-helix structure in the

Aβ(12-28) fragment is highly temperature dependent, in agreement with the expected behaviour (Shi et al., 2002). At low temperature, 3 °C, the fraction of PII-helix was estimated, using equation 25, to be 54%. Raising the tem-perature decreases the fraction of PII-helix and at 60°C the fraction of PII-helix has decreased to 25%.

The temperature dependence of the full length peptide as well as some shorter fragments thereof was studied in Paper IV. CD spectra show a clear PII-helix spectrum at low temperature. This holds especially true for the N-terminal fragments of the peptide. For the outermost N-terminal fragment Aβ(1-9) the fraction of PII-helix at 0°C was determined to be almost 60%. Increasing the temperature reduces the amount of PII-helix, and this struc-tural transition was modelled using a Zimm-Bragg analysis. This approach makes it possible to calculate both a transition temperature, the enthalpy change corresponding to the structural transition and the cooperativity from PII-helix population data.

The melting temperature, or the transition temperature, Ttrans, corresponds to the temperature when 50% of the peptide has a PII-helical secondary structure content. For the full length peptide Ttrans is 263K and for the shorter fragments Ttrans is higher, indicating that shorter fragments have PII-helices that are more stable. Introducing more flexibility to the chain also seems to affect the stability of the PII-helix. This was tested by studies of a variant Aβ peptide. The variant peptide fragment Aβ(12-28)G19G20, where the bulky phenylalanines are replaced by glycines, shows a less stable PII-helix secon-dary structure and the Ttrans is reduced from 283 to 267K compared to what is seen for the native form of Aβ(12-28). This may suggest that the side chains of the amino acids play a role in the stabilization of the extended PII-helix. The large side chains are kept apart, for steric reasons, and this favours an extended conformation.

The transition from PII to random coil has been studied for short model peptides and show non-cooperative transitions for peptides with length N = 1-3 (Ma and Wang, 2003; Chen et al., 2004). In the Aβ case the shortest

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fragment in our studies, the N-terminal Aβ(1-9), shows no cooperativity in the transition from PII-helix to random coil. The analysis assumes a two-state model where each residue can adopt one of two conformations, helical or random. The longer peptide fragments and the full length peptide exhibit some cooperativity in the structural transition. This cooperativity is however not high and the full length peptide has a cooperativity of 0.14 (a cooperativ-ity of 1 corresponds to no cooperativity) which is low compared to a typical value of 0.0002 for the highly cooperative transition from α-helix to coil exhibited by long chains. In the case of the α-helix to coil transition the co-operativity arises from the i,i+4 hydrogen bonds that stabilize the structure, but no stabilizing hydrogen bonds are present in the 31-helix. So, from where does the cooperativity arise? One hypothesis is that the weak cooperativity lies in bulky side chain interactions, in line with the argument outlined above. Another hypothesis is that the PII-helix forms a groove along the backbone, in which water may coordinate and form hydrogen bonding net-works that are energetically favourable. These hypotheses are just, hypothe-ses and the question has to be studied further.

The thermodynamics of the PII-helix-to-coil transition was studied by CD spectroscopy. The CD spectra of the different fragments of Aβ show an ap-proximate two-state transition with an isoelliptic point at 200-210 nm. A closer look at this isoelliptic point reveals that the point is more of a region suggesting that there might be more than two conformational states contrib-uting to the spectrum. Also, the fragment Aβ(25-35) shows a different be-haviour from that of the N-terminal fragments and the full length peptide: already at low concentrations this fragment adopts a thermally stable parallel β-sheet conformation. These two deviations from a simple two state model suggested that more information could be extracted by more high resolution methods. The enthalpy change in the PII to random coil transition has been studied for short model peptides and the results show very different values but are suggested to be in the order of -10 kJ mole-1 residue-1(Ma and Wang, 2003; Chen, 2004). However, for the full length Aβ the enthalpy change of the transition was estimated to ∆H = -6.8 ± 1.3 kJ mole-1 residue-1 in agree-ment with the enthalpy change of the shorter model peptides.

In order to study this structural transition in more detail NMR experi-ments were performed on two fragments of Aβ, Aβ(1-9) and Aβ(12-28) in Paper IV. These fragments were chosen because the N-terminal fragment has a high PII-helix propensity and the central fragment includes the impor-tant hydrophobic stretch. The changes in 3JHNHα couplings upon increasing the temperature indicated that the transition is in fact a three-state event: at low temperatures there is a high PII-helix propensity and a temperature in-crease is accompanied by a higher β-strand propensity of the peptide. In-creasing the temperature further induces a change in 3JHNHα couplings that corresponds to a transition towards random coil values. This may be ex-

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plained by an increase in thermal motions that mask the structural propensi-ties, as described above.

The results may have implications for peptide aggregation. At low tem-perature the peptide is more soluble than at higher concentration. The reason may be that the peptide is less prone to adopt a β-strand conformation at low temperatures, and therefor the correct contact points between peptides are not as easily found.

The temperature-induced structural changes of the peptide are also re-flected in the hydrodynamic radius. Translational diffusion measurements by PFG-NMR experiments were performed on the same fragment as the 3JHNHα couplings and are discussed in Paper II and IV. In Paper II we found that the hydrodynamic radius of Aβ(12-28) is smaller at low temperature than at room temperature. The RH at 1 °C was 96% of the RH at 25 °C. In Paper IV we studied this phenomenon further and the same pattern was observed for Aβ(12-28) which at low temperature has a RH that is 96% of the room tem-perature value. A series of experiments were performed at increasing tem-peratures. These experiments show that the hydrodynamic radius follows the same pattern as the 3JHNHα couplings. A three-state transition was found with an initially increasing RH to a maximum value at a certain temperature, and increasing the temperature further was found to reduce the RH again. This pattern was explained from a Monte Carlo calculation showing that the β-strand conformation is more extended and rigid than the PII-helix which is reflected in RH. However, the fraction of residues in PII-helix was never greater than 60% for the N-terminal fragment (1-9) and less than 55% in the central fragment (12-28). This suggests that there may be additional explana-tions to this behavior of the hydrodynamic radius.

To study the structure transitions further, in silico studies were performed on Aβ(1-9), as discussed in Paper VIII. The shorter fragment was selected for multiple reasons: the change in hydrodynamic radius was most signifi-cant for this peptide, the population of PII-helix was high in this fragment and finally the size of the peptide is very suitable for MD simulations.

The simulations show that at low temperatures there is a high propensity for PII-helix along the peptide chain, involving all residues, and with a total fraction of more than 50%. The high population of PII-helix is obtained when single residue dihedral angles are determined. When only structures with a persistence length longer than 2 residues are taken into account al-most no PII-helix secondary structure is present in the peptide. This is in agreement with the idea that PII-helix notion is an intrinsic amino acid resi-due property and the that PII-helix does not need to be looked upon as an overall conformation (Makowska et al., 2006).

The radius of gyration for Aβ(1-9) was calculated from the structures ob-tained from the MD simulation described in Paper VIII, and was calculated as the mass weighted mean distance to the centre of mass. The hydrody-namic radius can then easily be calculated from Rg using the relation

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Rg = 0.775 RH. Interestingly the simulated data mimics the experimental data quite well, in terms of the general behaviour of the peptide’s RH. At low temperature the ensemble average has a RH that is approximately 6.6 Å, in-creasing to 7 Å at room temperature and decreasing to 6.4 Å at high tem-perature. However, in the in silico case, this behaviour is not due to the β-strand being more extended than the PII-helix but because there are two structural conformations of Aβ(1-9), at low temperatures. This gives a bi-modal distribution of Rg as shown in Figure 1 in Paper VIII, where the population with the smaller radius corresponds to a hairpin-like structure and the population with a larger radius corresponds to an extended conformation. A similar hairpin structure has been shown in simulations of Aβ(12-28) and in experiments at low pH (Abe et al., 2002; Baumketner and Shea, 2006). The hairpin in the Aβ(1-9) is formed by a turn in the region comprising resi-dues 4-6 which corresponds well to the high mobility region suggested from our relaxation measurements and described above. This may explain the three state behaviour of the hydrodynamic radius. However, the 3JHNHα-couplings show the same pattern and these results are not directly explained by the presence of a hairpin conformation population.

Using the MD simulations in Paper VIII we also studied the stabilizing factors for the PII-helix. No direct correlation between the average number of hydrogen bonds from solvent water to backbone and PII-helix population of each residue could be observed. However, the total number of water hy-drogen bonded to the peptide backbone decreased significantly with raising the temperature. This may suggest that interactions between solvent water and peptide backbone are indeed involved in the stabilization of the PII-helix.

The structural propensities of Aβ corresponds to structure both in membrane mimicking media and in fibrils

The solution structure propensity model described above and shown in fig-ure 6 in Paper V does not represent a well defined secondary, or even less any tertiary, structure but merely represents residual local structural propen-sities. The structure propensities may however be important for peptide func-tion and malfunction. Unstructured proteins and peptides are very common and these often undergo structural transitions into well defined secondary structures when binding to a specific site where the peptide exhibits its bio-logical function (Bernadó et al., 2005). Other peptides, such as Aβ, exhibit its amyloidogenic and probably also its toxic function when having a more

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well defined structure than in aqueous solution. The residual structural pro-pensities of the inactive peptide seem to reflect the structure in the folded active peptide in many cases (Smith et al., 1996; Mohana-Borges et al., 2004).

The structural model described in Paper V has four regions with some re-sidual secondary structure propensity, two β-regions and two PII-helix re-gions, separated by two mobile regions. The structure of Aβ in fibrils has been investigated by solid-state NMR, H/D exchange experiments and selec-tive double Cysteine mutated Aβ. These studies have shown that two β-strands are formed and separated by a turn involving residues 26-30. The β-strands range from 17-25 and 31-40 and are aligned in an anti parallel orien-tation with respect to each other. The strands are held together by hydropho-bic interactions and in this conformation the peptides are stacked upon each other in a parallel orientation and are stabilized by intermolecular hydrogen bonds (Shivaprasad and Wetzel, 2004, 2006; Lührs et al., 2005; Sachse et

al., 2006; Petkova et al., 2006). There is a remarkably good agreement be-tween the solid state model and the structural propensities observed by us in terms of localization of the β-regions as well as the turn-region predicted from relaxation data. The β-strand structural propensities may contribute to the aggregation and amyloidogenic properties of Aβ. The structure, if any, of the N-terminal region 1-15 is not known.

Membrane mimicking media, such as SDS, induce structure of the Aβ-peptide. The induced structure is mainly composed of two α-helices. The helices are located in segments 16-24 and 29-38 and separated by a turn or kink. These segments are again in agreement with the regions that have β-strand propensity in the soluble monomer. These regions seem to be more prone to interact intra- or inter-molecularly than with solvent water.

To obtain further information on the Aβ peptide interaction with SDS mi-celles paramagnetic agents were used to determine the location of the pep-tide in the micelle. In Paper VII, the SDS-peptide interaction was studied. To study the size of the aggregate compared to the SDS micelle alone trans-lation diffusion measurements were performed. The results showed that, when measuring SDS diffusion it is of great importance to correct the ob-served SDS diffusion for obstruction effects and for the contribution from the free, non-micelle bound SDS molecules. The hydrodynamic radius of the SDS micelle alone was determined to be 22 Å.

When adding peptides to the micelles, the diffusion coefficient will be in-fluenced by several effects of unknown relative magnitudes, like a change in shape and in dynamics, so the exact hydrodynamic radius of a diffusing pep-tide-SDS micelle aggregate cannot be predicted with any precision.

If these effects are assumed to be small and neglected, we can estimate the change in hydrodynamic radius of the peptide-micelle complexes from the diffusion coefficients. The diffusion coefficient of the fragment Aβ(12-28) bound to SDS micelles and the diffusion coefficient of SDS micelles

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alone are the same. This unchanged diffusion behavior suggests that the pep-tide-micelle aggregate has the same size as the micelle. Also for Aβ(1-40) the peptide-micelle aggregate shows roughly the same hydrodynamic dimen-sions as the micelle alone. This suggests that the peptide is in a compact structure induced by the surrounding SDS molecules.

