1

Inhibition of μ-calpain;

Towards treatment of Rheumatoid Arthritis

Submitted by: Hala Issa September 2014

Supervised by: Rudolf K. Alleman

David Miller

2

Abstract

Rheumatoid arthritis (RA) is an autoinflammatory disease that would cause severe pain that

would progress to the inability to move. Neutrophils, which are activated by cytokines, migrate

to the site of inflammation via an amoeboid like movement facilitated by the action of μ-calpain,

a calcium dependant cysteine protease. Cytoskeletal proteins are among the popular substrates

for μ-calpain which by hydrolyzing the interdomain bonds can induce cell movement. Many

drugs are present nowadays in the market for the purpose of treating RA but most of them are

either expensive or invasive and too painful. Monohalogenated α-mercaptoacrylates, the most

potent calpain inhibitors are finding their way as effective small molecules drugs, which if they

were optimized to produce better pharmakokinetics, would be the next generation in RA

treatment along with other inflammatory and degenerative diseases. However, their mechanism

of action and their interactions with different enzyme domains must be elucidated first.

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1. Introduction

4

Calpain Family

Although calpains belong to the family of cysteine proteases, they are characteristically

distinguished by their dependence on physiological calcium influx for their activation. Calpains

are a wide family of cytosolic proteins consisting of 15 isoforms which are ubiquitous and tissue

specific 1 where they share the proteolytic triad Cys-His-Asn in common with other cysteine

proteases. However, the active site is not preformed and sterically blocked by a prosegment as in

the case of other cysteine proteases, instead Cys and His are ~ 7 A° apart, a distance too far to

elicit any reaction between the two residues and thus Ca2+

binding ultimately lead to closing

down this gap and forming the active site 2. Two major isoforms are found in human body

namely μ-calpain and m-calpain which get activated by micro (10-50 µM) and milli (0.2-0.35

mM) molar calcium concentrations in vitro respectively 3. Both major enzymes form a

heterodimer with two subunits; an identical shared small "regulatory" subunit (~ 30 kDa)

encoded by CAPNSI and different large "catalytic" subunit (~80 kDa) encoded by CAPNI and

CAPNII genes for μ- and m- calpain respectively 4. The degree of similarity between the two

large subunits is ~ 60% 5, 6

. Calpains are involved in modulating the function of their substrate

rather than digesting it 2.

Six domains define the structure of both calpains along with a short (~ 19 residues) 7N-terminal

α-helix present in the catalytic subunit where it functions to join the two subunits together The

protease domains DI-DII along with the C2-like domain DIII and the penta-EF (PEF-L) hand

calcium binding domain DIV are all present in the large subunit. The small one is composed of a

glycine rich DV, and another PEF (PEF-S) hand calcium binding domain DVI. DIV and DVI

both add some calmodulin like characteristics when they are present in calpain isoforms 2. The

small subunit along with some domains of the large subunit might be missing from the other 13

isoforms except for the catalytic domain.

The N-terminal α-helix in the catalytic subunit was found to be embedded through hydrophobic

interactions 6 into the PEF domain of the small subunit

8. As part of the activation process of the

enzyme, the helix autocleaved in order to lower Ca2+

requirements 9.

5

Figure (1): Schematic diagram showing the domain structure of human m-calpain 10

.

The catalytic triad residues are located at the interface of the CysPc domains DI and DII 2

(residue 29 to 356 in μ-calpain 11

) where the cysteine residue is located in DI and His and Asn

are found in DII. Crystal structure showed that the 10° distance is decreased upon the binding of

one calcium ion in each of the CysPc domains but it is maintained through the restraining effect

of the circular arrangements of domains 11

. The interaction of the anchor α-helix with PEF(S)

restricts the movement of DI 4. Thus this domain can bind two calcium ions.

C2-like domain, DIII, possesses an anti-parallel β-sheet sandwich structure and makes contact

with each of the other five domains of μ- and m-calpain and thus participates in linking the

CysPc domains to the PEF(L) 10

. It is also suggested that DIII is greatly involved in binding

phospholipids 10

which explains the ability of calpains to migrate from the cytosol to the plasma

membrane where they exert their function 4. An acidic loop in this domain interacts with basic

residues on DII thus adding more restrains to the formation of the active site cleft 4. A ~ 15

residue extended linker connects DIII with PEF(L) and plays an important role in calcium

induced conformational changes of calpains 2, 11

.

The N-terminal hydrophobic glycine rich DV is hard to see in crystal structures due to its highly

mobile conformations 4, 12

. This flexible nature might be exploited to attach the enzyme to other

molecules 4, 10

.

Heterodimerization of both subunits occurs through their C-terminal Ca2+

binding domains DIV

and DVI which are predominantly alpha helical 2 and interact with DI and DII to prevent

proteolytic activity 13

. The first four EF hands in both domains bind one Ca2+

each, while the

fifth hand is the one involved in dimerization 4.

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Activation of Calpains

Physiological calcium levels are in the range of 100 - 300 nM 14

and barely exceed 1 µM upon

induced signal. Thus a combination of events must take place to allow for the activation of

calpains to occur at such low Ca2+

concentrations. Binding of Ca2+

to PEF(L) and PEF(S) confers

slight conformational changes which are transmitted to the N-terminal anchor helix leading to its

dissociation and to DIII through the linker. These changes disrupt the electrostatic interactions

between DIII and DII 4 which subsequently allows for the formaiotn on the active site cleft by

the cooperative binding of two calcium ions to the CysPc domains 11

. Thus, a total of 10 Ca2+

ions bind across calpain domains where 4 calcium molecules bind in each of the PEF hands and

2 molecules bind in each of the CysPc domain (Figure 2).

Figure (2): 3D structure of inactive and active m-calpain showing the different domains of the

protein of both subunits; catalytic and regulatory (http://calpain.net/3dstructure/index.html)

Calpastatin

Inhibition and regulation of calpains takes place through their only known endogenous inhibitor

so far, calpastatin (70 kDa) 4. It is composed of four domains (I to IV)

5 and each domain is

furthermore divided into three subunits A, B, and C (Figure 3). Each of these subunits have a

specific binding preference towards calpain domains. Subdomains A and C which bind the

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PEF(L) and PEF(S) respectively through hydrophobic interactions have no inhibitory effect

when isolated whereas B which binds to the active sire cleft exhibited a slight inhibitory effect

on its own. Since calpastatin only binds activated calpains, then Ca2+

induced changes are

essential for this interaction to take place especially at the PEF level. Calpastatin is a substrate of

calpains and thus its degradation is implicated in the initiation of pathological events 6.

