Protein: Structure and Function

Books:

1. Lehninger Principle of Biochemistry ( by Nelson and Cox)

Amino acid

Properties:1. -carbon is bonded to four different groups except glycine2. -carbon is a chiral center3. Two possible stereoisomers (called enantiomers)4. Optically active (except glycine): rotate plan polarized light5. Can act as acids and bases

Amino acids in the human body

Essential amino acid

Non-essential amino acid

1. Leucine2. Isoleucine3. Valine4. Histidine5. Lysine

6. Methionine7. Phenylalanine8. Threonine9. Tryptophan

1. Alanine2. Arginine3. Asparagine4. Aspartic acid5. Cysteine6. Glutamic acid7. Glutamine

8. Glycine9. Proline10. Serine11. Tyrosine

Obtained from nutrition

Synthesized by the body

Non-polar, aliphatic R groups

Gly/G Ala/A Pro/P Val/V

Leu/L Ile/I Met/M

Polar, uncharged R groups

1. Cysteine forms dimercalled cystine

2. Disulfide residues are strongly hydrophobic

Ser/S Thr/T Cys/C

Asn/N Gln/Q

Positively charged R groups

Lys/K Arg/R His/H

Negatively charged R groups

Asp/D Glu/E

Aromatic R groups

1. Non-polar2. Hydrophobic3. Form H-bond4. Absorb UV light (280 nm)

Phe/F Tyr/Y Trp/W

pKa values for carboxyl and amino groups

Amino acid have characteristic titration curve1. Without ionisable R group

pI = (pK1 + pK2 )/2

pK1 = for acidpK2 = for base

2. With ionisable R group

pI = ?

2. With ionisable R group

pI = ?

1. All protein polymers are constructed from the same set of 20 amino acids.

2. Polymers of proteins are called polypeptides.

3. A protein consists of one or more polypeptides folded and coiled into a specific conformation

4. The physical and chemical characteristics of the R group determine the unique characteristics of a particular amino acid.

Protein

Peptide Bond

Properties of Peptide bond

1. Planar2. Double bond character (which prevent rotation

about this bond)3. Uncharged (helps to form tightly packed globular

structure4. Two conformations possible (cis and trans)5. All peptide bonds are in proteins are trans6. Other than peptide bonds in protein helps to take

many conformational structure7. Average half-life of peptide bond is 7 years

(intracellular condition)

Fully extended polypeptide chain

Both bond can rotate

and are zero

trans-Peptide group

cis-Peptide group

Peptide and Protein

Peptides: 1. Short polymers formed from the linking of (usually less

than or equal to 100) amino acids and comprise 2. Some of the basic components of human biological

processes, including enzymes, hormones, and antibodies.

Protein:1. A functional, polypeptide chain composed of at

least around fifty amino acids put together. 2. They play a critical role in biochemical reactions

within cells.

1. Simple protein: contain only amino acid residues2. Conjugated proteins: contain permanently associated chemical

components in addition to amino acids(a) Lipoproteins: contain lipid(b) glycoproteins: contain sugar(c) metalloprotein: contain specific metal

Types of Protein

Structure of Protein

I. The primary structure determines the folding of the polypeptide to give a functional protein

II. Polar amino acids (acidic, basic and neutral) are hydrophilic and tend to be placed on the outside of the protein.

III. Non-polar (hydrophobic) amino acids tend to be placed on the inside of the protein

IV. The possible conformations are very large

V. Most are useless, natural selection picks out the best

Primary Structure

1. Elongated2. Dynamic, heterogeneous3. Very rich in H-bond4. Every protein has unique amino acid sequence5. Sequence decide the mechanism of action6. Sequence determine the 3-D structure7. Sequence reveals about its evolutionary history

Primary structure of protein

1. Starting of the polypeptide chain : N-terminal (amino group)

2. End terminal of the polypeptide chain: C-terminal (carboxylic group)

Human insulin

1. Strong interactions(a) Covalent bonding(b) Ionic bonding(c) Resonance bonding

2. Weak Interaction(a) Van der Waals interaction

(i) Polar-polar (ii) Polar-non polar(iii) Non polar- non polar

(b) Hydrogen bonding

3. Effect of medium(a) Screening of field(b) Surface interaction(c) Hydrophobic environment(d) Hydrophilic environment

Molecular interactions

The polypeptide chain can fold into regular structures like:

1. Alpha helix2. Beta-sheet3. Turns and loops

These are called secondary structure which helps to form final 3-D structure

Secondary structure of proteinThe folding of the N-C terminals of the chain using many different interactions

Alpha-helix1. It is coiled structure stabilized by intra-chain H-bonds

between NH and CO groups (situated 4 residue ahead in the sequence) except end terminal groups

2. Pitch of alpha-helix 5.4 A

3. Both right handed and left handed helix are allowed , however right handed alpha-helices are energetically more favourable because there is less steric clash between the side chain and backbone

4. Content in the protein: alpha-helices may be 100%

5. Glycine, serine and threonine make amino terminal residue (N-cap) in alpha-helix

6. Glycine and asparagine makes carboxyl terminal (C-cap) of alpha-helix

Helical Wheel: Each residue can be plotted every 360/3.6=100 around a circle or spiral

Representation of -helices in protein

-helicesH bond between residues i, i+4

Rise per residue, d = 1.5

# of residues per turn, n = 3.6

Pitch of helix= n x d = 5.4

The -helix has a dipole moment

The dipole of a peptide unit. Numbers in boxes give the approximate fractional charges of the atoms of the peptide unit

The Dipoles of peptide units are aligned along the helical axis

Beta- sheet/strand1. It is stabilized by H-bonding between chains

2. This secondary structure may associated through side chain interactions and form super-secondary structure called motif

3. Beta-sheet is almost fully extended

4. The distance between adjacent amino acids along a beta-strand is roughly 3.5 A (in contrast to 1.5 A in alpha-helix)

5. A beta- sheet is formed by linking two pr more beta strands by H-bonds

6. Beta strand represented by broad arrows pointing in the direction of C-terminal

7. Beta sheet formation is important in fatty acid-binding proteins and lipid metabolism

-Strand

The side chain (green) are alternatively above and below the plane of the strand

NN

C

C

Anti-parallel -sheet

Adjacent -strand run in opposite direction. H-bond between NH and CO groups connect each amino acid on an adjacent strand and stabilize the structure

Parallel -sheet

Adjacent -strand run in same direction. H-bond between NH and CO groups connect each amino acid on one strand with two different amino acids on the adjacent strand

N

N

C

C

Mixed -sheet

Turns and Loops

1. Polypeptide chain can change direction with the help of turn and loops

2. CO group of residue i is H-bonded with NH group of residue i+3

3. This particular H-bond interaction stabilizes abrupt changes in the direction of polypeptide chain

4. Turn and loops connect alpha-helices and beta strand and allow a peptide chain to fold back on itself to make a compact structure

5. Loops often contain hydrophilic residues and are found on the protein surface

6. Turn or loops contain 5 residues or less

7. Beta turn connects different anti-parallel beta strands

Ramachandran plot

Phi ()

Psi ()

Phi () and Psi () rotate, allowing the polypeptide to assume its various conformations

some conformations of the polypeptide backbone result in steric hindrance and are disallowed

glycine has no side chain and is therefore conformationally highly flexible (it is often found in turns)

no stericclashes

permittedif atoms aremore closelyspaced

Tertiary structure of protein

Spatial arrangement of amino acid residues that are far apart in the sequence

1. This folding is sometimes held together by strong covalent bonds (e.g. cysteine-cysteine disulphide bridge)

2. Bending of the chain takes place at certain amino acids (e.g. proline)

3. Hydrophobic amino acids tend to arrange themselves insidethe molecule

4. Hydrophilic amino acids arrange themselves on the outside

Quaternary structure of protein

1. Polypeptide chains can assemble into multi sub-units

2. Sub units are spatially arranged

3. Helix-loop-helix: two helices connected by a turn

4. Coiled-coil: two alpha helices interact in parallel through their hydrophobic edge

5. Helix-bundle: several alpha-helices that associate in an anti-parallel manner

6. Beta-alpha-beta unit: two parallel beta strand linked to an intervening alpha helix by two loops

Levels of structure in proteins

Protein structure: overview

Structural element Description

primary structure amino acid sequence of protein

secondary structure helices, sheets, turns/loops

super-secondary structure (motif) association of secondary structures

domain self-contained structural unit

tertiary structure folded structure of whole protein includes disulfide bonds

quaternary structure assembled complex (oligomer) homo-oligomeric (1 protein type) hetero-oligomeric (>1 type)

