2011-12 Entry Cohort

DEPARTMENT OF CHEMISTRY

Advanced Research Project

FAP174 - Properties of a Chlamydomonas reinhardtii signalling protein

prepared by:

William Stockham Project Supervisor: Dr Avinash Kale, Dr Jacinta D’Souza, Dr Basir Ahmad

Date: 11/05/2015

Number of Words: Literature Review - 2955

Project report - 5299

FAP174 - Properties of a Chlamydomonas reinhardtii

signalling protein. Master’s Thesis by William Stockham

Supervised by : Dr Avinash Kale, Dr Jacinta D’Souza and Dr Basir Ahmad

The University of York and UMDAE CBS Mumbai

Submitted May 2015

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“Let us be a little humble; let us think that perhaps

the truth may not be entirely with us.”Jawaharlal Nehru

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Abstract.

The unfolding of flagellar associated protein 174 (FAP174) from Chlamydomonas reinhardtii was characterised by intrinsic and extrinsic fluorescence. Gibbs Free Energy of unfolding was found to be very low, which may explain the tendency of FAP174 to aggregate. Using a modified Gibbs-Helmholtz equation, the unfolding of FAP174 was found to be associated with a change in molar heat capacity. The model predicts cold unfolding below the freezing point of water. The melting point of FAP174 was measured at 96 different pH/NaCl solvent conditions. There is a clear relationship between NaCl concentration and melting temperature, which is explained in terms of ionic interactions with NaCl and the protein surface. Three FAP174 mutants have been shown to have unusual unfolding behaviour. Previous CD experiments showed that these mutants have well ordered secondary structure, however the work presented here shows that the tertiary structure of these proteins is disordered. It is suggested that the mutated residues VLV21, VLV25 and C46 play a role in maintaining the tertiary structure of FAP174. Intrinsic fluorescence suggests that tyrosine residues are exposed to a hydrophilic environment in the natively folded protein. A series of new experiments are suggested to further the understanding of the protein.

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Contents

Literature Review Introduction…………………………………………………………………………………………….…9Flagella as a ciliary model………………………………………………………………………………9Flagella Structure……………………………………………………………………………………….10The role of the central pair in flagella motion………………………………………………….……..11Identification of Flagella proteins………………………………………………………………………11Early Study of FAP174………………………………………………………………………………….13Biophysical properties of FAP174……………………………………………………………………..14Protein aggregation studies…………………………………………………………………………….15Studying Amyloid formation…………….………………………….…………….……….…….………16FAP174 as a model……….……………….……………….……………….……………….………….16Future goals……….……………….……………….……………….……………….…………………..17Conclusion……….……………….……………….……………….……………….……………….……17

Project reportIntroduction……….……………….……………….……………….……………….……………….…18

Materials and Methods E.coli transformation……….……………….……………….……………….……………….………20 Induction……….……………….……………….……………….……………….……………….…..20 Purification……….……………….……………….……………….……………….…………………20 Concentration estimation……….……………….……………….……………….……………….…21 Thermofluor Assay……….……………….……………….……………….……………….……..….21 Intrinsic Fluorescence……….……………….……………….……………….……………….…….22 Extrinsic Fluorescence - Chemical Denaturing……….……………….……………….………….22 Extrinsic Fluorescence - Thermal Denaturing……….……………….……………….……………23

Results and Discussion Transformation, Induction and Purification……….……………….……………….……………….24 Thermofluor Assay……….……………….……………….……………….……………….…….…..25 WT FAP174……….……………….……………….……………….……………….…………………26 FAP144 mutants……….……………….……………….……………….……………….……….…..29 Extrinsic Fluorescence - Chemical Denaturing……….……………….……………….…….…….31 Extrinsic Fluorescence - Thermal Denaturing……….……………….……………….……….……35 Extrinsic Fluorescence shift…………………………………………………………………………..41 Intrinsic Protein Fluorescence……….……………….……………….……………….……………..42 Recommended Further Work……….……………….……………….……………….……………….45Conclusion……….……………….……………….……………….……………….……………….…..47

Acknowledgments……….……………….……………….……………….……………….…………..49References……….……………….……………….……………….……………….……………….….50

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List of abbreviations.

AMY-1 - Associate of Myc 1 ANS - 8-Anilino-naphthalene-1-sulfonic acid [8-(Phenylamino)-1-naphthalenesulfonic acid] CD - Circular Dichroism (Spectroscopy) CHC - Citric acid, HEPES, CHES (buffer system) CHES - N-Cyclohexyl-2-aminoethanesulfonic acid DLS - Dynamic Light Scattering DOSY NMR - Diffusion Ordered Nuclear Magnetic Resonance (Spectroscopy) DMSO - Dimethyl sulfoxide DSC - Differential Scanning Calorimetry F - FluorescenceFAP174 - Flagella Associated Protein 174 FPLC - Fast Protein Liquid Chromatography GuHCl - Guanidinium Hydrochloride HEPES - 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid IC - Internal conversion IGEPAL CA-630 - Octylphenoxy poly(ethyleneoxy)ethanol IPTG - Isopropyl β-D-1-thiogalactopyranoside ISC - Intersystem Crossing LB- Lysogeny Broth MALDI-TOF - Matrix Assisted Laser Deapsorption Ionisation - Time of Flight (Mass spectrometry)MWCO- Molecular weight cut off MYCBP - Myc binding protein NiNTA - Nickel Nitrilotriacetic acid (functionalised agarose matrix) NPT - Neomycin phosphotransferase NR - Non-radiative decayP - Phosphorescence PI - Isoelectric Point PMSF- Phenylmethylsulfonyl fluoride POI - Protein of Interest qPCR - Quantitative Real Time Polymerase Chain Reaction (Instrument) RCF - Relative Centrifugal ForceSDS-PAGE - Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis SR - Solvent Relaxation TICT - Twisted Intramolecular Charge Transfer TNG - Tris, NaCl, Glycerol (buffer system) Tris - Tris(hydroxymethyl)aminomethane [2-Amino-2-(hydroxymethyl)-1,3-propanediol] VR - Vibrational relaxation WT - Wild Type

Notation. When discussing the substitution of one Amino acid for another in mutated proteins, the following notation convention is used: XnY Where X is the original amino acid, n is the position in the chain and Y is the new amino acid. For example VLV21AAA is Valine-Leucine-Valine, at the 21st position, replaced by Alanine-Alanine-Alanine.