Figure (3): Schematic representation of calpastatin domains 6

Physiological and Pathological Role of Calpains

Calpains are implicated in both health and disease states due to their versatile function and

abundance throughout the human body 6. Processes such as signal transduction, apoptosis, cell

motility 1, muscle homeostasis, and nerve growth

6 are highly dependent on balanced calpain

activation. Any defect in the homeostasis of calcium and thus imbalanced calpain activity can

progress into pathological and neurodegenerative disorders. Calpain 3 is majorly found in muscle

cells and thus any mutation that would result in a non functional enzyme leads to the

development of limb girdle muscular dystrophy 2A 2. Furthermore, substrates for calpain

include fodrin, p53, spectrin, talin, and fibronectin 6. Many of these substrates are involved in

cell migration, another important function carried out by calpains. Talin for example binds β-

integrin cytoplasmic domains and link them to actin filaments 15

which are also cleaved by

calpains 9 inflicting an amoeboid like movement on the migrating cell. Extrapolating these

findings to the movement of inflammatory cells during autoimmune diseases such as rheumatoid

arrthritis, neutrophils- the human body first line of defense 4- were found express μ-calpain

predominantly 16

. This suggest that targeting the inhibition of this calpain specifically can lead to

the development of therapeutic agents for the treatment of RA and other degenerative diseases

where calpain overactivation is implicated.

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Neutrophil Spreading and Migration

Neutrophils function by phagocytosis and the release of proteolytic enzymes against a pathogen

4. They are attracted to the site of inflammation through cytokines such as interlukin-17 (IL-17),

IL-8, interferon γ (IFNγ), and tumor necrosis factor 17

. These chemical signals help in the

activation and prolonged survival of neutrophils 17

. In the extravasation of neutrophils, they

undergo morphological change from spherical to flattened shape for faster spreading and motility

1, 4. The membrane of an unactivated neutrophils holds many wrinkles and maintains its shape

through membrane bound proteins 4. Actin, an intracellular protein, is linked to L-selectin and

β2-integrin- which are the membrane bound proteins of neutrophils- through ezrin and talin

respectively 4 both of which are μ-calpain substrates. On the other hand, release of cytokines and

immobilization of β2-integrin by surfaces provoke an increase in Ca2+

concentrations 4, 16

.

Activated μ-calpain is now driven towards the plasma membrane through its C2L domain and

initiate a proteolytic attack allowing the cell membrane to de-wrinkle and spread 4. Other

components of membrane bound proteins are involved in the forward movement of the

neutrophil.

Figure (4): activation, migration, and neutrophil spreading in response to the release of

cytokines (http://www.studyblue.com/notes/note/n/case-2/deck/2532289)

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Rheumatoid Arthritis

RA is a chronic inflammatory autoimmune disease that starts in small joints and progresses to

destruction of bone and cartilage 18

. The synovium is infiltrated by monocytes/macrophages,

rheumatoid arthritis synovial fibroblasts, T-cells and B-cells which are responsible for the release

of tumor necrosis factor α (TNF α) as well as other pro-inflammatory cytokines 18, 19

. This will

help recruiting more immune cells and ultimately lead to the formation of granular tissue known

as pannus 4. Neutrophils which are present specifically at the synovial fluid contribute to the

process of inflammation by releasing prostaglandins, proteolytic enzymes and reactive oxygen

species ROS 20

. These chemicals have detrimental effect on the joint and its surrounding and

contribute largely to the amount of damage caused during RA. Hardening of the surrounding

environment follows imposing difficulty in joint movement 4. Stopping the flow of these cells

towards the inflammation site is expected to reduce the destruction of the synovium and

eventually ease down the symptoms. Many of the available treatments nowadays are ineffective

in targeting the underlying reason of inflammation and depend mostly on relieving the symptoms

such as NSAIDS or involved in side effects (disease modifying anti-rheumatic drugs) 4. Other

approaches are invasive, extremely painful, and expensive. Inhibition of cytokines such as TNF

and Il-1 and transcription factors such as NF- κB proved efficient in resolving inflammation 21,

22. However, inhibiting some of these factors requires the inhibition of the phosphorylation step

and subsequently interfering with kinases several functions 22

. Thus, such treatments have to be

monitored closely.

Chemically Synthesized Calpain Inhibitors

Inhibition of calpain-mediated neutrophil migration by small-molecule drugs is an active search

in the field of pharmaceuticals 4. It aims to treat RA, and other diseases that result generally

from calpain overactivation such as Alzhiemer's disease, myocardial ischemia, and cancer 1, 23

.

Normally, some drugs were devised to target the active site of calpain, but these drugs were of

poor specificity because they interacted with active sites of other cysteine proteases nor between

μ- and m-calpain 4. Leupeptin (Ac-Leu-Leu-Arg-H), which is an example of peptidyl aldehyde

derived from Streptomyces reacts reversibly with the active site of calpains as well as other

cysteine proteases and it exhibited poor cell permeability 4, 24

. Modifications were made by the

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addition of lipophilic substituents to the N-terminus of leupeptin and the product was named

calpeptin 24

. Further modifications were undertaken to enhance permeability and specificity, but

since these compounds are aldehyde based, they were oxidized in the human body 4, 24

. Another

peptide based compound (compound 1) considered to be the most potent towards μ-calpain with

IC50 of 87 pM was synthesized and it is thought to function by chelation of Ca2+

instead of

binding to the active site 4. Peptidyl epoxysuccinate compounds are very popular irreversible

cysteine protease inhibitors but they lack selectivity towards calpains 4, 24

.

Lead (Pb2+

) and other nonpeptide organic compounds were also among the choices for inhibiting

calpains. Pb2+

is a candidate in replacing Ca2+

in a variety of physiological processes but it can

act as a non-competitive inhibitor 24

. α-mercaptoacrylates based inhibitors are the newest, most

selective, and cell permeable against calpains 4, 23, 24

. Two of them designated by PD150606 and

PD151746 are potent and reversible inhibitors of calpains with Ki of 0.21 µM and 0.37 µM

against μ- and m-calpain respectively 23

. They bind in the PEF(S) hydrophobic pocket, a

location where calpastatin region C initially binds 4, 6, 23

. Its mechanism depends on occupying

the calcium binding sites of PEF(S) and interrupting any conformational changes that would be

transmitted to the active site 23

.

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Structure activity relationship was further investigated to improve the potency and selectivity of

these α-mercaptoacrylates and this led to the development of compound (2) which added to the

previous characteristics by having enhanced potency 3 and it was successful in impairing

neutrophil spreading (Ki = 2-7 nM) 4. However, through studying the interactions of these

modified phenyl and indole analogues, it was determined that the volume of the pocket changes

with the size of the bound ring and the halogen attached which might be a reason for having an

increased affinity of indole derivatives to phenyl ones 1. Challenges to structures similar to α-

mercaptoacrylates is the presence of free sulfhydryls that can be oxidized inside the body and

thus reduce their bioavailability 3.