Our NMR-studies showed that Aβ in a SDS membrane mimicking envi-ronment adopts a mainly α-helical structure, and the two helical regions involve residues 16-24 and 29 -38, in good agreement with earlier studies on this system (Coles et al., 1998; Watson et al., 1998; Shao et al., 1999; Crescenzi et al., 2002; Lau et al., 2003). The position of Aβ in the mem-brane has been under some debate. Crescenzi et al suggested that the second, C-terminal helix is inserted into the membrane and the N-terminal helix is positioned on the surface of the membrane (Crescenzi et al., 2002). Addition of the paramagnetic ion manganese, Mn2+, was used by us to investigate the positioning of the peptide in the micelle. The results, discussed in depth in Paper VII, show directly that the C-terminal helix is protected from the manganese and covered by SDS molecules. The line broadening induced by the increased relaxation rate on the central helix is periodic. This periodicity is consistent with a helix located on the surface of the micelle and conse-quently some of the residues are protected while some are not.

Figure 13. Relative hydrophobicity of the Aβ fragment and the surrounding residues in APP. This shows clearly that the C-terminus of Ab is hydrophobic and indicates a central hydrophobic stretch. The suggested transmembrane segment is highlighted as a box and the cleavage sites for the proteolytic secretases are also indicated.

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The combined results from the structural studies, the paramagnetic relaxa-tion enhancement and the hydrodynamic data suggest a model for Aβ-SDS interaction described in Paper VII. This model is in agreement with the model suggested by Crescenzi et al. The studies of Aβ in a membrane mim-icking environment do not necessarily reflect the structure and positioning of Aβ in the membrane directly after degradation by γ-secretase. When study-ing the sequence of APP and the hydrophobicity of the residues, a membrane spanning region is suggested from residue 30 to 52 in Aβ nomenclature, see figure 13. In addition to this, membrane-release studies, where APP frag-ments of different lengths are inserted into a membrane and the fraction of released peptide is determined, show that the Aβ peptide (1-40 and 1-42) is rapidly released from the membrane, while longer segments of the trans-membrane APP have a larger fraction that remains inserted in the membrane (Unpublished data, personal communication with Nilsson IM). The N-terminal helix may be located at the surface of the membrane.

The structural and positioning studies of Aβ in micelles provides informa-tion on membrane interaction of the soluble, released Aβ, that has been sug-gested to form channels in biological membranes. It is debated whether the monomeric peptide inserts into membrane, if monomeric peptides aggregate on the membrane surface and then compromise the membrane integrity, or if Aβ oligomers bind to and induce ionic leakage (Bokvist et al., 2004; Kakio

et al., 2004; Demuro et al., 2005). The architecture of these channels is not known but Arispe et al. suggested a structure where the C-terminal helix is buried in the membrane and the N-terminal helix is located close to the membrane surface (Arispe, 2004).Using our results a similar model may be constructed, just using the intrinsic properties of the different helices, one hydrophobic and one amphipathic. This model is sketched out above in fig-ure 14.

Figure 14. Hypothetical Aβ pore architectures. The peptide may form pores by as-sembling into a barrel (A) formed by β-sheets. Alternatively the peptide-membrane interaction induces the formation of α-helices and the helical peptides then assemble into pores (B). The second model is in agreement with an amphipatic α-helix as found in our SDS-experiments.

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Metals such as copper and zinc interact with and alter the aggregation properties of Aβ

Metal binding to Aβ is suggested to be involved in the pathogenesis of Alz-heimer’s disease. Both APP and Aβ are suggested to participate in the nor-mal metal homeostasis system, as described in the text above. Copper and zinc interaction with Aβ peptide induces peptide aggregation if the metal concentration is high enough, >1:1 metal:peptide ratio and higher (Bush et

al., 1994a; Huang et al., 1997; Raman et al., 2005). The formed aggregate is suggested to be amorphous, and thus high concentrations of copper and zinc prevent fibril formation by formation of non-fibril aggregates (Brown et al., 1997; Yoshiike et al., 2001; House et al., 2004; Raman et al., 2005). On the other hand, binding of low concentrations of copper or zinc ions to the full length peptide Aβ(1-40), at less than 1:5 metal:peptide ratio, reduces the Aβ oligomeric stability and prevents aggregation, whether amorphous or fibril forming (Cardoso et al., 2005; Garai et al., 2006)

Figure 15. Aβ peptide aggregation measured using ThT fluorescence in the absence and presence of zinc and copper ions. In the absence of metal ions (open circles) the aggregation starts after a lagtime and is, when initiated, very fast. In the presence of 1:1 copper (open squares) the lagtime is somewhat prolonged and the aggregation rate is significantly slower. In the presence of zinc ions the lagtime is significantly shorter than in the apo-form of the peptide. The amount of fibrils formed differs between the samples, suggesting that metals reduce fibril formation, either by keep-ing the peptide in solution or by amorphous aggregation.

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The soluble monomeric peptide has a high affinity copper binding site in the N-terminus. The metal ion is suggested to coordinate in a planar configu-ration with the three histidines (6, 13 and 14) and the N-terminus or tyrosine 10 as ligands. N-terminal deletions alter the binding affinity, suggesting that Tyr10 is not involved in the copper binding. The peptide also has a zinc binding site involving the three histidines. The fourth ligand for zinc binding has recently been suggested to be Tyr10, Arg5, Glu11 or the N-terminus (Mekmouche et al., 2005; Zirah et al., 2006). Binding of a metal to the N-terminal part of Aβ highly affects the aggregation properties of the peptide, even though the regions that are involved in structure conversion upon ag-gregation are located in the central and C-terminal regions.

The formation of fibrils, rich in cross-β-structure, can be followed by ThT fluorescence, which is strongly enhanced upon binding to amyloid fibrils and provides a tool to study the kinetics of fibril formation. Aβ alone, at subcriti-cal concentrations shows a lag time followed by fast aggregation. In the presence of copper and zinc the lag time is significantly prolonged which may be due to the destabilization of oligomers brought about by metal ions (Figure 15) (Garai et al., 2006). Another feature shown in figure 15 is that the final concentration of fibrils is significantly reduced in the presence of zinc and copper. This may be due to increased amorphous aggregation in-duced by the metals.

In Paper VI 1H-13C and 1H-15N correlation NMR spectroscopy is used to study zinc binding to Aβ(1-40) at physiological pH. Metal binding induces a structural change in the peptide which is in intermediate exchange between the apo and the holoform on the NMR time scale. This causes line broaden-ing of the resonances affected by the binding. 1H-13C-HSQC gives more details than the 1H-15N-HSQC and shows that zinc binding involves the three histidines, 6, 13 and 14, and the N-terminal aspartic acid residue as ligands. The tyrosine has been suggested to be one of the ligands in metal coordina-tion but 1H-13C-HSQC of the aromatics shows that the tyrosine aromatic ring is unaffected by zinc while the imidazole rings of all histidines are strongly affected.

15N-relaxation studies reveal that metal binding induces increased order in the N-terminus, suggesting that the peptide folds around the ion. However, the high exchange rate and the low dissociation constant suggest that the folded structure is somewhat unspecific and flexible. It has been suggested that metal binding may produce stable peptide dimers or oligomers by His-His bridging. The translational diffusion measurements presented in Paper VI suggest that no such dimers are present, at least not in a population de-tectable by NMR under the conditions studied. Formation of dimers or oli-gomers may induce rapid aggregation leading to very large aggregates with line widths beyond the detection limits.

The binding of zinc to Aβ is very specific in terms of a binding site. In order to determine the affinity of metal to Aβ intrinsic Tyr 10 fluorescence

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was studied. The affinity for copper was determined by studying the para-magnettic copper induced fluorescence quenching. Zinc on the other hand is diamagnetic and consequently does not cause fluorescence quenching. To measure the zinc affinity we took advantage of the observation that copper and zinc seem to compete for the same binding site. Measuring copper quenching in the presence of increasing concentrations of zinc makes it pos-sible to indirectly calculate zinc affinity. Using this protocol the dissociation constants of copper and zinc binding to Aβ were determined and were found to be in the low micromolar range, with a somewhat stronger affinity for copper than for zinc. Kd for copper in phosphate buffer at pH 7.2 is 0.4 µM and for zinc 1.1 µM.

The data obtained from the high resolution NMR experiments lead to a model for zinc binding. This is sketched in figure 6 in Paper VI. No high resolution structure could be determined, due to intermediate exchange on the NMR time scale between the apo- and holoforms. Low peptide concen-tration as well as induced aggregation also makes it difficult to perform the experiments necessary for a reliable structure determination.

The N-terminal region of the Aβ peptide, with no metal bound to the pep-tide, has a high propensity for an extended PII-helix conformation, as pointed out above. The extended conformation, together with the presence of charged residues in the N-terminus, is proposed to help to keep the peptide soluble and protected from amorphous aggregation (Syme et al., 2002; Jarvet et al., 2003; Eker et al., 2004c). The concentration dependent metal effects on Aβ aggregation described above may be understood in the following simple model: Metal ions at high concentrations saturate the binding site(s) of Aβ and lower the electrostatic repulsion between the overall negatively charged Aβ peptides (net charge nominally -3 at pH 7). This effect dominates at high metal ion concentrations and explains the higher aggregation propensity under these conditions. Binding at a second binding site may be crucial. Assuming a second binding site that involves the charged residues Glu 22 and Asp 23, metal binding reduces intermolecular electrostatic repulsion, and this may facilitate interactions between the hydrophobic stretches of the peptide. This, in turn, may promote hydrophobic clustering and aggregation. The structure induction brought about by metal-induced folding of the N-terminus counteracts aggregation. This effect, masked at high metal ion concentrations, should dominate at low metal ion concentrations, thereby explaining the decreased aggregation propensity under these conditions. The extended conformation of the N-terminal region in the metal-free form of the peptide may facilitate fibril formation after peptide aggregation. In the metal-bound peptide, on the other hand, the folded N-terminus could interfere with fibril formation and this would explain the reduced fibril formation of metal-bound aggregated peptides.

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The toxic oligomers formed by Aβ are destabilized by metal binding (Garai et al., 2006). This may be explained by a model where the oligomers are micelle-like structures, which have been suggested by, among others, Sabaté et al. (Kayed et al., 2003; Sabaté and Estelrich, 2005). The formation of micelles requires amphipathic properties of the molecule, and this is the case of Aβ, as seen in figure 13. The micelle formation is also dependent on a peptide conformation that allows the hydrophobic regions to cluster in the centre of the micelle and with the hydrophilic tail pointing outward. Upon metal binding the conformational change of the hydrophilic tail may make the micelle state less energetically favourable.

Aβ binding of β-cyclodextrin involves aromatic residues and reduces aggregation

As pointed out above metals like copper and zinc reduce Aβ aggregation at low ionic concentrations due to induced structure and electrostatic effects in the N-terminal part of the peptide. This is somewhat surprising, since the metals induce structural and dynamic changes in the 1-15 region and this fragment of the peptide is known not to be amyloidogenic. The central hy-drophobic region, residues 17-21, seems to be important for aggregation and the formation of cross-β rich fibrils. Binding of a ligand to this region should influence the aggregation behavior of Aβ. The Alzheimer peptide has been shown to interact with cyclodextrin, and the interaction reduces fibril forma-tion at high concentrations of the cyclic oligosaccharide β-cyclodextrin (β-cd). In Paper III the peptide-β-cd interaction was studied using NMR. Both the full-length peptide and shorter fragments were studied at room tempera-ture and physiological pH.

Upon interaction, both the size and the shape of the interacting agents change and thus their hydrodynamic properties change. From diffusion data the dissociation constant, Kd, may be determined. The diffusion coefficient, Dobs, was determined over a range of total ligand ([L]0) or peptide ([H]0) concentrations. The obtained data was fitted to the following one-to-one binding equation:

(28)

A two-parameter fit with respect to Dcomplex and Kd using a range of at least three β-cd concentrations gives the desired parameter values. The diffusion constant of the free peptide (Dfree) has to be determined. In addition, the in-

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duced chemical shift changes were used to determine the binding site and to determine Kd, using diffusion data and chemical shift changes combined.