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Literature Review

Introduction

At the heart of the science of life is the study of proteins. Francis Crick’s central dogma1 shows how proteins are the cast in the grand drama of life, written in DNA, relayed with RNA to protein actors. Evolution has crafted proteins with niche properties that are perfectly adapted to their functions. Scientists are only just beginning to understand the subtle behaviour of proteins, and protein chemistry is an enormous and fast growing science. The advent of molecular simulations has given scientists unprecedented access to the world of these macromolecules however there are still many problems unsolved.

The aggregation problem is perhaps the most famous because of its far reaching implications in medicine2. The uncontrolled aggregation of proteins is a characteristic marker of a multitude of diseases and disorders3, 4, the most well known being Alzheimers disease, where the Tau protein forms regular ‘Amyloid fibrils’ in the brain. However there is no general model that can account for protein aggregation. Protein science suffers a chronic problem — no two proteins are exactly alike, and so predicting protein behaviour is an inherently inductive process. It is difficult to make generalisations about systems that are not general. Nevertheless, understanding the underlying chemistry of protein surfaces can shed light on their behaviour5. We can consider surface charges, hydrophobicity, folding stability and exposed residues to start to understand how proteins fold and interact.

The unicellular green algae Chlamydomonas reinhardtii, has been studied extensively as a model organism6. Many of its protein-protein interactions have been linked to their function, but their aggregation have not been studied. In many ways Chlamydomonas is the perfect model organism. It is photosynthetic, eukaryotic, and flagellated, and therefore relevant to many higher organisms. It can be viewed easily with a light microscope, and has had its genome sequenced and partially annotated. It is non-pathogenic and easy to grow, and has been used to understand processes as diverse as sperm motility7 and Hydrogen production. In this review and the subsequent report, the properties of a flagellar protein from C.reinhardtii are discussed in the context of protein aggregation and stability. This review considers primary literature, and by highlighting missing information makes recommendations about further study of this protein.

Flagella as a ciliary model

Cilia are hair like structures that protrude from the surface of cells into surrounding fluid. They have the ability to move in a variety of ways depending on their location and function8. They are involved in many biological processes in Homo sapiens and come in many types, from respiratory cilia which clear mucus from the oesophagus to the tails of sperm cells . Defects in cilia are the cause of several diseases and syndromes. For example polycystic kidney disease, Nephronophthisis, and Meckels syndrome have all been linked to mis or non functioning in cilia and are therefore classed as Cilliopthies9. For this reason research into the structure and regulation of human cilia is important for medical science. The technical and ethical difficulties of using human cell lines for research10 means. C. reinhardtii has become the major focus of research

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into human ciliopathies, Cilia and flagella are very closely related by both function and structure so algal flagella can be used to model mammalian cilia.

Flagella structure

The anatomy of the C.Reinhardtii flagella is relatively well understood (see Figure 1). It has two motile flagella used for locomotion, which extend from the basal body. Each flagella composed of nine outer doublet microtubule pairs and a central pair (CP) of microtubules. This is called the [9+2] architecture. Projections from each outer microtubule pair extend towards the central pair, and are called the radial spokes, composed of radial

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Figure 1. Left - Schematic diagram adapted form electron micrograph of C. reinhardtii, showing main structural features. The Flagella extends from the cell through the basal body. Right-Schematic cross section of the C.reinhardtii flagella, showing the 9 outer microtubule pairs, the central pair, the dynein arms and radial spokes. Adapted from Merchant et al11

spoke proteins (RSPs). Dynein motors on the outer microtubule pairs power the sliding of the microtubules which results in the flagella beating7.

The role of the central pair in flagella motion

The central pair is not present in all flagella/cilia. For example, the [9+0] arrangement of microtubules in the nodal cilia of developing embryos lack the central pair and radial spokes but are still motile7. The central pair and radial spokes may be required to convert simple symmetric beating into asymmetric waveforms required for motility (forward swimming). [9+2] flagella have planar large amplitude waveforms, whist [9+0] have low amplitude 3D helical waveforms. This means that [9+2] structures have more powerful planar beating strokes, which are suitable for intensive roles like swimming and sweeping mucus, whereas [9+0] perform more facile roles, like establishing left right asymmetry in the developing embryo12. The central pair and radial spoke assembly appears to have a complex regulatory role giving more sophisticated flagella motion. However, there is no clear model of the chemical regulation of flagella motion arising from the central pair.

Identification of Flagellar proteins

A report by Merchant et al11 studied the genome of C. reinhardtii and identified many previously unknown and unstudied genes associated with the flagella. D’Souza et al13 used BLAST (Basic Local Alignment Search Tool) software in an attempt to find homologous genes in other organisms. A protein called FAP174 (Flagella Associated Protein 174) from C.reinhardtii was found to be an ortholog of the human protein AMY-1 (Associate of Myc-1), also called MYCBP (Myc Binding Protein). This report will use the term AMY-1 for clarity. AMY-1 binds to the transcription factor Myc, but has a satellite function where it binds to AKAP1 (A-Kinase anchoring protein 1)14.

To understand the role of AMY1, we must look at a class of enzymes called kinases. These ubiquitous proteins phosphorylate substrates in cells as part of essential signalling pathways involved in almost all biological processes15. One class, known as cAMP dependent Protein Kinase A’s (PKA) are present in C. reinhardtii. PKAs are holoenzymes, consisting of 4 subunits, 2 regulatory and 2 catalytic16 (Figure 2a).

The catalytic subunit (PKAc) is where the phosphorylation reactions actually occur. The regulatory subunits RI and RII help coordinate the activity of PKA, and is the site of cAMP binding. In order to restrict phosphorylation reactions to specific sub cellular locations, PKA can bind to A Kinase Anchoring Protein (AKAP), which ensures phosphorylation events only occur where they are needed. AKAPs are proteins which exist to spatially organise phosphorylation reactions in cells. AKAPs bind to either RI, RII, or both (dual specificity AKAPs), and anchor PKA to organelles or other substructures in cells (Figure 2b)

When cAMP binds to the regulatory subunit, a conformational change takes place , releasing the active catalytic subunit, which then phosphorylates substrates as part of signalling pathways.

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PKA activity depends on the concentration of cAMP, so is regulated by the availability of cAMP in the cell. However there is a second level of control, involving the binding of regulatory proteins to RII and AKAP17, 18, 19(Figure 2c).