Physical parameters of protein

Size of the protein roughly between 1nm to 10nm

Persistence length of protein ranges between 0.3 nm to 0.8 nm

Elastic modulus of protein ranges between 1200 to 2000 pN/nm2

The protein structure must obey

1. The bond lengths and bond angles should be distorted as little as possible

2. No two atoms should approach one another more closely than is allowed by there van der Waals radii

3. The amide group must remain planar and in the trans configuration. This allows only rotation about the two bonds adjacent to the alpha-carbon

4. Some kind of non-covalent binding is necessary to stabilized a regular folding

Domain structures core is exclusively built from helices

Domain structures core comprises of antiparallel sheets, usually two sheets packed against each other

/ Domain structures made from combinations of -- motifs that form a predominantly parallel sheets surrounded by helices

Structure of proteins

Human plasma retinol binding protein. Retinol molecule (vitamin A) bound inside the barrel

Triosephosphateisomerase

Structure of proteins

Structure Function

Structure Mechanism

Structure Origins/Evolution

Structure-based Drug Design

Solving the Protein Folding Problem

Solving Protein Structures

Atomic resolution pictures of macromolecules X-ray Crystallography (first applied in 1961 - Kendrew & Perutz) NMR Spectroscopy (first applied in 1983 - Ernst & Wuthrich)

QHTAWCLTSEQHTAAVIWDCETPGKQNGAYQEDCAHHHHHHCCEEEEEEEEEEECCHHHHHHHCCCCCCC

Crystallographic structure of Myoglobin

10

(1958, Sir John Kendrew)

Protein Structure solved by X-ray crystallography

PDB contains 75000 structures mostly determined by X-ray crystallography and NMR. About 3-5 new structures per day

Total Yearly

Hemoglobin A: Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Hemoglobin S: Val-His-Leu-Thr-Pro-Val-Glu-Lys-

sticky patch causes hemoglobin S to agglutinate (stick together) and form fibers which deform the red blood cell

Importance of Protein StructureUsing electrophoresis, Pauling showed that individuals with sickle cell disease had a modified form of Hb

101 residues. each residue can assume three different conformations

the total number of structures would be 3100, which is equal to 5 1047

If it takes 10-13 s to convert one structure into another

the total search time would be 5 1047 10-13 s

which is equal to 5 1034 s, or 1.6 1027 years.

Protein folding: Levinthals paradox

The enormous difference between calculated and actual folding times is called Levinthal's paradox.

There should be some pathways for folding

Factors affecting protein folding

1. Space packingproteins are like liquid and gases instead of crystalline solidit helps in forming structure but space packing is not enough

2. Internal residue: Folding is directed mainly by internal residues not by surface residues. (Hydrophobic force-driven folding)

3. Protein structures are hierarchically organized

4. Protein structures are highly adaptable

5. Secondary structure can be context dependent and can be predicted by algorithms

6. Changing the fold of a protein

Speed limit of protein folding

For a single domain protein The approximate folding time (folding) is given by N/100 s

-protein fold faster than the protein or protein

folding = k exp(G/kBT)

Hydrophobic effect

Conformational entropy

Electrostatics

Hydrogen bonding

van der Waals interaction

Protein folding

The main driving force for folding water soluble globularprotein molecules is to pack hydrophobic side chains into the interior of the molecule , thus creating a HYDROPHOBIC CORE &HYDROPHILLIC SURFACE.

Problem- How to create such a hydrophobic core from a protein chain ???