The interaction has earlier been suggested to occur between the aromatic residues and the hydrophobic interior of β-cd. The Aβ peptide has four puta-tive aromatic binding sites, Phe4, Tyr10, Phe19 and Phe20. The results of our study show that if the phenylalanines at positions 19 and 20 in the frag-ment Aβ(12-28) are replaced with glycines, thus removing all aromatic side chains, no interaction occurs. This shows that the phenylalanines are neces-sary for β-cd -peptide interaction. For the central fragment Aβ(12-28) where the phenylalanines are present, interaction occurs and Kd = 3.8 mM. Induced chemical shift changes suggested that the interaction mainly involves phenylalanine 19. This binding is not strong, but it is in agreement with the fibril reducing activity of β-cd determined earlier. In the earlier study a 50% reduction of fibrils was seen at 5 mM cyclodextrin concentration (Yu, 2002).

When determining Kd of the full-length peptide cyclodextrin interaction, the combined results from induced chemical shift changes and diffusion data were consistent with a two-site binding involving Phe19 and Tyr10 with a total apparent dissociation constant of Kd = 4 mM. The individual Kd for the two sites was determined to be Kd

F

= 4.7 mM and Kd

Y

= 6.6 mM, respectively, if the sites are assumed to be independent. The outermost N-terminal aro-matic amino acid phenylalanine 4 did not seem to bind β-cyclodextrin with any measurable affinity. This was tested by showing that the fragment Aβ(1-9) does not bind β-cyclodextrin.

In the diffusion measurements, increasing amounts of β-cyclodextrin were added to the solution, introducing possible obstruction effects and/or viscosity changes which consequently bias the determination of the diffusion coefficient. This obstruction effect was studied in Paper III and it turned out that the effect is linear with β-cd concentration. This effect has been ac-counted for in all experiments in our study.

Other types of cyclodextrins were studied as well and showed no binding. This may be because the hydrophobic pocket of β-cyclodextrin has optimal dimensions for interaction with the aromatic side chains. The γ- and α-cyclodextrins have too large or too small cavities for interaction to occur.

The binding of a single phenylalanine to β-cyclodextrin has been deter-mined earlier by isothermal titration calorimetry and a dissociation constant of approximately 55 mM has been found (Aachmann, 2001). This was con-firmed in our study using NMR diffusometry. The interaction of di-phenylalanine was substantially stronger, Kd = 10 mM. Thus the interaction between the Aβ-peptide and cyclodextrin is stronger than the interaction between the aromatic ring alone and the β-cd, suggesting that the local envi-ronment of the aromatic residue contributes to the interaction. This is also supported by the fact that we could not detect interaction with the N-terminal phenylalanine at all. The aromatic Phe4 is surrounded by hydrophilic charged residues while the phelylalanines at positions 19 and 20 are part of

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the central hydrophobic cluster, and thus are located in a more hydrophobic environment (figure 16).

Figure 16. Location of the aromatic putative binding sites for β-cd and the relative hydrophobicity at the sites. The sites where we found binding in Tyr 10 and Phe 19 and/or 20 and they are all located in a somewhat hydrophobic environment while Phe 4 is surrounded by hydrophilic residues, and this may explain the weaker inter-action of β-cd with Phe 4.

Aβ binding of β-cd reduces stability and inhibits the formation of Aβ oli-gomers, just as metal binding does (Yu et al., 2002). The change in aggrega-tion properties brought about by metal interaction is caused by changes in the N-terminal region but in the case of β-cd it might be due to the interac-tion with the phenylalanines in the hydrophobic central stretch. The inhibi-tion by β-cd probably arises because β-cd covers the hydrophobic region and the hydrophilic side of β-cd readily interacts with the solvent water. Also, the β-cd bound in the hydrophobic region would sterically hinder peptide micelle formation.

The study presented in Paper III also shows that PFG-NMR diffusion measurements is a powerful tool for measuring weak interactions, even when the peptide and ligand have sizes of the same order. The combined use of diffusion results and induced chemical shift changes gave a high resolution picture of β-cd binding to Aβ.

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Outlook

In general terms, the aim of the different studies included in this thesis

has been to characterize structural and dynamic properties of the Aβ peptide that may have an impact on the molecular pathogenesis of AD. Even though not any of the studies or the sum of all studies provided any complete picture of the molecular mechanisms of peptide oligomerization and aggregation, new insights may be drawn from them, in the context of results from other laboratories.

Looking into the future, there are three areas that I would find very inter-esting to investigate further. First, the toxic mechanism of Aβ must be stud-ied further. The biophysical properties, such as structure and dynamics, of the Aβ-oligomer should be characterized. One major problem in such studies is the low concentration of Aβ that can be used to produce stable Aβ oli-gomers. Second, it is important to further characterize the membrane interac-tions. Is it the oligomer that interacts with the membrane and induces mem-brane leakage, or does the monomer binds to the membrane and then assem-ble into a membrane penetrating oligomer? How is the SDS-bound structure related to the peptide structure in a more realistic membrane mimicking en-vironment, such as vesicles, consisting of a true lipid bilayer? And how is that structure related to the reported channel properties of Aβ? Peptide inter-actions with a fast tumbling bicelle (mixed micelle) may better than micelles resemble the bilayer nature of a cell membrane. At the same time this mem-brane model system may provide possibilities to determine structure and dynamics of the membrane bound peptide using NMR.

The peptide, upon membrane interaction, has been suggested to form cation-specific channels; these may be studied by vesicle leakage experi-ments. Using leakage data the kinetics of both the leakage and membrane binding and peptide assembly may be estimated. Both these topics, the oli-gomers and the membrane interaction should be combined with further in-vestigations of the metal interactions of Aβ. How is the membrane interac-tion affected if the peptide is in an apo- or holoform? It has been shown that the induced structures when the peptide binds to a membrane differ in the presence or absence of metals. However, metals have also been shown to reduce oligomeric stability. The mechanism behind this would be of great importance to know in detail, because it would provide information on the oligomer stabilizing parameters. The structure of Aβ inserted in the mem-

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brane as a part of APP may be studied if a longer fragment is synthesized, including the whole trans membrane segment, i.e. Aβ(10-55).

Finally it would be very interesting to study other, similar unstructured, amyloid forming peptides to see if they also show some residual secondary structure propensities that are reflected in the function and malfunction of the peptides. Huntingtin, for example, shows a similar hydrophobicity pro-file as Aβ (Burke, 2003), and also the hydrophilic region in huntingtin has some extended properties, very similar to Aβ.

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Sammanfattning på svenska

Den här avhandlingen handlar om den amyloida β peptiden (Aβ), en peptid som återfinns i de proteinansamlingar, amyloida plack, som finns i hjärnbarken på patienter som har Alzheimers sjukdom. Aβ är en del av ett större, membranbundet, protein som kallas Alzheimers prekursor protein (APP), och i APP är Aβ den del av proteinet som förankrar proteinet i cellmembranet. APP bryts ner av enzymer och om en kombinerad degradering katalyserad av proteinet av β- och γ-sekretas sker så skapas Aβ. Efter degraderingen lämnar Aβ cellmembranet och är initialt en mestadels ostrukturerad monomer (peptiderna förekommer en och en), men aggregerar sedan och bildar giftiga oligomerer (aggregat av ett fåtal peptider). Sedemera aggregerar peptiderna till olösliga fibriller och plack. Sammansättnigen av Aβ till oligomerer och fibriller innebär en förändring av dess struktur, och den här avhandlingen handlar om den monomera peptidens strukturella och dynamiska egenskaper.

Vi har utfört diffusionsmätningar på Aβ, både hela peptiden och delar av den, och detta gjordes för att utröna om peptiden företrädesvis är monomer eller om den klumpar ihop sig parvis eller i oligomerer. Resultaten av dessa mätningar visar att peptiden är monomer vid låga koncentrationer, neutralt pH och rumstemperatur. Resultaten visar också att Aβ har hydrodynamiska egenskaper som indikerar att peptiden har en tendens till struktur, att peptiden inte beter sig som en helt ostrukturerad kedja av aminosyror.

Genom att använda NMR, kärnspinnresonansspektroskopi, och CD, cirkulär dikroism, kunde vi studera denna tendens till struktur hos Aβ-peptiden. Det visar sig att peptiden kan delas upp i 6 delar med olika typer av strukturpropensitet. I ena halvan av peptiden, den så kallade N-terminala delen har Aβ en tendens att anta en strukturtyp som kallas vänstervriden 31-helix, eller polyprolinlik helix typ II (PII). Det finns två sådana segment i de första 15-16 aminosyrorna och de är separerade av ett segment som är ostrukturerat och som har en hög rörlighet. I den andra delen av peptiden, i den C-terminala delen, har peptiden en tendens att anta en annan typ av struktur, så kallad β-flak struktur. Också i det här fallet är det två regioner som har denna egenskap och dessa är separerade av ett segment med hög rörlighet. Segmenten som har en tendens att anta en β-flakstruktur hos den monomera Aβ peptiden motsvarar direkt den β-flakstruktur som man kan finna hos Aβ i de olösliga fibrillerna, det är samma segment som antar β-flak i de peptider som bygger upp fibrillerna.

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Oligomerer av Aβ peptiden är troligen det som orsakar nervcellsdöden i Alzheimers sjukdom. Hur detta sker är inte klarlagt men mycket tyder på att Aβ binder till cellmembran och skapar hål i membranet och därigenom förstör cellmembranets barriärfunktion. För att studera hur Aβ peptiden interagerar med cellmembran genomförde vi studier av peptiden i SDS lösning. SDS är en detergent som bildar miceller, små sfäriska aggregat av SDS-molekyler och dess vattenavstötande del påminner om den vattenavstötande delen av ett cellmembran. I SDS-lösning antar Aβ en tredje typ av struktur, en så kallad α-helix, detta sker i samma segment där den antar β-flak i fibriller och i lösning. Det verkar som om dessa segment har en hög propensitet att anta en struktur. Våra resultat visade att peptiden binder till en micell och den ena α-helixen är begravd i micellen medan den andra ligger på ytan. Dessa resultat ledde till en hypotetisk struktur på en membranpor bildad av Aβ peptider.

Både Aβ och APP binder metalljoner såsom koppar och zink. När Aβ binder metalljoner så ändras lösligheten hos peptiden drastiskt på ett tämligen komplext sätt. Vid låga metallkoncentrationer motverkar metalljonerna aggregation och bildning av giftiga oligomerer men vid höga metalljonskoncentrationer så induceras aggregation i stället. Vi använde NMR och fluorescens för att studera hur Aβ binder tvåvärda zink- och kopparjoner. Vi kunde visa att Aβ binder zink med tre histidiner, i positionerna 6, 13 och 14 och den yttersta aminosyran aspartat. Sålunda viker sig peptiden runt zinkjonen och den N-terminala delen av peptiden får en ändrad struktur. Denna nya struktur motverkar troligen aggregation. Vi såg också tecken på ett andra bindningsställe, lokaliserat i den centrala delen av peptiden. När metalljonen binder till det andra stället minskar troligen lösligheten med aggregation av peptiden som följd.

En annan ligand som påverkar lösligheten för Aβ är sockret β-cyklodextrin (β-cd), en ringformad molekyl bestående av sju ihopbundna glukosmolekyler. Denna molekyl har visat sig kunnat minska fibrillbildning och också oligomerisering av Aβ. Vi använde NMR för att karakterisera hur Aβ och β-cd interagerar. Vi kunde visa att β-cd binder till två ställen på Aβ, dels till den centrala delen av peptiden och dels till aminosyraresten tyrosin på position 10. Den binder tämligen svagt och bindningskonstanten bestämdes med hjälp av diffusionsmätningar.