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Figure 2. a)Schematic diagram of Protein kinase A substructure. RI and RII are the regulatory subunits. PKAc is the catalytic Subunit. D/D is the docking/Dimerisation domain, where AKAP binds. cAMP is the cAMP binding site. C is the catalytic interface. b) Activity of PKA in the absence of an inhibitor. AKAP anchors PKA to the correct location in the cell. c) AMY1 acts as an inhibitor for PKA activity, by interfering with the release of the catalytic subunit. FAP174 is an orthologue of AMY1.

a)

b)

c)

AMY1 is an example of such a regulatory protein14, 20. It binds to the RII binding domain of AKAP through the hydrophobic face of a structurally conserved amphipathic helix21. Such helices form from an alternating repeating sequence of hydrophobic and hydrophilic residues (ΦΦΧΧ, where Φ is hydrophobic and X is hydrophilic). This results in a helix with one hydrophobic face and one hydrophilic face. The binding of AMY1 to AKAP disrupts the binding of PKA to AKAP, and regulates the activity of the enzyme in a process that is not well understood. FAP174 may play a similar role in C.reinhardii, it has been shown to exist as part of the central pair protein assembly.

Early study of FAP174

The primary sequence of FAP174 is shown below: 10 20 30 40 50MSESQKETFR KYLEQAGAID VLVKVLVQLY EEPSKPKTAL DYIKQCLGSP 60 70 80 90 TPAEYEAVVA ERDGLQKQLE EANQLIAELQ SRVQSLEAAA ETA

It is a 93 residue protein with a mass of 10,327 Da. Using the HeliQuest prediction programme, V.Rao et al13 predicted that the 16AGAIDVLVKVLVQLYEEP33 motif is a helix forming sequence capable of forming an amphipathic helix. This supports the view that FAP174 is an AMY-1 orthologue, since as well as primary sequence similarities shown in BLAST, it has secondary structural similarities. This amphipathic helix of FAP174 is proposed to be the site where it binds to an AKAP.

If this model were correct, we would expect to find AKAPs in C.reinhardtii with a strong affinity for FAP174. Two such AKAPs have been identified. Radial Spoke protein 3 (RSP-3) has been shown by Anne Roush Gaillard et al22 to be an AKAP by showing its interaction with PKA. This was done by performing overlay blot experiments with an isolated C. reinhardtii PKA RII domain. Another experiment by D’Souza et al13 isolated AKAP240, which was also shown to interact with a C. reinhardtii PKA RII domain. The interaction of these AKAPs with RII suggests that they play a role in the regulation of PKA activity in the C.reinhardtii flagella.

D’Souza, V. Rao 13 have shown that both RSP-3 and AKAP240 bind to FAP174. This was done in a co-purification experiment, where hexa-histidine tagged FAP174 was mixed with either RSP3 or AKAP 240 and purified using an NiNTA affinity column. RSP3 and AKAP240 bound strongly to FAP174 and were retained in the column.

In order to establish a link between flagella function and the expression of FAP17413, C.reinhardtii cells were deflagellated, and the expression of the fap174 gene monitored by measuring levels of FAP174 mRNA. When deflagellated by high pH, the expression of fap174 markedly increased compared to a control — ~27 fold increase in gene expression after 30 minutes. When deflagellated with CaCl2, the effect was even more pronounced — an approximate 65 fold increase in fap174 gene expression after 15 minutes.

These results indicate the importance of FAP174 in the flagella. The large increase in gene expression suggests that FAP174 is a key protein in the flagella, but does not tell us its role. However, its affinity with RSP3 and AKAP240, its similar primary structure to

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AMY1, and its predicted amphipathic helix, suggest it plays an important role in the regulation of PKA.

Biophysical properties of FAP174 Early biophysical characterisation of FAP174 by V.Rao and D’Souza13 revealed some interesting properties of the protein in vitro. During the purification of FAP174 it was observed on SDS-PAGE gels that the protein exists as both monomer and a dimer (Figure 3). This was confirmed by a MALDI TOF experiment (Figure 4), which detected a small but important dimer species.

The group suggested that the dimers appeared when an intermolecular disulfide bond linked two monomeric proteins. This could happen from the single cysteine residue at the 46th position. To confirm this, two experiments were conducted. First a titration experiment was conducted with the reducing agent DTT. DTT reduces Sulfur—Sulfur bonds to free thiol groups. The experiment showed that with increasing concentration of DTT there was a decreased concentration of the FAP174 dimer. This could be due to DTT reducing the sulfur—sulfur link between proteins in the dimer.

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Figure 3. Purification SDS PAGE gel of FAP174. Note the 2 bands, corresponding to the monomer and the dimer.

Incr

easin

g siz

e.

Dimer

Monomer

Monomer'FAP174''

Dimer'FAP174''

Monomer'Lysozyme'''

Dimer'Lysozyme'''

Figure 4. MALDI TOF mass spectrum of FAP174, showing peaks for the monomer and the dimer. Lysozyme was used as an internal standard. (From V.Rao13)

Secondly, a site directed mutagenesis experiment replaced the cysteine residue with an alanine residue to produce the C46A mutant. This mutant does not form dimers. This strengthens the view that the dimer is formed by a disulfide bridge. During this study it was observed that both the C46A and Wild type (WT) proteins form weakly interacting oligomers. An FPLC experiment detected multimeric species up to ~70kDa, suggesting hexameric aggregates form in solution [Fast Protein Liquid Chromatography (FPLC) is a gel filtration technique that separates proteins according to their physical size. Proteins elute in decreasing size order, and are detected by UV at 280nm].

These are weakly interacting as they do not appear in SDS gels, and there are no more free cysteine residues to form further disulfide bonds. This has hampered attempts to conduct conclusive NMR experiments, since spectra are poorly resolved and complex - the size limit for 2D protein NMR is around 35kDa. Two other mutants were synthesised, VLV21AAA and VLV25AAA. These were chosen to disrupt the helical binding domain. Preliminary results have suggested that these mutants form dimers more readily than WT FAP174. This could be due to misfolding of the protein, exposing the cysteine residue.

Circular Dichroism spectroscopy (CD) measures the percentage alpha helix and beta-sheet of a protein23. Briefly, the technique works by measuring the absorption of circularly polarised light by the sample. Different structures absorb circularly polarised light to different extents, resulting in transmitted light acquiring ‘left or right handed’ ellipticity. This is measured as a function of incident wavelength, and an algorithm predicts the secondary structure. V.Rao13 has shown that FAP174 and all three mutants have an almost 100% α-helical secondary structure. CD does not give information about the tertiary protein structure. This is consistent with in silico predictions using HeliQuest, which predicts the helical secondary structure of FAP174 and its mutants.

The observation that FAP174 forms small aggregates leads into the study of protein aggregation, which has major implications in many fields.

Protein Aggregation Studies

Protein aggregation processes are important in both medical and industrial settings24. Many diseases in humans have been associated with the aggregation of proteins, for instance Alzheimers disease is characterised by the formation of amyloid fibrils from unregulated aggregation of Tau protein in the brain4. Insulin dependent diabetes (Type 1) has been associated with protein aggregation in the beta cells of the pancreas, and patients with Parkinson’s disease typically have aggregates called Lewy’s bodies throughout the brain.