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Aim of the study

Calpains are associated with many physiological and pathological processes but the exact role of

the different forms are still to be elucidated. Inhibitors that can discriminate between the two

major isoforms recently been reported; monohalogenated mercaptoacrylic acids derivatives are

the most potent and selective inhibitors of μ- and m-calpain synthesized to date. Nevertheless,

information about their interaction with the different domains of calpain are limited. Moreover,

efforts continue to increase the selectivity of the inhibitors against one isoform over the other.

The aim of this work was to test the interactions of one of the potent inhibitor of calpains, (Z)-3-

(4-bromophenyl)-2-mercaptoacrylic acid, against the CysPc domain and PEF(L):PEF(S)

heterdimers. Combined with results of testing against homodimeric PEF(S) in previous studies,

this would allow us to better understand how these inhibitors interact with the various domains

of the enzyme and give insights into the molecular basis of selectivity.

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2. Material and

Methods

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Materials

Preparation of Luria-Bertani (LB) Media

For each 1 L of LB media; 10 g of tryptone, 5 g of yeast and 10 g of sodium chloride (NaCl)

were dissolved in deionized water (dH2O). The solution was a sterilised in an autoclave at 121

°C, 15 lb.(sq. in)-1

for 20 minutes

Preparation of LB Agar

5 g of tryptone, 2.5 g of yeast, 5.0 g of NaCl and 7.5 g of agar were transferred to 500 ml flask, ,

dH2O was added to a total volume of 500 ml. The solution was sterilized by autoclave at 121 °C,

15 lb.(sq. in)-1

for 20 minutes.

Depending on the antibiotic selectivity of the E.coli cells to be cultures, either ampicillin or

kanamycin were dissolved in the sterilised LB agar to a total concentration of 0.2 mg/ml and 0.1

mg/ml respectively. This was performed prior to plating the mixture.

Preparation of SDS-PAGE

Table ingredients for the preparation of 12.5 % sodium dodecyl sulfate polyacrylamide gel

(SDS-PAGE).

Resolving Stacking

H2O 3.4 mL 5.7 mL

Buffer 2.5 mL (pH 8.8) 2.5 mL (pH 6.8)

Acrylamide 4.0 mL 1.7 mL

10% SDS 100 µL 100 µL

APS (0.1 g/mL) 100 µL 100 µL

TEMED 15 µL 20 µL

Resolving buffer: 36.4 g of Tris-base was dissolved in 200 mL of dH2O to make a total

concentration of 1.5 M. The solution was adjusted to pH 8.8 and stored at 4 °C.

Stacking buffer: 15.8 g of Tris-HCl was dissolved in 200 mL of dH2O to make a total

concentration of 0.5 M. The solution was adjusted to pH 6.8 and stored at 4 °C.

10% (w/v) sodium dodecyl sulfate (SDS): 10 g of SDS was dissolved in 100 mL of dH2O.

15

0.1g/ml Ammonium persulfate (APS) :100 mg of APS was dissolved in 1 mL of ddH2O. . The

solution was prepared fresh prior to use.

Protein loading dye: 1.25 ml of 0.5 M Tris-HCl at pH 6.8, 2.5 mL glycerol, 0.2 mL

bromophenol blue, 2.0 ml of 10% SDS solution and 500 µL β-mercaptoethanol (β-ME) were

mixed with 3.55 ml of dH2O to achieve a total volume of 10 ml. The total concentrations were

62.5 mmol Tris-HCl, 25% glycerol, 0.6% (w/v) bromophenol blue, 2% SDS and 5% β-ME.

2% SDS and 5% β-ME.

Electrode Running Buffer: 10 × electrode running buffer (pH 8.3) was prepared by dissolving

30.3 g of Tris-base, 144.0 g of glycine, and 10.0 g of SDS in dH2O to a total volume to 1 L. 1 ×

electrode running buffer was prepared by diluting 100 mL of 10 × electrode running buffer with

900 mL of dH2O.

Protein Purification and Concentration Buffers

Non-imidazole Buffer: 2.42 g of Tris-base, 17.5 g of NaCl and 50 ml of glycerol were dissolved

in dH2O to a total volume of 1 L, providing final concentrations of 20 mM Tris-base, 0.3 M

NaCl and 5% (v/v) glycerol. The buffer was adjusted to pH 8.0.

Imidazole Buffer: 1.21 g of Tris-Base, 8.8 g of NaCl, 17 g of imidazole and 25 ml of glycerol

were dissolved in dH2O to a total volume of 0.5 L, providing final concentrations of 20 mM Tris-

base, 0.3 M NaCl, 0.5 M imidazole and 5% (v/v) glycerol. The solution was adjusted to pH 8.0.

10, 50, and 200 mM imidazole solutions were prepared by diluting 1, 5, and 20 ml of the 0.5 M

imidazole buffer, respectively, with the non-imidazole buffer to a total volume of 50 ml.

Dialysis Buffer: 4.84 g of Tris-base, 35 g of NaCl, 1.4 ml β-mercaptoethanol and 100 ml of

glycerol were dissolved in dH2O to a total volume of 2 L, providing final concentrations of 20

mM Tris-base, 0.3 M NaCl, 10 mM β-mercaptoethanol and 5% (v/v) glycerol. The solution was

adjusted to pH 8.0.

Protein Concentration Buffer (A): 1.21 g of TRIS-base, 8.75 g of NaCl, 25 mL of glycerol and

0.35 mL of β-ME were dissolved in dH2O to a total volume of 0.5 L, providing final

concentrations of 20 mM Tris-base, 0.3 M NaCl, 5% (v/v) glycerol and 10 mM β-ME. The

solution was adjusted to pH 8.0 and filtered.

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CD Buffer: 5 mM phosphate buffer was prepared using 0.77 ml of 1M dibasic phosphate

solution (K2HPO4) and 0.48 ml of 1M monobasic phosphate (KH2PO4) solution and mixed with

dH2O to a total volume of 250 ml. The solution was adjusted to pH 7.0, filtered and degassed

with the aid of a vacuum pump..

Size Exclusion Chromatography Buffer: 6.3 g Tris-HCl, 1.49 g ethylenediaminetetraacetic

acid disodium salt (EDTA), 0.7 ml β-ME and 17.5 g NaCl were dissolved in dH2O to a total

volume of 2 L, leading to final concentrations of 20 mM Tris-HCl, 2 mM EDTA, 5 mM β-ME

and 0.15 M NaCl. The solution was adjusted to pH 7.5, filtered and degassed with the aid of a

vacuum pump.