Den monomera Aβ peptiden är sålunda inte helt ostrukturerad utan har tendenser att anta strukturer och dessa strukturtendenser överensstämmer väl med de strukturer peptiden antar i aggregerat tillstånd och som bunden till SDS-miceller. De egenskaper som gör att peptiden formar fibriller är alltså mätbara redan hos den lösliga monomera peptiden. Den monomera strukturen kan vara avgörande för peptidens aggregationsegenskaper och då peptiden interagerar med en ligand som påverkar strukturen eller dess vattenavstötande egenskaper inverkar direkt på aggregationen av peptiden.

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Acknowledgements

The work on which this thesis is based in not one mans work and I have a number of persons to pay my gratitude to. In an acknowledgement section such as this it is impossible to cover all persons that, direct or indirect, has contributed to my years at the university resulting in this thesis. However, there are some people that I wish to mention in particular.

Astrid Gräslund, som först och främst vågade ge mig chansen till forskarutbildningen. Sedan dess har du inspirerat, och handlett mig på ett fantastiskt sätt. Du har också visat ett nästan oändligt tålamod med mina brister. Ditt sätt att leda institutionen skapar en atmosfär av frihet och kreativitet, där varje medarbetare och student förväntas ta ett eget ansvar, och det är mycket inspirerande.

Jüri Jarvet, som har lärt mig det jag kan om NMR och även andra typer av spektroskopi. Du har tålmodigt lyssnat på idéer och frågor, dumma som mindre dumma och tagit dem alla på allvar. I nästan alla projekt har vi samarbetat och samarbetet har alltid varit givande och lärorikt.

Lena Mäler, som alltid har kloka råd och insiktfulla åsikter om vetenskapligt arbete i allmänhet och om NMR i synnerhet.

Roberta Pierattelli and Lucia Banci, who teached me a lot about NMR spectroscopy and analysis of NMR data. You also gave me an insight into the world of paramagnetic and protonless NMR. Further, I am grateful that you made my stay at CERM in Firenze such a great time.

August Andersson, för stimulerande och givande diskussioner och debatter. Du ser alltid på ett problem från ett annat håll, så att det öppnas nya vägar till lösningar. Det har varit ett nöje att komma till kontoret varje dag.

Martin Dahlberg, för att du gett mig insikter i simuleringarnas värld och för din seriösa inställning.

Vladana Vukojevic and Georgy Bakalkin, for inspiring and interesting cooperation on the dynorphin project.

Torbjörn Astlind, för att alla spektrometrar och datorer fungerar på avdelningen. Jag upphör aldrig att imponeras av dina kunskaper.

Haidi Astlind, för att du alltid får det administrativa att flyta smidigt, och att du aldrig tröttnar på att hjälpa och svara på frågor.

Britt-Marie Olsson, för att laboratorierna fungerar och kemikalier och peptider inköps. Att du dessutom alltid sprider ett gott humör gör det roligare på institutionen.

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Peter Damberg, för dina kloka råd och dina kritiska kommentarer, och för att du delar med dig av dina kunskaper.

Jag vill också tacka övriga medförfattare, Göran Eriksson, Henrik Biverståhl, Jon Nordin, Ingrid Johansson, Marta Oleszczuk, Kamila Oglecka and Emma Lindahl. Henrik och Jon vill jag dessutom tillsammans med Evangelos Papadopoulos tacka för trevligt kontorssällskap.

And for great football discussions, indoor football games and glorious Friday port wine I wish to thank Vasco Castro and Loïc Hugonin. For in-teresting discussions and help I like to mention Jesper Lind, Elsa Bárány-Wallje, Joshua Hicks and Alex Peralvarez. I would also like to thank all people, present and former, at Biophysics for creating a great environment at the department.

Peter Brzezinski, för att du varit min bi-handledare under dessa år och att du tagit dig tid till detta.

Jonas Danielsson, att ha sin lillebror som vetenskaplig förebild och inspirationskälla är en ynnest. Att vi dessutom har delat intresset för Hammarby, både fotboll, handboll och bandy, gör det inte sämre. Slutligen vill jag nämna min familj, min närmsta likväl som min utökade, som stöttat mig genom alla dessa år. Mina barn, Axel, Alva och Ellen som hela tiden gör livet lätt att leva. Min mor och Torvald som varit där liksom mina systrar. Och Sara för att du står ut med mig, för att du inspirerar mig och för att du finns.

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References

Aachmann, F.L. (2001), NMR Spectroscopic Investigation of Cyclodextrin-Protein Interaction, Aalborg University, Aalborg

Aachmann, F.L., Otzen, D.E., Larsen, K.L. & Wimmer, R. (2003), "Structural background of cyclodextrin-protein interactions", Protein Eng, vol. 16, no. 12, pp. 905-912

Abe, H., Kawasaki, K. & Nakanishi, H. (2002), "pH-dependent aggregate forms and conformation of Alzheimer amyloid β-peptide(12-24)", J Biochem (Tokyo), vol. 132, no. 6, pp. 863-874

Alarcón, J.M., Brito, J.A., Hermosilla, T., Atwater, I., Mears, D. & Rojas, E. (2006), "Ion channel formation by Alzheimer's disease amyloid β-peptide (Aβ40) in unilamellar liposomes is determined by anionic phospholipids", Peptides, vol. 27, no. 1, pp. 95-104

Ali, F.E., Separovic, F., Barrow, C.J., Yao, S. & Barnham, K.J. (2006), "Copper and Zinc Mediated Oligomerisation of Aβ Peptides", Int J Pept Res Ther, vol. 12, no. 2, pp. 153-164

Alzheimer, A. (1907), "Über eine eigenartige erkrankung der hirnrinde", Allg Z Psychiatr, vol. 64, pp. 146-148

Arispe, N. (2004), "Architecture of the Alzheimer's Aβ P ion channel pore", J Membr Biol, vol. 197, no. 1, pp. 33-48

Arispe, N., Pollard, H.B. & Rojas, E. (1993), "Giant multilevel cation channels formed by Alzheimer disease amyloid β-protein [Aβ P-(1-40)] in bilayer membranes", Proc Natl Acad Sci U S A, vol. 90, no. 22, pp. 10573-10577

Arispe, N., Pollard, H.B. & Rojas, E. (1996), "Zn2+ interaction with Alzheimer amyloid β protein calcium channels", Proc Natl Acad Sci U S A, vol. 93, no. 4, pp. 1710-1715

Balbach, J.J., Ishii, Y., Antzutkin, O.N., Leapman, R.D., Rizzo, N.W., Dyda, F., Reed, J. & Tycko, R. (2000), "Amyloid fibril formation by Aβ 16-22, a seven-residue fragment of the Alzheimer'sβ-amyloid peptide, and structural characterization by solid state NMR", Biochemistry, vol. 39, no. 45, pp. 13748-13759

Barghorn, S., Nimmrich, V., Striebinger, A., Krantz, C., Keller, P., Janson, B., Bahr, M., Schmidt, M., Bitner, R.S., Harlan, J., Barlow, E., Ebert, U. & Hillen, H. (2005), "Globular amyloid β-peptide oligomer - a homogenous and stable neuropathological protein in Alzheimer's disease", J Neurochem, vol. 95, no. 3, pp. 834-847

Barnham, K.J., Cappai, R., Beyreuther, K., Masters, C.L. & Hill, A.F. (2006), "Delineating common molecular mechanisms in Alzheimer's and prion diseases", Trends Biochem Sci, vol. 31, no. 8, pp. 465-472

Baumketner, A. & Shea, J.E. (2006), "Folding Landscapes of the Alzheimer Amyloid-β (12-28) Peptide", J Mol Biol, vol. 362, no. 3, pp. 567-579

Bellingham, S.A., Ciccotosto, G.D., Needham, B.E., Fodero, L.R., White, A.R., Masters, C.L., Cappai, R. & Camakaris, J. (2004a), "Gene knockout of amyloid

Page 75: NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless misfolding diseases are an impor-tant field in medicine and pathophysiology and also

75

precursor protein and amyloid precursor-like protein-2 increases cellular copper levels in primary mouse cortical neurons and embryonic fibroblasts", J Neurochem, vol. 91, no. 2, pp. 423-428

Bellingham, S.A., Lahiri, D.K., Maloney, B., La Fontaine, S., Multhaup, G. & Camakaris, J. (2004b), "Copper depletion down-regulates expression of the Alzheimer's disease amyloid-β precursor protein gene", J Biol Chem, vol. 279, no. 19, pp. 20378-20386

Bernadó, P., Bertoncini, C.W., Griesinger, C., Zweckstetter, M. & Blackledge, M. (2005), "Defining long-range order and local disorder in native α-synuclein using residual dipolar couplings", J Am Chem Soc, vol. 127, no. 51, pp. 17968-17969

Bertini, I. & Luchinat, C. (1996), "NMR of Paramagnetic Substances", Coord Chem Rev, vol. 150, pp. 1-296

Bitan, G., Kirkitadze, M.D., Lomakin, A., Vollers, S.S., Benedek, G.B. & Teplow, D.B. (2003), "Amyloid β -protein (Abeta) assembly: Aβ 40 and Aβ 42 oligomerize through distinct pathways", Proc Natl Acad Sci U S A, vol. 100, no. 1, pp. 330-335

Blennow, K., de Leon, M.J. & Zetterberg, H. (2006), "Alzheimer's disease", Lancet, vol. 368, no. 9533, pp. 387-403

Bloch, F. (1946a), "Nuclear Induction", Phys Rev, vol. 70, pp. 460-474 Bloch, F., Hansen, W.W. & Packard, M. (1946b), "The Nuclear Induction

Experiment", Phys Rev, vol. 70, pp. 474-485 Bokvist, M., Lindström, F., Watts, A. & Gröbner, G. (2004), "Two types of

Alzheimer's β-amyloid (1-40) peptide membrane interactions: aggregation preventing transmembrane anchoring versus accelerated surface fibril formation", J Mol Biol, vol. 335, no. 4, pp. 1039-1049

Borreguero, J.M., Urbanc, B., Lazo, N.D., Buldyrev, S.V., Teplow, D.B. & Stanley, H.E. (2005), "Folding events in the 21-30 region of amyloid β-protein (Aβ) studied in silico", Proc Natl Acad Sci U S A, vol. 102, no. 17, pp. 6015-6020

Brendza, R.P., Bacskai, B.J., Cirrito, J.R., Simmons, K.A., Skoch, J.M., Klunk, W.E., Mathis, C.A., Bales, K.R., Paul, S.M., Hyman, B.T. & Holtzman, D.M. (2005), "Anti-Aβ antibody treatment promotes the rapid recovery of amyloid-associated neuritic dystrophy in PDAPP transgenic mice", J Clin Invest, vol. 115, no. 2, pp. 428-433

Brochard, F. & de Gennes, P. (1977), "Dynamical Scaling for Polymers in Theta Solvents", Macromolecules, vol. 10, no. 5, pp. 1157-1161

Brown, A.M., Tummolo, D.M., Rhodes, K.J., Hofmann, J.R., Jacobsen, J.S. & Sonnenberg-Reines, J. (1997), "Selective aggregation of endogenous β-amyloid peptide and soluble amyloid precursor protein in cerebrospinal fluid by zinc", J Neurochem, vol. 69, no. 3, pp. 1204-1212

Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C.M. & Stefani, M. (2002), "Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases", Nature, vol. 416, no. 6880, pp. 507-511

Burke, M.G. (2003), “Differential hydrophobicity drives self-assembly in Huntington’s disease”, Proc Natl Acad Sci U S A, vol. 100, no. 24, pp. 13928-13933

Bush, A.I. (2003a), "The metallobiology of Alzheimer's disease", Trends Neurosci, vol. 26, no. 4, pp. 207-214

Bush, A.I., Masters, C.L. & Tanzi, R.E. (2003b), "Copper, β-amyloid, and Alzheimer's disease: tapping a sensitive connection", Proc Natl Acad Sci U S A, vol. 100, no. 20, pp. 11193-11194

Page 76: NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless misfolding diseases are an impor-tant field in medicine and pathophysiology and also