Industrially, the production of proteins by over expression in genetically modified organisms like E.coli is often hampered by uncontrolled protein aggregation. These aggregates can form insoluble particles in cells called inclusion bodies24, 25. These may render the proteins inactive and require refolding back into their native state25, 26 This can be achieved by many strategies but is far from desirable. The formation of inclusion bodies does have its advantages, primarily since separation of these insoluble aggregates can easily be achieved using centrifugation or filtration. Protein aggregation is of great interest economically, scientifically, and medically, since many industries rely on the efficient production of useful proteins on large scales. The formation of inclusion bodies is not well understood, and the chemical process of

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amorphous aggregation is unclear24. Many aggregation processes have been shown to occur independently of primary sequence and secondary properties like hydrophobicity.24, 27

Studying amyloid formation

Amyloid Fibrils are polymeric threads of protein that can form spontaneously in the body. Research into the formation of amyloid fibrils in human proteins is intensive. This is because of the obvious need to understand the process of amyloid formation and its role in human disease3.

Much research has focused on the mechanism of amyloid formation in vivo. There are several models to account for amyloid formation in cells3, 5, 28, 29, but no general predictive model that describes whether proteins will form amyloid fibrils.

B. Ahmed et al 27, 30 have shown that changes in pH and percentage α-helicity contribute to the propensity of proteins to form amyloid like aggregates. They used intrinsic and extrinsic fluorescence, Dynamic Light Scattering (DLS) and CD to characterise the aggregation state of the protein HypF-N. By varying the pH, they were able to induce to protein to form amyloid fibrils, which were then studied by fluorescence. The used the dye Thioflavin T which binds to proteins aggregates in their extrinsic fluorescence studies. However they did not consider the role of salts like NaCl on the aggregation process, which can play a major role in protein stability and folding. In another paper, and subsequently31 the small molecules Curcumin and Kaempferol have been shown to inhibit amyloid formation in Hen Egg White Lysozyme (HEWL). The paper focuses on the kinetics of the effect, but does not give a molecular account of the inhibition process32.

FAP174 as a model

When producing FAP174 from genetically engineered E.coli no inclusion bodies are observed. It has been suggested that FAP174 could act as a model for protein aggregation processes, since it forms small aggregates but does not form inclusion bodies. Conditions under which FAP174 forms large oligomers could shed light on the formation of inclusion bodies in other proteins. This would also help expand on the work of B.Ahmed et al in understanding the aggregation of helical proteins27, by giving more insight into the chemical process of aggregation.

The regulation of PKA activity in flagella is not understood33. Understanding the role of FAP174 in this regulation would be a small but important step in understanding PKA activity.

Structural NMR study has been difficult due to FAP174 forming dynamic aggregates in vitro. To study the protein by NMR, conditions are required where the protein exists as a stable monomer. It is not known how FAP74 forms oligomers, and this is important if it is to be used as a model for future aggregation studies. It is not known why some proteins form inclusion bodies in cells whilst others do not.

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Future work • Study the oligomerisation profile of FAP174 by FPLC/Gel filtration• Expose FAP174 to different chemical conditions to establish a stable monomeric species• Characterise the proteins’ oligomerisation by Dynamic Light Scattering • Record NMR spectrum of monomeric FAP174 using 2D structural experiments • Study aggregation of FAP174 using extrinsic fluorescent dyes e.g. ANS, Nile red,

Thioflavin T• Try to induce Amyloid or higher aggregation states in FAP174 to understand aggregation

processes in other proteins - can FAP174 form amyloid fibrils? Do Curcumin and Kaempferol inhibit amyloid formation in FAP174?

• Study unfolding of FAP174 to understand its stability• Study kinetics and mechanism of these processes to understand the molecular basis for

aggregation.

Conclusion

FAP174 is a newly identified AMY-1 orthologue with some interesting characteristics. Previous work has focussed on its role in C.reinhardtii, but it can also be studied as part of the general field of protein science to understand why proteins behave as they do.

The two main directions of future study are as follows 1) To understand the regulation of signalling in the Chlamydomonas reinhardtii flagella, and by extension to understand human ciliary diseases, 2) As a model to understand aggregation of industrially and medically relevant proteins.

Future work should focus on gaining a deep basic understanding of the molecular properties of FAP174 and its aggregates. This could feed into work on protein aggregation in general, as well as be useful for understanding the biophysical properties of FAP174 itself.

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Project Report

IntroductionProteins of scientific or commercial interest are routinely synthesised using a

partially standardised expression protocol34. This is usually done with the bacteria Escherichia coli as the expression host and a modified semisynthetic DNA plasmid (containing the gene of interest) as the expression vector. Systems of this sort have become the paradigm of laboratory and commercial protein expression. However there are many issues with producing proteins in this way. Low yields, mis-folding, and aggregation of proteins are common problems that must be overcome24, 25, 26. More important is the concern that proteins in vitro do not behave in the same way as they do in vivo. The work presented here aims to clarify the solution phase behaviour in vitro of a flagella protein called FAP174 (Flagella associated protein 174) from the algae Chlamydomonas reinhardtii. FAP174 is an analogue of the human protein AMY1, and has been shown to bind to two A Kinase Anchoring Proteins (AKAP) called AKAP240 and RSP3. Its role in C.reinhardtii is not understood, but it is suggested that it plays a role in the regulation of Protein Kinase A in the flagella.

FAP174 has been shown to form disulfide dimers as well as weak dynamic aggregates of various sizes in vitro. These aggregates have made structural Nuclear Magnetic Resonance Spectroscopy unfeasible, since the dynamic nature of aggregation causes unmanageable broadening of NMR peaks. Until now no mechanism of aggregation of this protein has been suggested, nor have any solvent conditions been explored that reduce aggregation.

Genetic engineering has altered the entire field of molecular biology and biochemistry. It is routine to use organisms with recombinant or mutated or DNA to explore the properties of proteins. It is now relatively simple to produce small proteins like FAP174 in the lab, and purify them by exploiting the selection tags artificially appended to them. Here the pET28a plasmid was used to deliver the fap174 gene into BL21 (DE3) E.coli, which when induced using IPTG, expressed FAP174 with a hexa-histidine tag. This ‘homo-oligmeric’ amino acid motif allows for the easy separation of the protein of interest (POI)

This study makes particular use of easily accessible standard instrumentation to probe the behaviour of FAP174. For instance, experiments using a standard laboratory spectrofluorometer35, 36 can give quantitative information about the unfolding of proteins, including the thermodynamic parameters ΔH, ΔS, ΔG and ∆Cp. Intrinsic protein fluorescence occurs when amino acids relax from excited states, emitting detectable radiation which can be measured by standard UV-Visible Spectrofluorometer. Tryptophan, Tyrosine and Phenyl Alanine can all be used as internal fluorescence residues. Extrinsic fluorescence spectroscopy measures fluorescence spectra of dyes that interact in some way with proteins, and is used here to follow the unfolding of FAP174.