Protein Purification Buffer (A): 4.8 g Tris-base, 1.49 g of EDTA and 0.7 ml of β-ME were

dissolved in dH2O to a total volume of 2 L. The overall concentrations were 20 mM Tris-base, 2

mM EDTA, and 5 mM β-ME. The solution was adjusted to pH 8.0, filtered and degassed with

the aid of a vacuum pump.

Protein Purification Buffer (B): 4.8 g Tris-base, 1.49 g of EDTA, 120 g of NaCl and 0.7 mL of

β-ME were dissolved in dH2O with a total volume of 2 L. The overall concentrations were 20

mM Tris-base, 2 mM EDTA, 1 M NaCl and 5 mM β-ME. The solution was adjusted to pH 8.0,

filtered and degassed with the aid of a vacuum pump.

Protein Concentration Cacodylate Buffer: 0.31 g sodium cacodylate, 28 mg calcium chloride

(CaCl2) and 87.5 μl of β-ME were dissolved in dH2O to a total volume of 250 mL. The overall

concentrations were 5 mM sodium cacodylate, 1 mM CaCl2, and 5 mM β-ME.

Fluorometry Buffer: 0.788 g of Tris-HCl and 19 mg of EDTA were dissolved of dH2O with a

total volume of 0.5 L, the final concentrations were 20mM Tris-HCl and 0.1 mM EDTA. The

solution was adjusted to pH 7.4, and then filtered and degassed with the aid of a vacuum pump.

Cloning Strain: XL1-Blue ® super competent cells (Stratagene, USA) were used for the

preparation of plasmid constructs. These cells are highly proficient in replicating DNA and

produce high transformation efficiency ≥1 × 108 colony forming units per μg.

BL21-CodonPlus(DE3)-RP ® super competent cells (Stratagene, USA) were used for the

expression of the μ-calpain PEF(L) gene and the PEF(S) genes. They contain extra genes that

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encode for arginine and proline tRNAs, allowing for the efficient expression of proteins that

contain a large number of these amino acids.

Protocols

Transformation Protocol PEF(S), PEF(L), and CysPc

XL1-Blue ® super competent cells or BL21-CodonPlus(DE3)-RP ® were placed on ice to thaw.

1 µL of pET-28a-c for μ-calpain or pET21d for PEF(L) and PEF(S) was aliquoted into the cell

solution and mixed properly then incubated on ice for 20 minutes. The mixture was later

subjected to heat shock for 45 seconds at 40 °C and then placed on ice for a further 2 minutes. 1

mL of LB media was added to the mixture and then incubated on a shaker for 1 hour at 37° C.

After incubation, the mixture is centrifuged for 1 minute at 16100 RCF, supernatant discarded

and pellet re-suspended. The re-suspended pellet was transferred onto a selective agar plate

related to antibiotic resistance of the transformed cells. Plates were incubated overnight in the

oven at 37 °C.

PEF(S) and PEF(L) expression

200 µL of 50 mg/mL of ampicillin was transferred into 100 mL of LB media previously

prepared. One colony from the agar plate, prepared in the transformation protocol previously

mentioned was selected to inoculate the broth and the culture was incubated overnight at 37 °C

in a shaker at 150 rpm. 60 mL of the overnight culture was transferred into 3 L of ampicillin (1

μg/ml) selective LB media, which was incubated at 37 °C in a shaker set to 150 rpm. When an

optical density of 0.6 AU at 600 nm is reached isopropyl-β-D-1-thiogalactopyranoside (IPTG)

with total concentration of 0.5 mM was added to induce expression. The cultures were incubated

for at 25 °C and 37 °C for PEF(L) and PEF(S) respectively, in the shaker at 150 rpm for 5 hours.

The cells were harvested via centrifugation at 6080 RCF for 15 minutes. The supernatant was

discarded and the pellet retrieved and stored at -20 °C. During the 5 hour expression 1 mL

samples were taken from the cultures every hour, with the intial sample obtained prior to

induction. The samples were analysed by SDS-PAGE.

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Protein Purification (Ni-column and Anion Exchange Chromatography)

Purification of the PEF(L) and PEF(S) complex: the two frozen cell pellets were thawed on

ice, the pellet containing PEF(S) protein was re-suspended in 25 ml of non-imidazole buffer. The

re-suspended pellet was added to the thawed cell pellet containing PEF(L) and mixed

thoroughly. The homogenized cells were lysed by sonication on ice, the sonication program ran

for a total of 9 minutes, with repeating cycles 5 seconds sonication which was followed by 10

seconds of no sonication. The lysate was clarified by centrifugation for 30 minutes at 30310

RCF. The supernatant was isolated and filtered using a 0.2 μm syringe tip filter.

The filtered protein was transferred into a Ni-NTA column which had been equilibrated with the

non-imidazole buffer, the flow-through was collected and the column washed with the non-

imidazole buffer once more. Increasing concentrations of imidazole solutions, 10 mM, 50 mM,

200 mM and 500 mM, were applied to the column, with the eluted buffer collected each time.

The collected fractions were analysed by SDS-PAGE and the fractions that contained the desired

protein were pooled and dialysed into the dialysis buffer overnight at 4° C.

Purification of the PEF(S) complex: the frozen cell pellet was thawed on ice, the pellet

containing PEF(S) was re-suspended in 25 mL of protein purification buffer A. The cells were

lysed by sonication on ice, the sonication program ran for a total of 9 minutes, with repeating

cycles 5 seconds sonication which was followed by 10 seconds of no sonication. The lysate was

clarified by centrifugation for 30 minutes at 30310 RCF. The supernatant was isolated and

filtered using a 0.2 μm syringe tip filter. The filtered supernatant was applied to a Q-sepharose

anion exchange column attached to an FPLC machine, the column had been pre-equilibrated

with protein purification buffer A. The flow-through was collected prior to the purification

program was run. An increasing salt gradient was applied to the column, 0 M NaCl to 1 M NaCl,

over five column volumes (CV), the elution was monitored by UV analysis (280 nm). The eluent

was collected in 10 ml fractions. The fractions that corresponded to peaks on the UV trace, and

hence observed eluted proteins, were analysed with SDS-PAGE. The fractions that contained the

required protein were concentrated by Amicon, from 45 mL to ~ 15 mL for subsequent

purification by size exclusion chromatography.

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Size Exclusion Chromatography

The concentrated PEF(S) was loaded onto Superdex 75 (320 mL CV) which was pre-equilibrated

with size exclusion chromatography buffer. 1.5 CV of the size exclusion chromatography buffer

was passed through the column with 10 ml fractions collected and eluent was monitored at 280

nm. The fractions that corresponded to an increase in absorbance at 280 nm were analysed by

SDS-PAGE. Fractions that contained the desired protein were pooled together and concentrated.