76

Bush, A.I., Pettingell, W.H., Multhaup, G., d Paradis, M., Vonsattel, J.P., Gusella, J.F., Beyreuther, K., Masters, C.L. & Tanzi, R.E. (1994a), "Rapid induction of Alzheimer Aβ amyloid formation by zinc", Science, vol. 265, no. 5177, pp. 1464-1467

Bush, A.I., Pettingell, W.H., Paradis, M.D. & Tanzi, R.E. (1994b), "Modulation of Aβ adhesiveness and secretase site cleavage by zinc", J Biol Chem, vol. 269, no. 16, pp. 12152-12158

Cameron, K.S. & Fielding, L. (2001), "NMR diffusion spectroscopy as a measure of host-guest complex association constants and as a probe of complex size", J Org Chem, vol. 66, no. 21, pp. 6891-6895

Camilleri, P., Haskins, N.J. & Howlett, D.R. (1994), "Cyclodextrin interacts with the Alzheimer amyloid β-A4 peptide", FEBS Lett, vol. 341, no. 2-3, pp. 256-258

Cardoso, S.M., Rego, A.C., Pereira, C. & Oliveira, C.R. (2005), "Protective effect of zinc on amyloid-β 25-35 and 1-40 mediated toxicity", Neurotox Res, vol. 7, no. 4, pp. 273-281

Carr, H.Y. & Purcell, E.M. (1954), "Effects of Diffusion on Free Precession in Nuclear Magnetic Resonance Experiments", Phys Rev, vol. 94, no. 3, pp. 630-638

Carrotta, R., Manno, M., Bulone, D., Martorana, V. & San Biagio, P.L. (2005), "Protofibril formation of amyloid β-protein at low pH via a non-cooperative elongation mechanism", J Biol Chem, vol. 280, no. 34, pp. 30001-30008

Carson, J.A. & Turner, A.J. (2002), "β-amyloid catabolism: roles for neprilysin (NEP) and other metallopeptidases?", J Neurochem, vol. 81, no. 1, pp. 1-8

Castronuovo, G., Elia, V., Fessas, D., Giordano, A. & Velleca, F. (1995), "Thermodynamics of the interaction of cyclodextrins with aromatic and α, ω-amino acids in aqueous solutions: a calorimetric study at 25 oC", Carbohydr Res, vol. 272, no. 1, pp. 31-39

Chellgren, B.W. & Creamer, T.P. (2004), "Effects of H2O and D2O on polyproline II helical structure", J Am Chem Soc, vol. 126, no. 45, pp. 14734-14735

Chen, G., Chen, K.S., Knox, J., Inglis, J., Bernard, A., Martin, S.J., Justice, A., McConlogue, L., Games, D., Freedman, S.B. & Morris, R.G. (2000), "A learning deficit related to age and β-amyloid plaques in a mouse model of Alzheimer's disease", Nature, vol. 408, no. 6815, pp. 975-979

Chen, K., Liu, Z. & Kallenbach, N.R. (2004), "The polyproline II conformation in short alanine peptides is noncooperative", Proc Natl Acad Sci U S A, vol. 101, no. 43, pp. 15352-15357

Chen, R.F. (1967), "Fluorescence Quantum Yields of Tryptophan and tyrosine", Anal Lett, vol. 1, pp. 35-42

Chen, Y.R. & Glabe, C.G. (2006), "Distinct Early Folding and Aggregation Properties of Alzheimer Amyloid-β Peptides Aβ40 and Aβ42: STABLE TRIMER OR TETRAMER FORMATION BY Aβ42", J Biol Chem, vol. 281, no. 34, pp. 24414-24422

Chimon, S. & Ishii, Y. (2005), "Capturing intermediate structures of Alzheimer's β-amyloid, Aβ(1-40), by solid-state NMR spectroscopy", J Am Chem Soc, vol. 127, no. 39, pp. 13472-13473

Chiti, F. & Dobson, C.M. (2006), "Protein misfolding, functional amyloid, and human disease", Annu Rev Biochem, vol. 75, pp. 333-366

Coles, M., Bicknell, W., Watson, A.A., Fairlie, D.P. & Craik, D.J. (1998), "Solution structure of amyloid β-peptide(1-40) in a water-micelle environment. Is the membrane-spanning domain where we think it is?", Biochemistry, vol. 37, no. 31, pp. 11064-11077

Page 77: NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless misfolding diseases are an impor-tant field in medicine and pathophysiology and also

77

Crescenzi, O., Tomaselli, S., Guerrini, R., Salvadori, S., D'Ursi, A.M., Temussi, P.A. & Picone, D. (2002), "Solution structure of the Alzheimer amyloid β-peptide (1-42) in an apolar microenvironment. Similarity with a virus fusion domain", Eur J Biochem, vol. 269, no. 22, pp. 5642-5648

Curtain, C.C., Ali, F., Volitakis, I., Cherny, R.A., Norton, R.S., Beyreuther, K., Barrow, C.J., Masters, C.L., Bush, A.I. & Barnham, K.J. (2001), "Alzheimer's disease amyloid-β binds copper and zinc to generate an allosterically ordered membrane-penetrating structure containing superoxide dismutase-like subunits", J Biol Chem, vol. 276, no. 23, pp. 20466-20473

Curtain, C.C., Ali, F.E., Smith, D.G., Bush, A.I., Masters, C.L. & Barnham, K.J. (2003), "Metal ions, pH, and cholesterol regulate the interactions of Alzheimer's disease amyloid-β peptide with membrane lipid", J Biol Chem, vol. 278, no. 5, pp. 2977-2982

Damberg, P., Jarvet, J. & Gräslund, A. (2001), "Accurate measurement of translational diffusion coefficients: a practical method to account for nonlinear gradients", J Magn Reson, vol. 148, no. 2, pp. 343-348

Danielsson, J., Jarvet, J., Damberg, P. & Gräslund, A. (2005), "The Alzheimer β-peptide shows temperature-dependent transitions between left-handed 31-helix, β-strand and random coil secondary structures", FEBS J, vol. 272, no. 15, pp. 3938-3949

de Gennes, P.G. (1979), Scaling Concepts in Polymer Physics, Cornell University Press, Ithaca, NY

Dehner, A. & Kessler, H. (2005), "Diffusion NMR spectroscopy: folding and aggregation of domains in p53", Chembiochem, vol. 6, no. 9, pp. 1550-1565

Demuro, A., Mina, E., Kayed, R., Milton, S.C., Parker, I. & Glabe, C.G. (2005), "Calcium dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers", J Biol Chem, vol. 280, no. 17, pp. 17294-17300

Dickerson, T.J. & Janda, K.D. (2003), "Glycation of the amyloid β-protein by a nicotine metabolite: a fortuitous chemical dynamic between smoking and Alzheimer's disease", Proc Natl Acad Sci U S A, vol. 100, no. 14, pp. 8182-8187

Ding, L., Chen, K., Santini, P.A., Shi, Z. & Kallenbach, N.R. (2003), "The pentapeptide GGAGG has PII conformation", J Am Chem Soc, vol. 125, no. 27, pp. 8092-8093

Dobson, C.M. (2003), "Protein folding and misfolding", Nature, vol. 426, no. 6968, pp. 884-890

Dobson, C.M. (2004), "Protein chemistry. In the footsteps of alchemists", Science, vol. 304, no. 5675, pp. 1259-1262

Eker, F., Griebenow, K., Cao, X., Nafie, L.A. & Schweitzer-Stenner, R. (2004a), "Tripeptides with ionizable side chains adopt a perturbed polyproline II structure in water", Biochemistry, vol. 43, no. 3, pp. 613-621

Eker, F., Griebenow, K., Cao, X., Nafie, L.A. & Schweitzer-Stenner, R. (2004b), "Preferred peptide backbone conformations in the unfolded state revealed by the structure analysis of alanine-based (AXA) tripeptides in aqueous solution", Proc Natl Acad Sci U S A, vol. 101, no. 27, pp. 10054-10059

Eker, F., Griebenow, K. & Schweitzer-Stenner, R. (2004c), "Aβ(1-28) fragment of the amyloid peptide predominantly adopts a polyproline II conformation in an acidic solution", Biochemistry, vol. 43, no. 22, pp. 6893-6898

Espeseth, A.S., Xu, M., Huang, Q., Coburn, C.A., Jones, K.L., Ferrer, M., Zuck, P.D., Strulovici, B., Price, E.A., Wu, G., Wolfe, A.L., Lineberger, J.E., Sardana, M., Tugusheva, K., Pietrak, B.L., Crouthamel, M.C., Lai, M.T., Dodson, E.C.,

Page 78: NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless misfolding diseases are an impor-tant field in medicine and pathophysiology and also

78

Bazzo, R., Shi, X.P., Simon, A.J., Li, Y. & Hazuda, D.J. (2005), "Compounds that bind APP and inhibit Abeta processing in vitro suggest a novel approach to Alzheimer disease therapeutics", J Biol Chem, vol. 280, no. 18, pp. 17792-17797

Evin, G. & Weidemann, A. (2002), "Biogenesis and metabolism of Alzheimer's disease Aβ amyloid peptides", Peptides, vol. 23, no. 7, pp. 1285-1297

Ferreira, S.T., Stella, L. & Gratton, E. (1994), "Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue", Biophys J, vol. 66, no. 4, pp. 1185-1196

Fitzkee, N.C. & Rose, G.D. (2004), "Reassessing random-coil statistics in unfolded proteins", Proc Natl Acad Sci U S A, vol. 101, no. 34, pp. 12497-12502

Flöck, D., Colacino, S., Colombo, G. & Di Nola, A. (2006), "Misfolding of the amyloid β-protein: a molecular dynamics study", Proteins, vol. 62, no. 1, pp. 183-192

Flory, P.J. (1988), Statistical Physics of chain Molecules, Hansens Publishers Frederickson, C.J., Koh, J.Y. & Bush, A.I. (2005), "The neurobiology of zinc in

health and disease", Nat Rev Neurosci, vol. 6, no. 6, pp. 449-462 Gaggelli, E., Kozlowski, H., Valensin, D. & Valensin, G. (2006), "Copper

homeostasis and neurodegenerative disorders (Alzheimer's, prion, and Parkinson's diseases and amyotrophic lateral sclerosis)", Chem Rev, vol. 106, no. 6, pp. 1995-2044

Garai, K., Sengupta, P., Sahoo, B. & Maiti, S. (2006), "Selective destabilization of soluble amyloid β oligomers by divalent metal ions", Biochem Biophys Res Commun, vol. 345, no. 1, pp. 210-215

Ghiso, J. & Frangione, B. (2002), "Amyloidosis and Alzheimer's disease", Adv Drug Deliv Rev, vol. 54, no. 12, pp. 1539-1551

Gong, Y., Chang, L., Viola, K.L., Lacor, P.N., Lambert, M.P., Finch, C.E., Krafft, G.A. & Klein, W.L. (2003), "Alzheimer's disease-affected brain: presence of oligomeric Aβ ligands (ADDLs) suggests a molecular basis for reversible memory loss", Proc Natl Acad Sci U S A, vol. 100, no. 18, pp. 10417-10422

Görter, C.J. (1936), "Paramagnetic Relaxation", Phys Rev, vol. 3, no. 6, pp. 503-514 Gralle, M., Oliveira, C.L., Guerreiro, L.H., McKinstry, W.J., Galatis, D., Masters,

C.L., Cappai, R., Parker, M.W., Ramos, C.H., Torriani, I. & Ferreira, S.T. (2006), "Solution conformation and heparin-induced dimerization of the full-length extracellular domain of the human amyloid precursor protein", J Mol Biol, vol. 357, no. 2, pp. 493-508

Greenfield, N. & Fasman, G.D. (1969), "Computed circular dichroism spectra for the evaluation of protein conformation", Biochemistry, vol. 8, no. 10, pp. 4108-4116