The Thermofluor Assay is an ingenious technique that exploits the mainstream Quantitative Real Time Polymerase Chain Reaction (qPCR) instrument. It is used to measure the melting temperature (Tm) of a protein by observing changes in fluorescence intensity at λmax of emission from the fluorescent dye called SYPRO® Orange. This dye binds to the hydrophobic residues of a protein as they get exposed on unfolding. The dye fluorescence increasing markedly on protein binding, and clearly shows the progress of

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thermal unfolding. The advantage of this system comes with the ease with which many different solvent conditions can be screened in a short space of time. Indeed in this experiment, 96 different pH and NaCl combinations were screened in little over an hour, generating large volumes of data which can reveal information about the stability of the protein.

Three mutants of FAP174 are also briefly studied here. These mutants, C64A, VLV21AAA and VLV25AAA, were synthesised by site directed mutagenesis in a previous experiment. These mutants were designed to disrupt the binding of FAP174 to AKAP240 and RSP3, proteins that are fundamental to the action of FAP174. This study tries to understand if these proteins are suitable for further biochemical work.

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Materials and Methods

E. coli Transformation

E.coli cells were transformed to express hexa-histidine tagged Wild Type (WT), C46A, VLV21AAA and VLV25AAA FAP174 using the following protocol. Lysogeny Broth (LB) was prepared from 5gdm-3 NaCl, 5gdm-3 yeast extract and 10gdm-3 Tryptone in deionised H2O.

The pCBS-3 plasmid (pET28a containing fap174) 100ngμl-1, 1μl was added to 100μl of competent BL21 (DE3) E.coli cells, and incubated at 0℃ for 60 mins, then at 42℃ for 90s (heat shock), and at 0℃ for 5 mins (cold shock). The sample was warmed to room temperature over 5 mins, then 1 ml LB was added and the sample incubated at 37℃ for 60 mins to allow the Kanamycin resistance phenotype to develop. The sample was centrifuged at 5204 x g (RCF) for 5 mins to collect the transformant cells, the supernatant was removed and the cells were resuspended in 1 ml LB . The culture was then spread onto a 22.5μg dm-3 Kanamycin/LB Agar plate, and incubated at 37℃ for 16 hours. 16 colonies were selected and grid spread onto a 22.5μg dm-3 Kanamycin/LB Agar plate to produce 16 colonies of clones. A single colony was then used for subsequent study to ensure genetic homogeneity of transformant cells. Untransformed BL21 (DE3) E.coli were grown on LB/agar and 22.5μg dm-3 Kanamycin/LB Agar as controls to ensure that the cells were a) healthy and able to grow on LB, and b) not already resistant to kanamycin. Induction

A small quantity of transformant clones were inoculated into 10 ml of LB as a seed colony for induction. This was incubated with shaking at 36℃ for 16 hours. 2 ml of the seed culture was added to 500 ml 22.5μg dm-3 Kanamycin in LB, and incubated at 36℃ until an OD600 > 0.5 was reached (approximately 4-5 hours). IPTG (1M, 0.5 ml) was added to give a final concentration of 1mM. The sample was incubated for 5 hours at 37℃. The culture was then centrifuged at 5031 x g (RCF) for 10 mins to remove the supernatant, and the cell pellets pooled and frozen at -80℃. To confirm over-expression an SDS-PAGE was run using a 5% stacking gel and a 15% resolving gel, at 50 V and 9090 VV respectively.

Purification

Purification of FAP174 and its mutants was carried out by NiNTA affinity chromatography as described below. The following solutions were prepared prior to purification and stored at 4℃

TNG buffer : 50mM Tris, 30 mM NaCl, 10%Glycerol (Propan-1.2.3-triol), adjusted to pH 7.4 with 30% HCl(aq) in deionised H2O Lysis buffer/Wash buffer 1: 0.5% IGEPAL CA-630 (surfactant), 20mM Imidazole,10mM β-Mercaptoethanol (2-Hydroxy-1-Ethanthiol) reduces disulfide bonds, 1mM PMSF (Phenylmethylsulfonyl fluoride) (protease inhibitor) in TNG bufferWash buffer 2 : 20mM Imidazole, 10mM β-Mercaptoethanol, 1mM PMSF, in TNG bufferElution buffers :1mM PMSF, 10mM β-Mercaptoethanol and various imidazole concentrations according to elution order:

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Elution 1 (E1) - 100mM Imidazole Elution 2 (E2) - 200mM Imidazole Elutions 3a, 3b, 3c (E3a,b,c) - 300mM Imidazole

All procedures were carried out at 4℃ to avoid protein degradation by protease activity. 6 ml Ni-NTA agarose matrix resin was washed with lysis buffer to remove ethanol, and allowed to equilibrate with the buffer for 1 hour. Transformant cells were resuspended in 50 ml of lysis buffer, and sonicated on ice at 50% intensity over 15 periods of 2 mins with 2 minute rests to allow the solution to cool. The lysis mixture was centrifuged at 18,514 x g (RCF) for 15 minutes, and the supernatant was mixed with the Ni-NTA agarose matrix resin and incubated for 1 hour to allow the tagged proteins to bind to the resin.

The resin mixture was then loaded into a 25 ml column. The first fraction collected was the crude ‘flow through’. The resin was then washed sequentially with Wash 1, Wash 2, E1, E2, E3a, E3b and E3c, and these fractions collected separately. Fractions E1-E3c were then dialysed with a 10 kDa MWCO regenerated cellulose dialysis tube against a 60mM pH 7.4 Sodium Phosphate buffer for 2 days to remove imidazole, glycerol, β-Mercaptoethanol and PMSF. An SDS PAGE was run to confirm purification, using a 5% stacking and 15% resolving gel, at 50 V and 90 V respectively. Concentration estimation

The concentration of eluted proteins was calculated using the Bradford assay37. Bradford reagent (0.2 ml) mixed with small volumes of protein sample (depending on sample concentration, typically 5-20μl to ensure signal falls within the calibration range). The samples were incubated at room temperature for 5 mins, then the absorption at 595nm measures, and compared to a calibration curve from Bovine Serum Albumin (BSA). Samples were concentrated to ~2mgml-1 using a 5kDa MWCO centrifuge concentrator. Samples were aliquoted and frozen at -20 ℃.