Analytical Size Exclusion Chromatography

The PEF(L):PEF(S) complex was evaluated with analytical size exclusion chromatography. A

500 μL sample at a concentration of 1.5 mg/mL was applied to an analytical Superdex 75 (CV =

26 mL), pre-equilibrated with size exclusion chromatography buffer (vide infra). 1.5 CV of the

buffer was run and absorbance was monitored at 280 nm. Protein standards (1 mg/mL) with

different molecular weights were analysed with the column at these were BSA (MW 66,400),

PEF(S) (MW 20,125), and trypsin (MW 23,300)

Protein Concentration

After the PEF(L):PEF(S) protein was dialysed into the dialysis buffer it was concentrated using

spin column (MWCO 10,000). In the process equal amounts of buffer concentration A and

protein were added and centrifuged for 30 minutes at 4000 rpm. PEF(S) was concentrated in a

similar manner except the buffer of choice was the cacodylate buffer

UV-vis Protein Concentration Determination or UV Absorption

Protein concentration was determined using the UV-Vis spectroscopy absorption technique. For

each of the selected wavelengths (205, 210, 215, and 220 nm) 995 µL of sodium sulfate buffer

was first added to a 1 cm cuvette and the reading was zeroed prior to the addition of 5 µL of

sample and measuring again. Dilution factor was thus 200.

Protein concentration was calculated according to this formula:

A= ϵ*C*l

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* Where A is absorbance, ϵ is the molar absorption coefficient , C is the protein concentration,

and l is the cell pathlength (l = 1).

C= A/ ϵ* dilution factor

Wavelength(nm) Molar Absorption Coefficient (ϵ)

Absorbance (A.U)

Concentration (A/ ϵ)*dilution factor

205 31 0.5506 3.56

210 20 0.394 3.94

215 15 0.2837 3.78

220 11 0.1957 3.56

Average Concentration for PEF(L):PEF(S) heterdoimer. 3.71 (mg/mL)

Wavelength(nm) Molar Absorption Coefficient (ϵ)

Absorbance (A.U)

Concentration (A/ ϵ)*dilution factor

205 31 2.280 14.7

210 20 1.714 17.4

215 15 1.338 17.84

220 11 0.964 17.5

Average Concentration for PEF(S) 16.795 (mg/mL)

To get the concentration in M Concentration (mg/mL)/M.W of protein

3.71/43000 = 86.3 μM for the heterodimer

16.8/20000= 840 μM for the PEF(S)

CD spectroscopy:

For the purpose of running the protein on CD, its concentration had to be diluted down to 20 µM.

In the process, 225 µl and 292 µl of CD buffer was added to 75 µL PEF(L):PEF(S) complex and

PEF(S) respectively. The CD cuvette has a total volume of 350 µL and path length of 0.1 cm.

The mean residue ellipticity (MRE) was calculated according to the following formula:

ΘMRE= θ/10*n*c*l

* where θ is the CD signal, n is the number of backbone peptides 369 in the case of

PEF(L):PEF(S) complex and 173 in the case of PEF(S), c is the protein concentration, and l is

the path length.

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PEF(S) Crystallization

Crystallisation precipitant: 50 mM sodium cacodylate, 12.5% PEG3350 and 20 mM CaCl2 at

pH 7.0.

To the perform crystallization experiment; the protein was diluted to 10 mg/mL using the

cacodylate buffer. The hanging drop technique was employed where 1 mL of crystallization

precipitant was transferred into the tray wells, the hanging drops are composed of 3 µL of the

protein solution, 1 µl of dH2O and 3 µl of precipitant. The wells were sealed and left to grow at

20 °C.

Fluorescence spectroscopy

Before the start of the procedure the luminescence spectrometer was validated by dH2O. Samples

were 3 ml of volume. PEF(S) and PEF(L):PEF(S) complex were diluted down to 2.5 µM by the

addition of 17.8 µl into 2944.7 µl and 87.2 µl into 2807 µl of fluorometry buffer respectively.

Stock solutions of 50 mM ANS (1-Anilinonaphthalene-8-Sulfonic Acid) and 5 mM inhibitor

were used to prepare 1 mM ANS and 0.5 mM inhibitor. 7.5 µl of ANS were first added to the

cuvette making its final concentration 2.5 µM. Fluorescence signal was recorded, and then 30 µl

of 100 mM CaCl2 were pipetted into the mixture to have a final concentration of 1 mM.

Excitation of ANS took place at 370 nm, and emission at 470 nm. Spectra were measured over a

range of 380 to 610 nm. Increasing gradient of the inhibitor (Z)-3-(4-bromophenyl)-2-

mercaptoacryilic acid were added until a concentration of 10 µM was reached and the spectra

were monitored continuously. For proper interpretation of the graphs within these

concentrations, the PEF(S) concentration was divided by two.

22

3. Results and

Discussion

23

PEF(L) and PEF(S) expression

For the purpose of protein expression, BL21-CodonPlus(DE3)-RP E.coli expression cells were

transformed with pET21d vector containing either PEF(S) or PEF(L). 60 ml of the overnight

culture was transferred into 3 L of ampicillin selective media (1 µg/ml) and incubated on a

shaker (150 rpm) at 37 °C until an optical density of 0.6 AU at 600 nm was reached. Induction

with 0.5 mM of isopropyl-β-D-1-thiogalactopyranoside (IPTG) followed, and the expression was

carried out for four hours. Each hour, 1 ml sample from the culture was collected in order to be

analyzed on sodium dodycyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

Expression of PEF(L) and PEF(S) took place at 25 °C and 37 °C respectively. SDS-PAGE was

performed to determine if protein expression was successful (Figure 5).

Figure (5): SDS polyacrylamide gel visualization indicating the presence of PEF(S) (left) with a

MW of 20,000 and PEF(L) (right) with a MW 23,000.

The remaining cells were harvested by centrifugation at 4 °C (6080 RCF) for 15 minutes. The

supernatant was discarded and the pellet was retrieved and stored at -20 °C for further

purification.

24

Purification of PEF(S)

Anion exchange chromatography

Frozen cell pellet containing PEF(S) was thawed on ice, resuspended in protein purification

buffer (A) and then lysed by sonication. The lysate was centrifuged (30 minutes, 30310 RCF)

and crude extract containing the desired protein was isolated and filtered using 0.2 µm syringe

tip filter in preparation for anion exchange chromatography.

Ion exchange chromatography uses a charged resin that will bind strongly to an oppositely

charged test substrate and then the latter will be eluted upon increase in salt concentration

flowing through the resin 25

. The filtered extract was loaded onto a Q-sepharose anion exchange

chromatography attached to an FPLC machine. The flow-through was collected before the start

of the purification program and then the column was washed with increasing gradient of salt

from 0 M NaCl to 1 M NaCl over five column volume (CV) to elute the bound components.