Hahn, E.L. (1950), "Spin Echoes", Phys Rev, vol. 80, no. 4, pp. 580-594 Halle, B. & Davidovic, M. (2003), "Biomolecular hydration: from water dynamics

to hydrodynamics", Proc Natl Acad Sci U S A, vol. 100, no. 21, pp. 12135-12140

Hardy, J. & Selkoe, D.J. (2002), "The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics", Science, vol. 297, no. 5580, pp. 353-356

Harper, J.D. & Lansbury, P.T. (1997), "Models of amyloid seeding in Alzheimer's disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins", Annu Rev Biochem, vol. 66, pp. 385-407

Page 79: NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless misfolding diseases are an impor-tant field in medicine and pathophysiology and also

79

Hartley, D.M., Walsh, D.M., Ye, C.P., Diehl, T., Vasquez, S., Vassilev, P.M., Teplow, D.B. & Selkoe, D.J. (1999), "Protofibrillar intermediates of amyloid β-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons", J Neurosci, vol. 19, no. 20, pp. 8876-8884

Hetényi, C., Szabó, Z., Klement, E., Datki, Z., Körtvélyesi, T., Zarándi, M. & Penke, B. (2002), "Pentapeptide amides interfere with the aggregation ofβ-amyloid peptide of Alzheimer's disease", Biochem Biophys Res Commun, vol. 292, no. 4, pp. 931-936

Hou, L. & Zagorski, M.G. (2006), "NMR reveals anomalous copper(II) binding to the amyloid Aβ peptide of Alzheimer's disease", J Am Chem Soc, vol. 128, no. 29, pp. 9260-9261

House, E., Collingwood, J., Khan, A., Korchazkina, O., Berthon, G. & Exley, C. (2004), "Aluminium, iron, zinc and copper influence the in vitro formation of amyloid fibrils of Aβ42 in a manner which may have consequences for metal chelation therapy in Alzheimer's disease", J Alzheimers Dis, vol. 6, no. 3, pp. 291-301

Hsia, A.Y., Masliah, E., McConlogue, L., Yu, G.Q., Tatsuno, G., Hu, K., Kholodenko, D., Malenka, R.C., Nicoll, R.A. & Mucke, L. (1999), "Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models", Proc Natl Acad Sci U S A, vol. 96, no. 6, pp. 3228-3233

Huang, X., Atwood, C.S., Moir, R.D., Hartshorn, M.A., Vonsattel, J.P., Tanzi, R.E. & Bush, A.I. (1997), "Zinc-induced Alzheimer's Aβ1-40 aggregation is mediated by conformational factors", J Biol Chem, vol. 272, no. 42, pp. 26464-26470

Ishima, R. & Torchia, D.A. (2000), "Protein dynamics from NMR", Nat Struct Biol, vol. 7, no. 9, pp. 740-743

Jacob, J., Baker, B., Bryant, R.G. & Cafiso, D.S. (1999), "Distance estimates from paramagnetic enhancements of nuclear relaxation in linear and flexible model peptides", Biophys J, vol. 77, no. 2, pp. 1086-1092

Jacobson, B., Anderson, W. & Arnold, J. (1954), "A Proton Magnetic Resonance Study of the Hydration of Deoxyribonucleic Acid", Nature, vol. 173, pp. 772-773

Janus, C., Pearson, J., McLaurin, J., Mathews, P.M., Jiang, Y., Schmidt, S.D., Chishti, M.A., Horne, P., Heslin, D., French, J., Mount, H.T., Nixon, R.A., Mercken, M., Bergeron, C., Fraser, P.E., St George-Hyslop, P. & Westaway, D. (2000), "Aβ peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease", Nature, vol. 408, no. 6815, pp. 979-982

Jarvet, J., Damberg, P., Danielsson, J., Johansson, I., Eriksson, L.E. & Gräslund, A. (2003), "A left-handed 3(1) helical conformation in the Alzheimer Aβ(12-28) peptide", FEBS Lett, vol. 555, no. 2, pp. 371-374

Kagan, B.L. (2005), "Amyloidosis and protein folding", Science, vol. 307, no. 5706, pp. 42-3 author reply 4

Kakio, A., Yano, Y., Takai, D., Kuroda, Y., Matsumoto, O., Kozutsumi, Y. & Matsuzaki, K. (2004), "Interaction between amyloid β-protein aggregates and membranes", J Pept Sci, vol. 10, no. 10, pp. 612-621

Karplus, M. (1959), "Contact Electron-Spin Coupling of Nuclear Magnetic Moments", J Chem Phys, vol. 30, no. 1, pp. 11-15

Karplus, M. (1963), "Vicinal Proton Coupling in Nuclear Magnetic Resonance", J Am Chem Soc, vol. 85, pp. 2870-2871

Karr, J.W., Akintoye, H., Kaupp, L.J. & Szalai, V.A. (2005), "N-Terminal deletions modify the Cu2+ binding site in amyloid-β", Biochemistry, vol. 44, no. 14, pp. 5478-5487

Page 80: NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless misfolding diseases are an impor-tant field in medicine and pathophysiology and also

80

Kayed, R., Head, E., Thompson, J.L., McIntire, T.M., Milton, S.C., Cotman, C.W. & Glabe, C.G. (2003), "Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis", Science, vol. 300, no. 5618, pp. 486-489

Klein, W.L., Krafft, G.A. & Finch, C.E. (2001), "Targeting small Aβ oligomers: the solution to an Alzheimer's disease conundrum?", Trends Neurosci, vol. 24, no. 4, pp. 219-224

Kohn, J.E., Millett, I.S., Jacob, J., Zagrovic, B., Dillon, T.M., Cingel, N., Dothager, R.S., Seifert, S., Thiyagarajan, P., Sosnick, T.R., Hasan, M.Z., Pande, V.S., Ruczinski, I., Doniach, S. & Plaxco, K.W. (2004), "Random-coil behavior and the dimensions of chemically unfolded proteins", Proc Natl Acad Sci U S A, vol. 101, no. 34, pp. 12491-12496

Kourie, J.I., Henry, C.L. & Farrelly, P. (2001), "Diversity of amyloid β protein fragment [1-40]-formed channels", Cell Mol Neurobiol, vol. 21, no. 3, pp. 255-284

Kowalewski, J. & Mäler, L. (2006), Nuclear Spin Relaxaxion in Liquids: Theory, Experiments, and Applications, Taylor & Francis, Boca Raton

Kowalik-Jankowska, T., Ruta, M., Wisniewska, K. & Lankiewicz, L. (2003), "Coordination abilities of the 1-16 and 1-28 fragments of β-amyloid peptide towards copper(II) ions: a combined potentiometric and spectroscopic study", J Inorg Biochem, vol. 95, no. 4, pp. 270-282

Lakowicz, J.R. & Weber, G. (1973), "Quenching of fluorescence by oxygen. A probe for structural fluctuations in macromolecules", Biochemistry, vol. 12, no. 21, pp. 4161-4170

Lashuel, H.A., Hartley, D., Petre, B.M., Walz, T. & Lansbury, P.T. (2002), "Neurodegenerative disease: amyloid pores from pathogenic mutations", Nature, vol. 418, no. 6895, p. 291

Lau, T.-l., Barnham, K.J., Curtain, C.C., Masters, C.L. & Separovics, F. (2003), "Magnetic Resonance Studies of β-Amyloid Peptides", Aust J Chem, vol. 56, pp. 349-356

Lesné, S., Koh, M.T., Kotilinek, L., Kayed, R., Glabe, C.G., Yang, A., Gallagher, M. & Ashe, K.H. (2006), "A specific amyloid-β protein assembly in the brain impairs memory", Nature, vol. 440, no. 7082, pp. 352-357

Levitt, M.H. (2001), Spin Dynamics Basics of Nuclear Magnetic Resonance, John Wiley & Sons, LTD, Chichester

Lifshitz, I., Grosberg, A. & Khokhlov, A. (1978), "Some problems of the statistical physics of polymer chains with volume interaction", Rev Mod Phys, vol. 50, no. 3, pp. 683-713

Lin, H., Bhatia, R. & Lal, R. (2001), "Amyloid β protein forms ion channels: implications for Alzheimer's disease pathophysiology", FASEB J, vol. 15, no. 13, pp. 2433-2444

Lipari, G. & Szabo, A. (1982), "Model-free Approach to the Interpretation of Nuclear Magnetic Resonance Relaxation in Macromolecules. 1. Theory and Range of Validity", J Am Chem Soc, vol. 104, no. 17, pp. 4546-4559

Lippens, G., Sillen, A., Smet, C., Wieruszeski, J.M., Leroy, A., Buée, L. & Landrieu, I. (2006), "Studying the natively unfolded neuronal Tau protein by solution NMR spectroscopy", Protein Pept Lett, vol. 13, no. 3, pp. 235-246

Liu, S.T., Howlett, G. & Barrow, C.J. (1999), "Histidine-13 is a crucial residue in the zinc ion-induced aggregation of the Aβ peptide of Alzheimer's disease", Biochemistry, vol. 38, no. 29, pp. 9373-9378

Longsworth, L.G. (1953), "Diffusion Measurements, at 25 oC, of Aqueous Solutions of Amino Acids, Peptides and Sugars", J Am Chem Soc, vol. 75, pp. 5705-5709

Page 81: NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless misfolding diseases are an impor-tant field in medicine and pathophysiology and also

81

Lovell, M.A., Robertson, J.D., Teesdale, W.J., Campbell, J.L. & Markesbery, W.R. (1998), "Copper, iron and zinc in Alzheimer's disease senile plaques", J Neurol Sci, vol. 158, no. 1, pp. 47-52

Lue, L.F., Kuo, Y.M., Roher, A.E., Brachova, L., Shen, Y., Sue, L., Beach, T., Kurth, J.H., Rydel, R.E. & Rogers, J. (1999), "Soluble amyloid beta peptide concentration as a predictor of synaptic change in Alzheimer's disease", Am J Pathol, vol. 155, no. 3, pp. 853-862

Lührs, T., Ritter, C., Adrian, M., Riek-Loher, D., Bohrmann, B., Döbeli, H., Schubert, D. & Riek, R. (2005), "3D structure of Alzheimer's amyloid-β(1-42) fibrils", Proc Natl Acad Sci U S A, vol. 102, no. 48, pp. 17342-17347

Ma, K., Clancy, E., Zhang, Y., Ray, D., Wollenberg, K. & Zagorski, M. (1999), "Residue-Specific pKa Measurements of the β-Peptide and Mechanism of pH-Induced Amyloid Formation", J Am Chem Soc, vol. 121, no. 38, pp. 8698-8706

Ma, K. & Wang, K. (2003), "Malleable conformation of the elastic PEVK segment of titin: non-co-operative interconversion of polyproline II helix,β-turn and unordered structures", Biochem J, vol. 374, no. PT 3, pp. 687-695

Maccioni, R.B., Muñoz, J.P. & Barbeito, L. (2001), "The molecular bases of Alzheimer's disease and other neurodegenerative disorders", Arch Med Res, vol. 32, no. 5, pp. 367-381

Makowska, J., Rodziewicz-Motowidlo, S., Baginska, K., Vila, J.A., Liwo, A., Chmurzynski, L. & Scheraga, H.A. (2006), "Polyproline II conformation is one of many local conformational states and is not an overall conformation of unfolded peptides and proteins", Proc Natl Acad Sci U S A, vol. 103, no. 6, pp. 1744-1749

Marchesi, V.T. (2005), "An alternative interpretation of the amyloid Abeta hypothesis with regard to the pathogenesis of Alzheimer's disease", Proc Natl Acad Sci U S A, vol. 102, no. 26, pp. 9093-9098

Masters, C.L., Cappai, R., Barnham, K.J. & Villemagne, V.L. (2006), "Molecular mechanisms for Alzheimer's disease: implications for neuroimaging and therapeutics", J Neurochem, vol. 97, no. 6, pp. 1700-1725

Matsunaga, Y., Fujii, A., Awasthi, A., Yokotani, J., Takakura, T. & Yamada, T. (2004), "Eight-residue Aβ peptides inhibit the aggregation and enzymatic activity of Aβ42", Regul Pept, vol. 120, no. 1-3, pp. 227-236