Thermofluor assay (Differential Scanning Fluorometry)

CHC buffer system was prepared as described previously38. A 96 well qPCR plate was used to screen many buffer conditions in each experiment. Each well was loaded with 5μl 1mgml-1 protein solution, 5μl 100x SYPRO® orange protein dye in DMSO, and 10μl of CHC buffer/NaCl according to scheme 1 below. After mixing the samples were left to equilibrate at 4℃ for 1 hour. A BioRad qPCR instrument was used to measure the fluorescence intensity of each well, λex = 497nm and λem = 520 nm, between 25 - 95℃ at 1℃ intervals, allowing 1 min at each temperature for any conformational changes to take place.

�21

Intrinsic Fluorescence

FAP-174 was diluted with a) 60mM pH7.4 Sodium Phosphate buffer to a final concentration of 20μM, b) 7.4M GuHCl in 60mM pH 7.4 Sodium Phosphate buffer a final concentration of 20μM. Samples were incubated at 4℃ for one hour. Spectra were recorded at room temperature, using a 1.4 ml quartz cuvette, path length 10 mm x 4 mm with 4 polished windows, λex = 280nm and λem from 290-450 nm. Entrance and exit slits were both set at 5nm.

Extrinsic Fluorescence - Chemical denaturing

A 2.18 x10-3 moldm-3 stock ANS solution was prepared and filtered with a Millipore syringe filter. The concentration was measured using UV-Vis absorption (λmax = 372nm, ε=7880 dm3mol-1cm-1). 13 FAP174 samples were prepared in various concentrations of GuHCl with a 5:1 ANS:FAP174 molar ratio according to Table 1 below. Samples were incubated at 4℃ for one hour. Spectra were recorded at room temperature, using a 1.4 ml quartz cuvette, path length 10 mm x 4 mm with 4 polished windows, λex = 380nm and λem from 400-600 nm. Entrance and exit slits were both set at 5nm.

�22

pH

4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 10

0

25

50

100

250

500

1000

2000

[NaCl] /mM

Scheme 1. Representation of 96 well qPCR plate used for Thermofluor Assay. Each well has a unique combination of [NaCl] and pH.

Extrinsic Fluorescence - Thermal denaturing

A solution of 20μM FAP174, 100μM ANS in 60 mM pH7.4 Sodium Phosphate buffer was prepared. Samples were incubated at 4℃ for one hour. Fluorescence spectra were recorded from 25-95℃ at 5℃ intervals, using a 1.4 ml quartz cuvette, path length 10 mm x 4 mm with 4 polished windows, λex = 380nm and λem from 400-600 nm. Entrance and exit slits were both set at 5nm.

Table 1. Concentrations of reagents used in GuHCl denaturing experiments

[GuHCl]/M

[FAP174]/μM

[ANS]/μM

0 20 100

0.5 20 100

1 20 100

1.5 20 100

2 20 100

2.5 20 100

3 20 100

3.5 20 100

4 20 100

4.5 20 100

5 20 100

5.5 20 100

6 20 100

�23

Results and Discussion

Transformation, induction and purification

Transformation of BL21 (DE3) E.coli was confirmed by its successful growth on substrates containing the antibiotic Kanamycin. This represents the selectable phenotype of the pET 28a plasmid vector, where only transformant cells contain the gene for Kanamycin resistance. The controls showed that the untransformed cells being used were healthy because they grew successfully on LB/agar medium, and did not already contain the Neomycin Phosphotransferase (NPT) Kanamycin resistance gene as they did not grow on LB/Kanamycin/agar medium.

Induction was confirmed by SDS-PAGE as shown in Figure 5. There is a dark band at around 12kDa which corresponds to the molecular weight of FAP174 and its mutants. This is higher than the MW of the 93 amino acids that code FAP174 since it includes a thrombin cleavage site, a hexa-histidine tag and other associated amino acids. The control here is from uninduced transformant cells i.e. these cells contain the fap174 gene but are not expressing FAP174 since IPTG was not added. It is interesting to note the lower level of expression of all other proteins in

induced cells compared to the control lane. This is because induced cells over-express the protein of interest, neglecting the transcription and translation of ‘normal’ proteins, which are produced at a largely diminished rate. This process contributes to the early cell death of induced transformants, and is why E.coli cannot be used to continuously express proteins.

�24

C C46A VLV21AAA VLV25AAA MW Ladder

Figure 5.Induction SDS-PAGE of 3 FAP174 mutants (C46A, VLV21AAA and VLV25AAA), with and unindused control (C) and a molecular weight marker (MW).Induction bands are clearly visible in the induced lanes.

Induction bands

Purification with an NiNTA affinity column isolates 6 x Histadine tagged proteins from the myriad untagged proteins present in the E. coli cell lysate. Purification was also confirmed using SDS-PAGE (Figure 6).

The presence of 2 bands in the main elution fractions E1-E3c represent the FAP174 monomer and dimer. Previous work13 attributes the formation of the dimer to an intermolecular disulfide bond between the single cysteine residues at the 46 position. Lane E3a is the first wash with 300mM imidazole, and contains the highest concentration of FAP174. The broad monomer band is due to the gel being overloaded with a large excess of protein. This is done intentionally, in order to try to resolve any contaminants that may be present in low concentrations.

Thermofluor Assay

The thermofluor assay is designed as an efficient method to compare how different solvent conditions affect the behaviour of a protein. It gives both quantitive information (Tm) and qualitative information based on the shape of the melting curves.

�25

SP CP FT E1 E2 E3a E3b E3c

Figure 6. Purification SDS PAGE gel of Wild Type FAP174 using NiNTA affinity chromatography. SP is the supernatant from lysed cells, CP is the Cell Pellet after supernatant was removed, FT is the Flow Through, which is the first elution of the column. E1-E3a are the subsequent elution fractions, using increasing imidazole concentrations.

Dimer bands

Monomer bands

Wild Type FAP174

There is insufficient space here to include all thermofluor assay spectra, however Figure 7 below shows an example of a typical spectrum in order to illustrate the method used to derive the melting point of FAP174

�26

20 30 40 50 60 70 80 90 100 110

200

400

600

800

1000

1200

1400

Flu

ores

cenc

e In

tens

ity/ a

.u

T/ oC

64

20 30 40 50 60 70 80 90 100 110-80

-60

-40

-20

0

20

40

60

80

dF/d

T

T/ oC

a)

b)

Figure 7. Typical thermofluor spectrum of wild Type FAP174. a) Fluorescence intensity variation caused by the binding of SYPRO® Orange dye to newly exposed hydrophobic patches.b) The first derivative reveals the inflection point of the Fluorescence spectrum, which is at the protein melting temperature

Figure 7 is a typical WT FAP174 spectrum, reveals that the protein is properly folded as it shows a clear unfolding transition39. This supports previous CD data that showed WT FAP174 had a well ordered helical secondary structure. This is unsurprising, as we would expect a wilt type protein to have a well ordered 3D structure in order to carry out its role in the cell.