?Protein folding

Compact (in general)Defined structure

Insulin

Molten Globule

1. Secondary structure that is present in a native protein forms within a few microsecond

2. This is because of hydrophobic collapse3. It is larger by (5-15%) in size of native conformations4. Side chains are not ordered/packed5. Structure fluctuation is much larger 6. Not thermodynamically stable

Folding protein moves over energy surface from unfolded to folded state:

unfolded

folded

Energy landscape governs folding

100

k BT

Deg

ree

of

nativ

enes

s

H= bond stretching + bending of angles + Bond rotations + van der waals interaction + electrostatic interaction

Folding is a complex process

Misfolding

Macromolecules must fold into correct shape to function properly: Structure Function

Disease(non-native structures)

Many diseases with large impacts involve protein misfolding:

Alzheimers Disease A peptide

Parkinsons Disease -synuclein

Creutzfeldt-Jakob disease Prion

Huntingtons Disease Huntingtin protein

Amyotrophic Lateral Sclerosis Superoxide dismutase

Type II Diabetes Amylin

Misfolding and aggregation are complex

nativelyfolded

amyloid fibrils

Many different species and steps involved in misfolding and aggregation:

protein synthesis unfolded

ribosome

partiallyunfolded

partially-unfoldedand aggregated

disorderedaggregates

degradedfragments

non-native structuredaggregates

misfolded

Chiti & Dobson, Annu. Rev. Biochem., (2006)

Force-extension curvesMove traps apart at constant rate to stretch handles and apply force to a single-molecule:

50 mM MOPS, 200 mM KCl, pH 7.0

unfolding

stretching handles

molecule unfoldsrefolding

Apparently two-state folding

molecule unfolds

Reconstructing landscapes from FECsBased on Jarzysnki equality for relating equilibrium free energy to non-equilibrium work:

Hummer & Szabo, PNAS (2001)

Many applications, never validated experimentally

Forc

e

Distance

Work

But energy is dissipated!

WGeqm

Recover equilibrium energy fromfluctuations in work done:

Prob

abili

ty

Work

Geqm

W

Jarzynski, Phys. Rev. Lett. (1997)

Jarzynski equality:

=

TkW

TkG

B

eqmnon

B

eqm expexp

Method to calculate free energy profile G(x)

Reconstructing energy landscape profilesUsing equilibrium probability distributions:

Exte

nsio

n (n

m)

Time (s)

Prob

abili

ty d

ensit

y

( ) ( )expB

G xP x

k T

( ) ( )logBG x k T P x =

Thousands oftransitions!

Extension (nm)

Free energy (kJ/mol)

Invert

Deconvolution to remove effects of compliant handles:

Prob

abili

ty

Extension (nm)

P(x)

deconvolution

Residual

Free

ene

rgy

(kJ/

mol

)

Extension (nm)

G(x)

raw data

Woodside et al., Science (2006)

parameter-free modelfor folding

Woodside et al., PNAS (2006)

Slow and time consuming experiments

Computational approach for protein folding

1. Energy minimization(a) Steepest(b) Conjugated gradient

2. Monte Carlo Simulation(a) Random Sampling(b) Stimulated annealing

3. Molecular dynamics(a) Compute conformational change(b) Calculate trajectories at thermal condition and fond

the ensemble averaged physical quantity

Denaturants high temperatures

- cause protein unfolding, aggregation

low temperatures- some proteins are sensitive to cold denaturation

heavy metals (e.g., lead, cadmium, etc.)- highly toxic; efficiently induce the stress response

proteotoxic agents (e.g., alcohols, cross-linking agents, etc.)

oxygen radicals, ionizing radiation- cause permanent protein damage

chaotropes (urea, guanidine hydrochloride, etc.)- highly potent at denaturing proteins;often used in protein folding studies

Protein folding/unfolding

Force spectroscopy

AFM Optical Tweezers

Protein-Protein Interaction NetworksYeast ~6000 proteins, ~3 interactions per protein, i.e. ~>20,000 interactions. Humans ~100,000 interactions

Nat. Biotechnol. 18, 12571261 (2000)

Which two proteins will interact?

AND, which will not?

The ANSWER lies in the nature of theinteracting surfaces

A-B, A-C forms poorly matched surfaces, few weak bonds are formed, broken apart by thermal motion

A-D offers well matched surfaces, enough noncovalent bonds are formed to create a stable interface

Forces driving protein-protein interaction

Long-range attractive interactions

electrostatic steering

Short-range non-covalent forces:

Hydrophobic interactions

van der Waals attraction

Hydrogen bonds

Ion pairs

Other factors:

Shape and charge complementarity

Secondary structure

Amino acid composition

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