Matsuyama, K., El-Gizawy, S. & Perrin, J.H. (1987), "Thermodynamics of Binding of Aromatic Amino Acids", Dru Dev Ind Pharm, vol. 13, pp. 2687-2691

Maynard, C.J., Bush, A.I., Masters, C.L., Cappai, R. & Li, Q.X. (2005), "Metals and amyloid-β in Alzheimer's disease", Int J Exp Pathol, vol. 86, no. 3, pp. 147-159

McLean, C.A., Cherny, R.A., Fraser, F.W., Fuller, S.J., Smith, M.J., Beyreuther, K., Bush, A.I. & Masters, C.L. (1999), "Soluble pool of Aβ amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease", Ann Neurol, vol. 46, no. 6, pp. 860-866

Mekmouche, Y., Coppel, Y., Hochgräfe, K., Guilloreau, L., Talmard, C., Mazarguil, H. & Faller, P. (2005), "Characterization of the ZnII binding to the peptide amyloid-β 1-16 linked to Alzheimer's disease", Chembiochem, vol. 6, no. 9, pp. 1663-1671

Miller, W.G. & Goebel, C.V. (1968), "Dimensions of protein random coils", Biochemistry, vol. 7, no. 11, pp. 3925-3935

Millet, O., Loria, P.J., Kroenke, C.D., Pons, M. & Palmer III, A.G. (2000), "The Static Magnetic Field Dependence of Chemical Exchange Linebroadening Defines the NMR Chemical Shift Time Scale", J Am Chem Soc, vol. 122, pp. 2867-2877

Page 82: NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless misfolding diseases are an impor-tant field in medicine and pathophysiology and also

82

Miura, T., Suzuki, K., Kohata, N. & Takeuchi, H. (2000), "Metal binding modes of Alzheimer's amyloid β-peptide in insoluble aggregates and soluble complexes", Biochemistry, vol. 39, no. 23, pp. 7024-7031

Mohana-Borges, R., Goto, N.K., Kroon, G.J., Dyson, H.J. & Wright, P.E. (2004), "Structural characterization of unfolded states of apomyoglobin using residual dipolar couplings", J Mol Biol, vol. 340, no. 5, pp. 1131-1142

Molina, J.A., Jiménez-Jiménez, F.J., Aguilar, M.V., Meseguer, I., Mateos-Vega, C.J., González-Muñoz, M.J., de Bustos, F., Porta, J., Ortí-Pareja, M., Zurdo, M., Barrios, E. & Martínez-Para, M.C. (1998), "Cerebrospinal fluid levels of transition metals in patients with Alzheimer's disease", J Neural Transm, vol. 105, no. 4-5, pp. 479-488

Momot, K.I. & Kuchel, P.W. (2004), "Convection-compensating PGSE experiment incorporating excitation-sculpting water suppression (CONVEX)", J Magn Reson, vol. 169, no. 1, pp. 92-101

Moore, S.A., Huckerby, T.N., Gibson, G.L., Fullwood, N.J., Turnbull, S., Tabner, B.J., El-Agnaf, O.M. & Allsop, D. (2004), "Both the D-(+) and L-(-) enantiomers of nicotine inhibit Aβ aggregation and cytotoxicity", Biochemistry, vol. 43, no. 3, pp. 819-826

Mukrasch, M.D., Biernat, J., von Bergen, M., Griesinger, C., Mandelkow, E. & Zweckstetter, M. (2005), "Sites of tau important for aggregation populate β-structure and bind to microtubules and polyanions", J Biol Chem, vol. 280, no. 26, pp. 24978-24986

Muresan, Z. & Muresan, V. (2006), "Neuritic deposits of amyloid-β peptide in a subpopulation of central nervous system-derived neuronal cells", Mol Cell Biol, vol. 26, no. 13, pp. 4982-4997

Murphy, R.M. (2002), "Peptide aggregation in neurodegenerative disease", Annu Rev Biomed Eng, vol. 4, pp. 155-174

Nelson, R., Sawaya, M.R., Balbirnie, M., Madsen, A.Ø., Riekel, C., Grothe, R. & Eisenberg, D. (2005), "Structure of the cross-β spine of amyloid-like fibrils", Nature, vol. 435, no. 7043, pp. 773-778

Nilsson, M. & Morris, G.A. (2006), "Correction of systematic errors in CORE processing of DOSY data", Magnet Reson Chem, vol. 44, no. 7, pp. 655-660

Okamoto, T., Takeda, S., Murayama, Y., Ogata, E. & Nishimoto, I. (1995), "Ligand-dependent G protein coupling function of amyloid transmembrane precursor", J Biol Chem, vol. 270, no. 9, pp. 4205-4208

Ono, K., Hasegawa, K., Naiki, H. & Yamada, M. (2004), "Curcumin has potent anti-amyloidogenic effects for Alzheimer's β-amyloid fibrils in vitro", J Neurosci Res, vol. 75, no. 6, pp. 742-750

Pauling, L., Corey, R.B. & Branson, H.R. (1951), "The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain", Proc Natl Acad Sci U S A, vol. 37, no. 4, pp. 205-211

Petkova, A.T., Yau, W.M. & Tycko, R. (2006), "Experimental Constraints on Quaternary Structure in Alzheimer's β-Amyloid Fibrils", Biochemistry, vol. 45, no. 2, pp. 498-512

Phinney, A.L., Drisaldi, B., Schmidt, S.D., Lugowski, S., Coronado, V., Liang, Y., Horne, P., Yang, J., Sekoulidis, J., Coomaraswamy, J., Chishti, M.A., Cox, D.W., Mathews, P.M., Nixon, R.A., Carlson, G.A., St George-Hyslop, P. & Westaway, D. (2003), "In vivo reduction of amyloid-β by a mutant copper transporter", Proc Natl Acad Sci U S A, vol. 100, no. 24, pp. 14193-14198

Price, W.S. (1997), "Pulsed-Field Gradient Nuclear Magnetic Resonance as a Tool for Studying Translational Diffusion : Part 1. Basic Theory", Magnetic Resonance: an Educational Journal, vol. 9, no. 5, pp. 299-336

Page 83: NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless misfolding diseases are an impor-tant field in medicine and pathophysiology and also

83

Purcell, E.M., Torrey, H.C. & Pound, R.V. (1946), "Resonance Absorption by Nuclear Magnetic Moments in a Solid", Phys Rev, vol. 69, no. 1-2, pp. 37-38

Qin, X.R., Abe, H. & Nakanishi, H. (2002), "NMR and CD studies on the interaction of Alzheimer β-amyloid peptide (12-28) with β-cyclodextrin", Biochem Biophys Res Commun, vol. 297, no. 4, pp. 1011-1015

Quist, A., Doudevski, I., Lin, H., Azimova, R., Ng, D., Frangione, B., Kagan, B., Ghiso, J. & Lal, R. (2005), "Amyloid ion channels: A common structural link for protein-misfolding disease", Proc Natl Acad Sci U S A, vol. 102, no. 30, pp. 10427-10432

Raman, B., Ban, T., Yamaguchi, K., Sakai, M., Kawai, T., Naiki, H. & Goto, Y. (2005), "Metal ion-dependent effects of clioquinol on the fibril growth of an amyloid β peptide", J Biol Chem, vol. 280, no. 16, pp. 16157-16162

Redfield, A.G. (1957), "On the Theory of relaxation Processes", IBM J, vol. 1, pp. 19-31

Redfield, A.G. (1965), "The Theory of Relaxation Processes", Adv Magnet Reson, vol. 1, pp. 1-32

Religa, D., Strozyk, D., Cherny, R.A., Volitakis, I., Haroutunian, V., Winblad, B., Naslund, J. & Bush, A.I. (2006), "Elevated cortical zinc in Alzheimer disease", Neurology, vol. 67, no. 1, pp. 69-75

Riek, R., Güntert, P., Döbeli, H., Wipf, B. & Wüthrich, K. (2001), "NMR studies in aqueous solution fail to identify significant conformational differences between the monomeric forms of two Alzheimer peptides with widely different plaque-competence, Aβ(1-40)(ox) and Aβ(1-42)(ox)", Eur J Biochem, vol. 268, no. 22, pp. 5930-5936

Rucker, A.L. & Creamer, T.P. (2002), "Polyproline II helical structure in protein unfolded states: lysine peptides revisited", Protein Sci, vol. 11, no. 4, pp. 980-985

Rucker, A.L., Pager, C.T., Campbell, M.N., Qualls, J.E. & Creamer, T.P. (2003), "Host-guest scale of left-handed polyproline II helix formation", Proteins, vol. 53, no. 1, pp. 68-75

Sabaté, R. & Estelrich, J. (2005), "Evidence of the existence of micelles in the fibrillogenesis of β-amyloid peptide", J Phys Chem B Condens Matter Mater Surf Interfaces Biophys, vol. 109, no. 21, pp. 11027-11032

Sabo, S.L., Ikin, A.F., Buxbaum, J.D. & Greengard, P. (2001), "The Alzheimer amyloid precursor protein (APP) and FE65, an APP-binding protein, regulate cell movement", J Cell Biol, vol. 153, no. 7, pp. 1403-1414

Sachse, C., Xu, C., Wieligmann, K., Diekmann, S., Grigorieff, N. & Fändrich, M. (2006), "Quaternary Structure of a Mature Amyloid Fibril from Alzheimer's Aβ (1-40) Peptide", J Mol Biol, vol. 362, no. 2, pp. 347-354

Salomon, A.R., Marcinowski, K.J., Friedland, R.P. & Zagorski, M.G. (1996), "Nicotine inhibits amyloid formation by the β-peptide", Biochemistry, vol. 35, no. 42, pp. 13568-13578

Santhoshkumar, P. & Sharma, K.K. (2004), "Inhibition of amyloid fibrillogenesis and toxicity by a peptide chaperone", Mol Cell Biochem, vol. 267, no. 1-2, pp. 147-155

Saunders, M., Wishnia, A. & Kirkwood, J. (1957), "The Nuclear Magnetic Resonance Spectrum of Ribonuclease", J Am Chem Soc, vol. 76, pp. 3289-3290

Schwarzman, A.L., Tsiper, M., Gregori, L., Goldgaber, D., Frakowiak, J., Mazur-Kolecka, B., Taraskina, A., Pchelina, S. & Vitek, M.P. (2005), "Selection of peptides binding to the amyloid β-protein reveals potential inhibitors of amyloid formation", Amyloid, vol. 12, no. 4, pp. 199-209

Page 84: NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless misfolding diseases are an impor-tant field in medicine and pathophysiology and also

84

Schweitzer-Stenner, R., Eker, F., Griebenow, K., Cao, X. & Nafie, L.A. (2004), "The conformation of tetraalanine in water determined by polarized Raman, FT-IR, and VCD spectroscopy", J Am Chem Soc, vol. 126, no. 9, pp. 2768-2776

Selkoe, D.J. (1998), "The cell biology of β-amyloid precursor protein and presenilin in Alzheimer's disease", Trends Cell Biol, vol. 8, no. 11, pp. 447-453

Selkoe, D.J. (2003), "Folding proteins in fatal ways", Nature, vol. 426, no. 6968, pp. 900-904

Selkoe, D.J. & Podlisny, M.B. (2002), "Deciphering the genetic basis of Alzheimer's disease", Annu Rev Genomics Hum Genet, vol. 3, pp. 67-99

Shao, H., Jao, S., Ma, K. & Zagorski, M.G. (1999), "Solution structures of micelle-bound amyloid β-(1-40) and β-(1-42) peptides of Alzheimer's disease", J Mol Biol, vol. 285, no. 2, pp. 755-773

Shi, Z., Woody, R.W. & Kallenbach, N.R. (2002), "Is polyproline II a major backbone conformation in unfolded proteins?", Adv Protein Chem, vol. 62, pp. 163-240

Shivaprasad, S. & Wetzel, R. (2004), "An intersheet packing interaction in Aβ fibrils mapped by disulfide cross-linking", Biochemistry, vol. 43, no. 49, pp. 15310-15317