Figure 8 shows that FAP174 is least stable under high salt and extreme pH conditions. Again this is to be expected considering that C.reinhardtii is a mesophile that grows best in neutral pH and low NaCl concentration.

Using the online Bioinformatics tool ProtParam40, the theoretical isoelectric point (PI) was calculated for FAP174 as pH 4.61. This is the pH at which the overall charge of the protein is zero. This is an important consideration since most conditions studied here, and certainly physiological conditions, are far higher than this calculated value. This means the protein is negatively charged under these conditions.

There is a strong dependence of Tm on [NaCl]. There are 3 main effects that could be influencing the stability of the protein41, 42, 43 : Electrostatic screening, the Hofmeister effect, and site specific binding. These may all play an important role in determining the stability of FAP174. Electrostatic screening is a strong effect which occurs when ions are attracted to charged patches

�27

0 1000 2000 3000 4000

54

56

58

60

62

64

66

Tm

/ o C

[NaCl]

pH4 pH4.5 pH5 pH5.5 pH6 pH6.5 pH7 pH7.5 pH8 pH8.5 pH9 pH10

Figure 8. Protein melting Temperature (Tm) vs NaCl concentration at 12 different pH conditions. There is a clear dependance stability on the salt concentration, (see discussion). The pH dependance is less clear, other than in the 2 outliers at pH 10 and 4, where the melting temperature is considerably lower than at other conditions.

0 500 1000 1500 2000

[NaC]/mM

on the protein surface. These ions can cover the charged patch, and screen it from its usual intramolecular interactions with other charged patches on the protein surface. The Hofmeister series orders ions according to hydration affinity. It predicts that only ions of matching hydration affinity will interact strongly. From this we can predict that Na+

ions will interact weakly with the negatively charged carboxylate side chains of FAP174 due to a mismatch in hydration affinity. However there may be stronger interactions between Cl- ions and non polar or non charged residues because of their similar hydration affinities, and these will probably play a large role in determining the protein stability. As pH increases, the negative charge on FAP174 also increases, and the surface properties will be dominated by the negatively charged carboxylate side chains from glutamic and aspartic acid. These side chains are strongly hydrated and we might expect this to influence the unfolding stability. See ‘Extrinsic fluorescence — thermal denaturing’ below. The values of Tm derived from the thermofluor assay do not only reflect the unfolding of FAP174. The oligomeric state of the protein is also important, since oligomers will dissociate before protein unfolding (this was shown by Rayleigh scattering in a separate experiment not included here). This means that Tm may reflect the additive effect of changing oligomeric state and unfolding.

This may explain the high Tm observed at pH 4 with 0M NaCl. The protein is near to its PI, and has been observed to precipitate under these condition. This is because the overall net charge on the protein is zero or near zero, so two protein molecules can easily approach each other and interact, forming non specific aggregates which can act as nucleation sites for precipitation.

At low pH it is likely that the tertiary structure is highly disrupted, as intramolecular electrostatic interactions are hampered due to the protonation of the negatively charged residues (Asp and Glu). Therefore we may expect Tm to be low as FAP174 is already in a highly disordered state. However, the high melting point observed may be an indication that the protein has aggregated, and the heat applied is being used to break apart these aggregates.

At or near the PI (low pH) we would also expect the solubility of the protein to increase with increasing NaCl, and this may reflect the decrease in Tm with increasing [NaCl]. Attractive interactions can take many forms, including ion pair interactions, cation — π interactions, π stacking, the hydrophobic effect and Van der Waals attractions, and these may all be disrupted by high concentrations of NaCl.

At the highest NaCl concentration there is a small increase in Tm under some pH conditions. This may be due to the ‘salting out’ effect, where protein solubility decreases at high salt concentrations. Briefly, the molecular basics for this effect is that the high ionic strength of the solution causes ions to interact strongly with the solvent water molecules. This interaction decreases the interaction with the dissolved protein, and protein-protein interactions become more favourable. In a sense the high salt concentration decreases the concentration of water by decreasing availability of water to interact with the protein surface.

�28

FAP174 Mutants

The three FAP174 mutants were designed in order to disrupt the formation of dimers (C46A) and the structure of the amphipathic helix (VLV21AAA and VLV25AAA). Previous CD data13 has suggested that the mutants are all helical, with a well ordered secondary structure. The thermofluor data from this study offers new and surprising insights into the tertiary structure of the FAP174 mutants. As Figure 9 shows, none of the three mutants give sharp transitions in the thermofluor spectra. This indicates that the proteins are ‘not well behaved’, or are unstructured. Additionally SYPRO® Orange fluorescence in experiments using the VLV21AAA mutant is an order of magnitude higher than for either the Wild Type or the C46A mutant. This may suggest that this mutant has refolded in such a way as to highly expose hydrophobic residues on the protein surface, allowing SYPRO® Orange to bind and fluoresce with high intensity. VLV25AAA also has a high initial fluorescence intensity, suggesting the exposure of hydrophobic residues, but to a lesser extent than the VLV21AAA mutant. It does not show any melt transition, and therefore may also have a disordered tertiary structure.

For the C46A mutant, the initial fluorescence signal intensity is comparable to the wild type protein, however it does not display any easily discernible unfolding transition. Thus we can conclude that despite having an intact helical secondary structure, the tertiary structure of the C46A mutant is highly disrupted and probably misfolded or fully denatured at the temperatures studied.

It should be noted that the thermofluor Assay relies entirely on the changing affinity of SYPRO® orange for the protein surface. It may give misleading results for well folded proteins with hydrophobic surfaces. Complimentary experiments should be used to conform this result. These results may explain a previous observation that the VLV mutants more readily form covalent dimers. The mutant proteins may be disordered in such a way as to expose the cysteine side chains increasing the kinetic availability of these groups to form bonds. Due to the significant variation in these spectra, the inconsistency in their tertiary structure, and the probability of misfolding, the FAP174 mutants were not studied further here. X-ray crystallography or structural NMR of these mutants could give interesting information on their 3D structure.