Absorption was monitored at 280 nm and fractions of 10 ml were collected. Seven fraction

exhibited a peak on the UV trace were isolated for later analysis on SDS-PAGE to determine

which eluent contained the desired protein (Figure 6). Four of these fractions (20, 21, 22, 23)

showed the correct band position corresponding to the molecular weight (MW) of PEF(S).

Figure (6): SDS-PAGE after anion exchange chromatography showing the four fractions (20,

21, 22, 23) with correct band position corresponding to MW of ~ 20,000, where M = marker, CE

= crude extract, FT = flow-through, and the numbers correspond to the fractions collected that

showed the presence of a band

M FT CE 23 22 21 20

25

Size exclusion Chromatography

A total volume of ~ 40 ml was obtained as pure protein from anion exchange chromatography.

The protein was further concentrated to ~ 15 ml using amicon ultrafiltration membrane that has a

molecular weight cut off (MWCO) of 10,000. The concentrated protein underwent purification

using size exclusion chromatography or gel permeation chromatography so that larger protein

molecules elute faster. The column used was Superdex 75 (320 ml) and it was connected to an

FPLC machine. Size exclusion chromatography buffer was used to wash the column (1.5 CV),

and fractions showing increased absorbance at 280 nm were collected and analysed on SDS-

PAGE (Figure 7). After confirmation of the desired protein, the fractions were pooled together

and concentrated using

Figure (7): SDS-PAGE of fractions obtained after size exclusion chromatography showing the

desired protein of the proper band size (MW 20,000) with CE = crude extract, FT = flow-

through, BSA = bovine serum albumin and PEF(L,S) complex were used instead of protein

marker.

Purification of PEF(L):PEF(S) complex

Ni-NTA column purification

An ice thawed PEF(S) cell pellet was resuspended in 25 ml non-imidazole buffer and mixed

thoroughly with PEF(L) cell pellet. Lysing, centrifuging, and filtering procedures were the same

as those performed for PEF(S).

The filtered product was transferred to Ni-NTA column for purification. This technique utilizes

the strong affinity of nickel towards His-tagged proteins and thus the protein of choice will not

CE PEF(S) FT PEF(L,S) BSA

26

be washed away with the flow-through. Nickel is bound to resin via nitroloacetic acid (NTA)

chelation. 1 CV of non-imidazole buffer was washed through then increasing concentration of

the imidazole buffer (10 mM, 50 mM, 200 mM, and 500 mM) were applied to the column and

flow-through were collected at each time. Increasing the gradient of imidazole buffer will serve

to elute down the bound His-tagged protein. The collected fractions were analyzed by SDS-

PAGE (Figure 8), and those fractions with the appropriate band sizes were pooled together and

dialysed into the dialysis buffer overnight at 4 °C. This will help remove impurities and any

remaining imidazole in protein by diffusion. Products were run on SDS-polyacrylamide gel for

further confirmation of the product size (Figure 9).

Figure (8): SDS-PAGE analysis of the fraction products from Ni-NTA column with the small box

showing the desired protein of the proper band size for both PEF(L) protein MW 23,000 and

PEF(S) protein MW 20,000 in the first elution with 200 mM imidazole buffer, CE = crude

extract before Ni-NTA column, M = marker, W = wash with non-imidazole buffer, 10 & 50 &

200 = increasing gradient of imidazole buffer with flow-through from 200 mM collected at three

separate fractions ( 15 ml each).

200 200 200 50 10 W M CE

(3) (2) (1)

27

Figure (9): SDS-PAGE analysis of the pure product obtained after dialysing PEF(L):PEF(S)

complex in dialysis buffer overnight. The two bands correspond to PEF(L) (MW 23,000) and

PEF(S) (MW 20,000)

Protein concentration

Protein concentration of both PEF(L):PEF(S) complex and PEF(S) were determined after further

concentration using a spin column with MWCO of 10,000. Protein concentration buffer (A) and

cacodylate buffer were used in this process to concentrate PEF(L):PEF(S) complex and PEF(S)

respectively. The final concentration of both proteins was determined using UV-Vis

spectroscopy at different wavelengths (205, 210, 215, 220). PEF(L):PEF(S) complex and PEF(S)

had a concentration of 86.3 µM (3.71 mg/ml) and 840 µM (16.795 mg/ml) respectively.

M

PEF(L)

PEF(S)

28

Characteristics of PEF(L):PEF(S) complex and PEF(S)

Circular Dichroism (CD) spectroscopy

Circular dichroism spectroscopy is a valuable tool for the determination of the structure of

proteins whether they form α-helix, β-sheet, or any other unorganized structure 26

. Spectra

obtained from CD measurements can be compared to those obtained from other methods such as

X-ray crystallography or NMR and sometimes it is an important step before proceeding to these

methods, especially if the protein understudy is a multi-domain enzyme. Moreover,

conformational changes in proteins as a result of ligand binding and protein foldings can also be

detected by CD spectroscopy 27

.

CD spectroscopy was performed for both proteins to check if they form α-helical structure 1.

Samples were prepared by diluting PEF(L):PEF(S) and PEF(S) to 20 µM using the CD buffer

and CD was run between 190 and 350 nm. The resulting spectra for both proteins recorded two

minima at 208 nm and 222 nm which is consistent with α-helical secondary structure 27

(Figures

10,11).

Figure (10): The CD spectrum of the homodimer PEF(S) with peak minima at 222 nm and 208

nm is consistent with α-helical secondary structure.

-2000

-1000

0

1000

2000

3000

190 210 230 250 270 290 310 330 350

θ M

RE/

de

g cm

2 d

mo

l-1

wavelength/ nm

29

Figure(11):The CD spectrum of the heterodimer PEF(L):PEF(S) complex with peak minima at

222 nm and 208 nm is consistent with α-helical secondary structure.

The uneven peak at 190 nm of the PEF(L):PEF(S) complex was due to the fact that this PEF(L)

portion of the protein is only soluble in buffers that contain salt. Purification and concentration of

PEF(S) homodimer on the other hand did not require a salt containing buffer, thus the CD

spectra recorded an even peak at 190 nm.