Shivaprasad, S. & Wetzel, R. (2006), "Scanning cysteine mutagenesis analysis of Aβ-(1-40) amyloid fibrils", J Biol Chem, vol. 281, no. 2, pp. 993-1000

Sipe, J.D. & Cohen, A.S. (2000), "Review: history of the amyloid fibril", J Struct Biol, vol. 130, no. 2-3, pp. 88-98

Smet, C., Leroy, A., Sillen, A., Wieruszeski, J.M., Landrieu, I. & Lippens, G. (2004), "Accepting its random coil nature allows a partial NMR assignment of the neuronal Tau protein", Chembiochem, vol. 5, no. 12, pp. 1639-1646

Smith, D.P., Smith, D.G., Curtain, C.C., Boas, J.F., Pilbrow, J.R., Ciccotosto, G.D., Lau, T.L., Tew, D.J., Perez, K., Wade, J.D., Bush, A.I., Drew, S.C., Separovic, F., Masters, C.L., Cappai, R. & Barnham, K.J. (2006), "Copper-mediated amyloid-β toxicity is associated with an intermolecular histidine bridge", J Biol Chem, vol. 281, no. 22, pp. 15145-15154

Smith, L.J., Fiebig, K.M., Schwalbe, H. & Dobson, C.M. (1996), "The concept of a random coil. Residual structure in peptides and denatured proteins", Fold Des, vol. 1, no. 5, pp. R95-106

Sparks, D.L. & Schreurs, B.G. (2003), "Trace amounts of copper in water induce β-amyloid plaques and learning deficits in a rabbit model of Alzheimer's disease", Proc Natl Acad Sci U S A, vol. 100, no. 19, pp. 11065-11069

Stejskal, E.O. & Tanner, J.E. (1965), "Spin Diffusion Measurements: Spin Echoes in the Presence of a Time-Dependent Field Gradient", J Chem Phys, vol. 42, pp. 288-292

Stellato, F., Menestrina, G., Serra, M.D., Potrich, C., Tomazzolli, R., Meyer-Klaucke, W. & Morante, S. (2006), "Metal binding in amyloid β-peptides shows intra- and inter-peptide coordination modes", Eur Biophys J, vol. 35, no. 4, pp. 340-351

Storey, E. & Cappai, R. (1999), "The amyloid precursor protein of Alzheimer's disease and the Aβ peptide", Neuropathol Appl Neurobiol, vol. 25, no. 2, pp. 81-97

Syme, C.D., Blanch, E.W., Holt, C., Jakes, R., Goedert, M., Hecht, L. & Barron, L.D. (2002), "A Raman optical activity study of rheomorphism in caseins, synucleins and tau. New insight into the structure and behaviour of natively unfolded proteins", Eur J Biochem, vol. 269, no. 1, pp. 148-156

Syme, C.D., Nadal, R.C., Rigby, S.E. & Viles, J.H. (2004), "Copper binding to the amyloid-β (Aβ) peptide associated with Alzheimer's disease: folding,

Page 85: NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless misfolding diseases are an impor-tant field in medicine and pathophysiology and also

85

coordination geometry, pH dependence, stoichiometry, and affinity of Aβ-(1-28): insights from a range of complementary spectroscopic techniques", J Biol Chem, vol. 279, no. 18, pp. 18169-18177

Syme, C.D. & Viles, J.H. (2006), "Solution (1)H NMR investigation of Zn(2+) and Cd(2+) binding to amyloid-β peptide (Aβ) of Alzheimer's disease", Biochim Biophys Acta, vol. 1764, no. 2, pp. 246-256

Szejtli, J. (1998), "Introduction and General Overview of Cyclodextrin Chemistry", Chem Rev, vol. 98, no. 5, pp. 1743-1754

Tanzi, R.E., Moir, R.D. & Wagner, S.L. (2004), "Clearance of Alzheimer's Aβ peptide: the many roads to perdition", Neuron, vol. 43, no. 5, pp. 605-608

Taylor, B.M., Sarver, R.W., Fici, G., Poorman, R.A., Lutzke, B.S., Molinari, A., Kawabe, T., Kappenman, K., Buhl, A.E. & Epps, D.E. (2003), "Spontaneous aggregation and cytotoxicity of the β-amyloid Aβ1-40: a kinetic model", J Protein Chem, vol. 22, no. 1, pp. 31-40

Terzi, E., Hölzemann, G. & Seelig, J. (1997), "Interaction of Alzheimer β-amyloid peptide(1-40) with lipid membranes", Biochemistry, vol. 36, no. 48, pp. 14845-14852

Tickler, A.K., Smith, D.G., Ciccotosto, G.D., Tew, D.J., Curtain, C.C., Carrington, D., Masters, C.L., Bush, A.I., Cherny, R.A., Cappai, R., Wade, J.D. & Barnham, K.J. (2005), "Methylation of the imidazole side chains of the Alzheimer disease amyloid-β peptide results in abolition of superoxide dismutase-like structures and inhibition of neurotoxicity", J Biol Chem, vol. 280, no. 14, pp. 13355-13363

Tjernberg, L.O., Näslund, J., Lindqvist, F., Johansson, J., Karlström, A.R., Thyberg, J., Terenius, L. & Nordstedt, C. (1996), "Arrest of β-amyloid fibril formation by a pentapeptide ligand", J Biol Chem, vol. 271, no. 15, pp. 8545-8548

Tokuyama, M. & Oppenheim, I. (1994), "Dynamics of hard-sphere suspensions", Phys Rev, E Stat Phys Plasmas Fluids Relat Interdiscip Topics, vol. 50, no. 1, pp. R16-R19

Vassar, R., Bennett, B.D., Babu-Khan, S., Kahn, S., Mendiaz, E.A., Denis, P., Teplow, D.B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M.A., Biere, A.L., Curran, E., Burgess, T., Louis, J.C., Collins, F., Treanor, J., Rogers, G. & Citron, M. (1999), "Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE", Science, vol. 286, no. 5440, pp. 735-741

Walsh, D.M. & Selkoe, D.J. (2004), "Deciphering the molecular basis of memory failure in Alzheimer's disease", Neuron, vol. 44, no. 1, pp. 181-193

Wang, J., Dickson, D.W., Trojanowski, J.Q. & Lee, V.M. (1999), "The levels of soluble versus insoluble brain Aβ distinguish Alzheimer's disease from normal and pathologic aging", Exp Neurol, vol. 158, no. 2, pp. 328-337

Wangsness, R.K. & Bloch, F. (1953), "The Dynamical Theory of Nuclear Induction", Phys Rev, vol. 89, no. 4, pp. 728-739

Ward, R.V., Jennings, K.H., Jepras, R., Neville, W., Owen, D.E., Hawkins, J., Christie, G., Davis, J.B., George, A., Karran, E.H. & Howlett, D.R. (2000), "Fractionation and characterization of oligomeric, protofibrillar and fibrillar forms of β-amyloid peptide", Biochem J, vol. 348 Pt 1, pp. 137-144

Ware, W.R. (1962), "Oxygen Quenching of Fluorescence in Solution: An Experimental Study of the Diffusion Process", J Phys Chem, vol. 66, pp. 455-458

Watson, A.A., Fairlie, D.P. & Craik, D.J. (1998), "Solution structure of methionine-oxidized amyloid beta-peptide (1-40). Does oxidation affect conformational switching?", Biochemistry, vol. 37, no. 37, pp. 12700-12706

Page 86: NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless misfolding diseases are an impor-tant field in medicine and pathophysiology and also

86

Wennerström, H., Lindblom, G. & Lindman, B. (1974), "Theoretical aspects on the NMR of Quadropolar Ionic Nuclei in Micellar Solutions and Amphiphillic Liquid Crystals", Chem Scr, vol. 6, pp. 97-103

Westerman, M.A., Cooper-Blacketer, D., Mariash, A., Kotilinek, L., Kawarabayashi, T., Younkin, L.H., Carlson, G.A., Younkin, S.G. & Ashe, K.H. (2002), "The relationship between Aβ and memory in the Tg2576 mouse model of Alzheimer's disease", J Neurosci, vol. 22, no. 5, pp. 1858-1867

White, A.R., Du, T., Laughton, K.M., Volitakis, I., Sharples, R.A., Xilinas, M.E., Hoke, D.E., Holsinger, R.M., Evin, G., Cherny, R.A., Hill, A.F., Barnham, K.J., Li, Q.X., Bush, A.I. & Masters, C.L. (2006), "Degradation of the Alzheimer disease β-peptide by metal-dependent up-regulation of metalloprotease activity", J Biol Chem, vol. 281, no. 26, pp. 17670-17680

Whittington, S.J. & Creamer, T.P. (2003), "Salt bridges do not stabilize polyproline II helices", Biochemistry, vol. 42, no. 49, pp. 14690-14695

Wilkins, D.K., Grimshaw, S.B., Receveur, V., Dobson, C.M., Jones, J.A. & Smith, L.J. (1999), "Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques", Biochemistry, vol. 38, no. 50, pp. 16424-16431

Wirmer, J., Peti, W. & Schwalbe, H. (2006), "Motional properties of unfolded ubiquitin: a model for a random coil protein", J Biomol NMR, vol. 35, no. 3, pp. 175-186

Woody, R.W. (1992), "Circular Dichroism and Conformation of Unordered Polypeptides", Adv Biophys Chem, vol. 2, pp. 37-79

Xu, Y., Shen, J., Luo, X., Zhu, W., Chen, K., Ma, J. & Jiang, H. (2005), "Conformational transition of amyloid β-peptide", Proc Natl Acad Sci U S A, vol. 102, no. 15, pp. 5403-5407

Yang, D.S., McLaurin, J., Qin, K., Westaway, D. & Fraser, P.E. (2000), "Examining the zinc binding site of the amyloid-β peptide", Eur J Biochem, vol. 267, no. 22, pp. 6692-6698

Yang, J.T. & Foster, J.F. (1954), "Changes in the Intrinsic Viscosity and Optical Rotation of Bovine Plasma Albumin associated with acid binding", J Am Chem Soc, vol. 76, no. 6, pp. 1588-1595

Yoshiike, Y., Tanemura, K., Murayama, O., Akagi, T., Murayama, M., Sato, S., Sun, X., Tanaka, N. & Takashima, A. (2001), "New insights on how metals disrupt amyloid β-aggregation and their effects on amyloid-β cytotoxicity", J Biol Chem, vol. 276, no. 34, pp. 32293-32299

Yu, J., Bakhos, L., Chang, L., Holterman, M.J., Klein, W.L. & Venton, D.L. (2002), "Per-6-substituted β-cyclodextrin libraries inhibit formation of β-amyloid-peptide (Aβ)-derived, soluble oligomers", J Mol Neurosci, vol. 19, no. 1-2, pp. 51-55

Zimm, B.H. & Bragg, J.K. (1959), "Theory of the Phase Transition between Helix and Random Coil in Polypeptide Chains", J Chem Phys, vol. 31, no. 2, pp. 526-535

Zirah, S., Kozin, S.A., Mazur, A.K., Blond, A., Cheminant, M., Ségalas-Milazzo, I., Debey, P. & Rebuffat, S. (2006), "Structural changes of region 1-16 of the Alzheimer disease amyloid β-peptide upon zinc binding and in vitro aging", J Biol Chem, vol. 281, no. 4, pp. 2151-2161

Zirah, S., Stefanescu, R., Manea, M., Tian, X., Cecal, R., Kozin, S.A., Debey, P., Rebuffat, S. & Przybylski, M. (2004), "Zinc binding agonist effect on the recognition of the β-amyloid (4-10) epitope by anti-β-amyloid antibodies", Biochem Biophys Res Commun, vol. 321, no. 2, pp. 324-328

Page 87: NMR studies of the amyloid ββββ-peptide190048/FULLTEXT01.pdf · 2009-02-27 · Nevertheless misfolding diseases are an impor-tant field in medicine and pathophysiology and also

87