Analytical size exclusion chromatography

Analytical size exclusion chromatography is used on a more quantitave scale in determining

purity and size of a molecule, and thus the presence of dimers, oligomers, or monomers 28

. Both

proteins were assessed using this method (Figure 12, a-d), but the main goal of this procedure

was to determine the heterogenity of PEF(L):PEF(S) complex. Thus PEF(S) was used as a

standard protein (MW 20,125) along with BSA (66,400) and trypsin (23,300) at a concentration

of 1 mg/ml while the protein under investigation was at 1.5 mg/ml concentration. Superdex 75

(CV = 26) connected to an FPLC machine and pre-equilibrated by size exclusion

chromatography buffer was injected with 500 µl of sample. Results pertaining to the absorbance

of proteins analyzed where plotted on graphs against volume and they showed that

PEF(L):PEF(S) complex eluted at 11.53 ml which is after the elution of BSA (10.19 ml) and

around the same volume of PEF(S) (11.59 ml). Interestingly, since PEF(S) is known to form a

-8000

-6000

-4000

-2000

0

2000

4000

6000

8000

190 210 230 250 270 290 310 330 350

θ M

RE

/ d

eg c

m2

dm

ol-1

Wavelength/ nm

30

dimer 1 making its total mass around 40,000, and thus consistent with the approximate mass of

the complex which is around 43,000.

0.00

50.00

100.00

150.00

200.00

250.00

300.00

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

Ab

sorb

ance

(2

80

nm

)/ m

AU

Volume / ml

Trypsin (MW 23,000)

Trypsin (MW 23,000)

A

0.00

50.00

100.00

150.00

200.00

0.00 5.00 10.00 15.00 20.00 25.00

Ab

sorb

ance

(2

80

nm

)/m

AU

Volume/ml

PEF(S) (MW 20,125)

PEF(S) (MW 20,125)

B

0.00

50.00

100.00

150.00

200.00

250.00

0.00 5.00 10.00 15.00 20.00 25.00 30.00

Ab

sorb

ance

(2

80

) n

m/m

AU

Volume / ml

BSA (MW 66,400)

BSA (MW 66,400)

C

31

Figure (12): analytical size exclusion chromatogram for a) trypsin (MW 23,300), b) PEF(S)

(MW 20,000), c) BSA (MW 66,400), and d) PEF(L):PEF(S)(MW 43,000)

Conclusion

Challenges were implicated in maintaining the solubility of the PEF(L) domain which was

overcome by adding salt to the buffer and thus prevent it from precipitation. PEF(S) on the other

hand is very stable in solution, and thus it was isolated and purified with higher concentrations

than the complex.

The previously mentioned methods are very useful tools in determining structural characteristics

of proteins. Analytical size exclusion chromatography allows the correlation between the

molecular weights of standard proteins with the investigated molecule. Thus by monitoring

absorbance vs. elution volume of the investigated molecules, it is possible to easily determine the

mass of the protein and whether it forms a monomer or a multimer. Circular dichroism data on

the other hand was consistent with the previously mentioned data regarding the α-helical

structure of both proteins and it was in agreement with X-ray crystallography of PEF(S) 1.

0.00

50.00

100.00

150.00

200.00

250.00

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

Ab

sorb

ance

(2

80

nm

)/ m

AU

Volume/ ml

PEF(L):PEF(S) MW (43,000)

PEF(L):PEF(S) MW (43,000)

D

32

Interaction of (Z)-3-(4-bromophenyl)-2-mercaptoacryilic acid with PEF(S)

and PEF(L):PEF(S) complex

Previous studies have determined that α-mercaptoacrylic acid derivatives are able to interact with

PEF(S) domain through florescence studies and X-ray crystallography of the inhibitor and the

domain complex 1, 3, 23

. However, not much is known about the binding and interaction of these

compounds with PEF(L). It is thought that the inhibitors would react in the same manner with

because it is a calcium binding domain as well and it contains the hydrophobic groove to where

the α-mercaptoacrylic acid derivatives bind in PEF(S). However, the difference in inhibition

constants between the two major enzymes can also be attributed to this domain since they have

different catalytic subunit 1.

For this purpose, a fluorescent displacement assay adapted from previous study 23

was used to

determine whether (Z)-3-(4-bromophenyl)-2-mercaptoacryilic acid would interact with PEF(L)

in the same manner as it does with PEF(S). In this assay, 1-anilinonaphthalene- 8-sulfonic acid

(ANS) (Figure 13), is a fluorescent molecule with a modest binding affinity against the studied

domains was used. It has an absorption maximum at 370 nm and emission minimum at 470 nm.

2.5 μM of protein, 2.5 μM ANS, 0.1 mM EDTA and 20 mM Tris-HCl at pH 7.4, in a total

volume of 3 ml were initially incubated together and the fluorescence spectra were recorded. 1

mM of CaCl2 was later added to the sample and then followed by increasing concentrations of

the inhibitor while continuously monitoring the spectra between 380 nm and 610 nm (Figure 14).

33

0

50

100

150

200

250

300

350

400

380 430 480 530 580

Flu

ore

sce

nce

/FU

Wavelength/nm

PEF(S)-ANS

PEF(S)-ANS-Ca2+

0.1 μM inhibitor

0.5 μM inhibitor

1.0 μM inhibitor

1.5 μM inhibitor

2.0 μM inhibitor

3.0 μM inhibitor

4.0 μM inhibitor

5.0 μM inhibitor

6.0 μM inhibitor

7.0 μM inhibitor

9.0 μM inhibitor

10 μM inhibitor

A

0

100

200

300

400

500

600

380 430 480 530 580

Flu

ore

sce

nce

/FU

Wavelength/nm

PEF(L):PEF(S) complex- ANS

PEF(L):PEF(S) complex- ANS-Ca2+

0.1 μM inhibitor

0.5 μM inhibitor

1.0 μM inhibitor

1.5 μM inhibitor

2.0 μM inhibitor

3.0 μM inhibitor

4.0 μM inhibitor

5.0 μM inhibitor

6.0 μM inhibitor

7.0 μM inhibitor

9.0 μM inhibitor

10 μM inhibitor

B

34

Figure (14):Fluorescence spectra of the fluorescent probe ANS bound toPEF(S) (A) and

PEF(L):PEF(S)complex (B and C). Initial spectra were obtained of 3 ml solutions containing 2.5

μM of protein, 2.5 μM ANS, 0.1 mM EDTA and 20 mM Tris-HCl at pH 7.4, followed by a second

set of spectra with the addition of 1.0 mM CaCl2. Increasing concentrations of the inhibitor ((Z)-

3-(4-bromophenyl)-2-mercaptoacrylic acid) were added (0.1, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0,

7.0, 9.0, and 10.0). Excitation = 370 nm and emission =470 nm.

Upon the addition of CaCl2, a substantial spike was observed in the spectra, indicating the rapid

activation of both domains. Interestingly, a difference in the strength of the signal between

PEF(S) and PEF(L):PEF(S) complex was recorded. That could be attributed to the binding

affinity of the fluorescent probe towards the two proteins. Increasing concentration of the

inhibitor were added (0.1, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 9.0, and 10.0) and the

fluorescence signal was observed to gradually fall down because the displaced probe would be

quenched by the solution. The decrease in signal was not happening at the same pace. Smaller

concentrations of the inhibitor were found to be more effective on the PEF(L):PEF(S) complex

rather than the PEF(S) homodimer, where it was enough to add 7 µM of the inhibitor to the

heterodimeric complex to decrease the fluorescence beyond the point that was recorded without

the addition of Ca2+

. However, not even 10 µM were able to bring down the fluorescent signal in

PEF(S) beyond zero Ca2+

point.

PEF(S) crystallization

0

50

100

150

200

250

300

350

400

450

500

550

600

380 430 480 530 580

Flu

ore

sce

nce

/FU

Wavelenght/nm

PEF(L):PEF(S)-ANS

PEF(L):PEF(S)-ANS-Ca2+ 0.1 μM inhibitor

0.5 μM inhibitor

1.0 μM inhibitor

1.5 μM inhibitor

2.5 μM inhibitor

3.0 μM inhibitor

C

35

Hanging-drop method was used in crystallizing PEF(S) where the crystallization precipitant was

pipetted in excess to the volume of the protein drop ( 1000 µl to 7 µl) into the tray well. The

protein drop contained 3 µl of 10 mg/ml PEF(S), 3 µl of crystallization precipitant, and 1 µl of

dH2O and was placed on the surface above the precipitant. Although crystals were left to grow at

20 °C, there was not enough time to check if any were formed.

Conclusion

The interactions between PEF(L):PEF(S) complex and (Z)-3-(4-bromophenyl)-2-

mercaptoacryilic acid were studied for the first time during this work. Although the inhibitor was

successful in displacing ANS, which suggests that it interacts with calcium binding sites in both

domains, results suggest that in contrary to what was believed, there is some differences in the

interaction of the inhibitor with PEF(L) and PEF(S). Several reasons might be causing this

discrepancy. First of all, it can be attributed to the use of a fluorescent probe different than the

conventional TNS. Moreover, the salt concentration of PEF(L):PEF(S) complex might also play

a role in the binding and interaction of the protein with the inhibitor. One way to eliminate this

possibility is to concentrate PEF(S) with salt and then re-run the assay.

Protein crystallography can give a handful of information about the specific interactions taking

place as it was proved from previous studies 1. Success in crystallizing PEF(L) with α-

mercaptoacryilic acid derivatives can be a cornerstone in answering all the mysterious questions.

36

4.Conclusion and

Future Work

37

Conclusion

α-mercaptoacrylate drivatives represent promising candidates for the development of therapeutic

drugs for treatment of neurodegenerative and inflammatory diseases where the overactivation of

calpain is pronounced. PD150606 which is considered to be the precursor for the synthesis of

these compounds was first thought to involve the unmodifeid sulfhydryl and carboxylic acid in

inhibiting calpains and a speculative role in Ca2+

ion chelation 23

. However, a very recent study

suggests that these functional groups might not be important for the overall inhibition process,

which would lead to more freedom in the synthesis mechanism 1. Moreover, binding of these

inhibitors to a flexible hydrophobic pocket in PEF(S) can also pave the way for improved

inhibitors with larger rings and halides.

The newly synthesized monohalogenated α-mercaptoacrylic acids represent the most potent

inhibitors so far, yet they have been tested for their inhibition only on PEF(S) domain of

calpains. Since this domain is identical in the two major isoforms, the discrepancy between the

inhibition constants must be due to interactions with another domain. Because these chemical

compounds interfere with the calcium binding sites, the goal of this work was to test one of these

inhibitors, Z)-3-(4-bromophenyl)-2-mercaptoacryilic acid, against the other two known calcium

binding sites of the enzyme; PEF(L) and CysPc domains.

Several attempts were undertaken to try and clone the CysPc domain, but unfortunately the

ligation step always failed. Due to the lack of time, the procedure was overlooked towards the

final days of this work. Focus was directed to the expression, isolation and purification of

PEF(L), which was successful. However, the concentration of the heterodimer PEF(L):PEF(S)

was not high enough to proceed to protein crystallization; but the inhibitor interactions were

nevertheless studied using fluorescence spectroscopy. Although this method is not as accurate as

other techniques, but it was evident through the results obtained that there was difference in

interactions between PEF(L) and PEF(S) domains .

38

Future Work

As mentioned earlier, α-mercaptoacrylate drivatives have the ability to bind to calcium binding

sites, which μ- and m- calpains have two more identified domains so far other than the PEF(S).

These two domains remain poorly studied but can hold many important answers especially to the

difference in inhibition constants of the two major isoforms. Future work in this area must

include a full understanding of how these inhibitors interact with all of the domains of the

enzyme. It was already proved that there might be a chance for difference in interactions between

the inhibitor and PEF(L) and thus more data has to be collected and extensively analyzed for this

domain and for the CysPc domain. Understanding how these compounds function against calpain

will elucidate more information about the role of calpain in health and disease state, and

eventually it will lead the way to more improved and selective coumpounds with better

understanding of their mechanism of action.

39

5. References

40

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20. Parslow, T. G.; Stites, D. P.; Terr, A. I.; Imboden, J. B., Medical Immunology. 10th edition ed.; McGraw-Hill: 2001. 21. Firestein, G. S., Evolving concepts of rheumatoid arthritis. Nature 2003, 423 (6937), 356-61. 22. Smolen, J. S.; G; [uuml]; Steiner, n., Therapeutic strategies for rheumatoid arthritis. Nature Reviews Drug Discovery 2003, 2 (6), 473-488. 23. Wang, K. K.; Nath, R.; Posner, A.; Raser, K. J.; Buroker-Kilgore, M.; Hajimohammadreza, I.; Probert, A. W., Jr.; Marcoux, F. W.; Ye, Q.; Takano, E.; Hatanaka, M.; Maki, M.; Caner, H.; Collins, J. L.; Fergus, A.; Lee, K. S.; Lunney, E. A.; Hays, S. J.; Yuen, P., An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective. Proc Natl Acad Sci U S A 1996, 93 (13), 6687-92. 24. Donkor, I. O., A survey of Calpain Inhibitors. 2000. 25. Dean, J.; Jones, A.; Holmes, D.; Reed, R.; Weyers, J.; Jones, A., Practical Skills in Chemistry. 2002. 26. Circular Dichroism: Studies of Proteins. 2014. 27. Kelly, S. M.; Jess, T. J.; Price, N. C., How to study proteins by circular dichroism. Biochim Biophys Acta 2005, 1751 (2), 119-39. 28. Size‐exclusion Chromatography. http://www.els.net/WileyCDA/ElsArticle/refId-a0002676.html.