�29

�30

VLV25AAA

T/℃

C46A

Fluo

resc

ence

Inte

nsity

/a.u

T/℃

VLV21AAA

Fluo

resc

ence

Inte

nsity

/a.u

T/℃

Fluo

resc

ence

Inte

nsity

/a.u

Figure 9. Sample thermofluor data for 3 FAP174 mutants. Each of the spectra were recorded in a 0M NaCl in CHC buffer. Note the very high fluorescence intensity for the VLV21AAA mutant. None of the mutants show sharp melting transition, implying they are poorly or mis-folded. NB Y-axis scales vary for each mutant.

VLV25AAA

T/℃

C46A

Fluo

resc

ence

Inte

nsity

/a.u

T/℃

VLV21AAA

Fluo

resc

ence

Inte

nsity

/a.u

T/℃

Fluo

resc

ence

Inte

nsity

/a.u

Extrinsic fluorescence - Chemical Denaturing

GuHCl is a Chaotropic salt which strongly disrupts hydrogen bonding, hydrophobic effects and Van der Waals attractions in aqueous solutions. It is widely used as a protein denaturant, since it disrupts the specific interactions required for protein structural integrity. Figure 10 shows the variation in ANS (an extrinsic fluorescent dye) fluorescence with GuHCl concentration.

Measuring the fluorescence spectrum of ANS as a function of a protein denaturant gives information about the thermodynamic stability of the protein44, 45, 46. Importantly ΔG0 (the free energy of unfolding in the absence of a denaturant) can be calculated using the linear Extrapolation method described here.

�31

400 450 500 550 600-10

0

10

20

30

40

50

60

70

80

Flu

ores

cenc

e In

tens

ity/a

.u.

Wavelength/nm

Figure 10. Extrinsic ANS Fluorescence Spectra of FAP174 at various GuHCl concentrations. denotes the spectrum recorded with 0M GuHCl, with fluorescence intensity decreasing at a function of GuHCl

The Fluorescence signal from a sample is the sum of the signal contributions from all the micro-states present in solution: �

Where S is the observed signal, si is the signal from micro state i, and xi is the mole fraction of micro state i. For a 2 state system composed of a native (folded) and unfolded protein, this becomes

Where N and U denote the native and unfolded states respectively. Since we assume only 2 micro states are present, the sum of the mole fractions will equal unity.

Substituting (4) into (2) gives

Rearranging gives

Finally yielding an expression for the mole fraction of unfolded protein

�32

S = xisi∑ (1)

S = xNsN + xUsU (2)

xN = 1− xu (4)

xU = S − sNsU − sN

(8)

S − sN = xu(sU − sN) (7)

S = sN − xusN + xUsU (6)

S = (1− xu)sN + xUsU (5)

xN + xU = 1 (3)

The equilibrium between two state is described by the following equation:

Where K is the equilibrium constant, and [U] and [N] are the concentrations of native and unfolded states respectively. Substituting equation (4) into equation (9) gives

Substituting equation (8) into equation (10) yields

Which simplifies to

In this case the measured signal is the fluorescence intensity, I, so we can replace S with I:

Where I is the observed intensity, IN is the intensity with the native protein and IU is the intensity with the unfolded protein.

�33

K = [U ][N ]

= xUxN

(9)

K = xU1− xU

(10)

K =( S − sNsU − sN

)

1− ( S − sNsU − sN

)

K = sN − SS − sU

(11)

(12)

K = IN − II − IU

(13)

Finally substituting this into the expression for free energy change gives a relationship between fluorescence intensity and the free energy of unfolding.

Where ΔG is the free energy of unfolding, R is the universal gas constant (=8.314JK-1mol-1), and T is the temperature in degrees kelvin. A plot of ΔG vs [GuHCl] (Figure 11) yields a linear plot from which we can extrapolate the value of ΔG back to the Y axis, and estimate the value of ΔG0,

according to equation 16 below

Where [D] is the denaturant concentration, and m is a measure of how much ∆G is dependant on [D] i.e. d∆G/d[D]. m is a unique property of a protein and has been linked to its surface properties55. The extrapolated value of ΔG0 =5.01kJmol-1 is rather low, but also consistent with other experiments (see below).

�34

ΔG = −RTInK

ΔGU = −RTIn( IN − II − IU

) (15)

(14)

ΔG = ΔG0−m⋅[D] (16)

!∆G!=!%2.9432[D]!+!5.0108!R²!=!0.9634!

%14!

%12!

%10!

%8!

%6!

%4!

%2!

0!

2!

4!

6!

0! 1! 2! 3! 4! 5! 6!

∆G/kJm

ol)1+

[GuHCl]/M+

Figure 11. The linear extrapolation of Gibbs free anergy vs [GuHCl] for FAP174 at 298K in pH 7.460mM sodium phosphate buffer. The Y intercept represents a prediction of ∆G for unfolding of FAP174 in the absence of Guanidinium HCl. The m value of 2.9 kJMmol-1 can be related to the change in Accessible Surface Area (∆ASA)55.

Extrinsic Fluorescence - Thermal Denaturing

The thermal denaturing of FAP174 in the presence of ANS at pH 7.4 was also measured using fluorescence, and analysed in a similar manner. Figure 12 shows the fluorescence spectra taken at different temperatures (denaturing, increasing temperature).

�35

400 425 450 475 500 525 550 575 6000

20

40

60

80

100

120

140

160

Flu

ores

cenc

e In

tens

ity/ a

.u.

Wavelength/ nm

Figure 12. Extrinsic Fluorescence spectra of 20μM FAP174 with ANS from 298K (denoted ) to 368K. Fluorescence intensity decreases with temperature as the protein unfolds and ANS binding affinity decreases. There is a marked red shift due to changing ANS environment (see main text)

The usual method for calculating the enthalpy and entropy changes in a temperature dependent system uses the van’t Hoff plot of lnK vs 1/T according to the Van’t Hoff equation (equation 17), and calculating the gradient which = -ΔH/R. However, as Figure 13 shows, such a plot is non-linear for FAP174, which indicates the system cannot be treated with this simple model. In fact a nonlinear van’t Hoff plot indicates that ∆H and ∆S are not constant47.

�36

lnK = − ΔHRT

+ ΔSR

(17)

y"="1E+07x2"+"93854x"+"157.54"R²"="0.999"

+3"

+2"

+1"

0"

1"

2"

3"

4"

5"

6"

0.00275" 0.0028" 0.00285" 0.0029" 0.00295" 0.003" 0.00305" 0.0031" 0.00315" 0.0032" 0.00325" 0.0033"

lnK$ $

1/T(K)$

Figure 13. The van’t Hoff plot for the thermal denaturation of WT FAP174. The non linear nature implies a change in heat capacity associated with the reaction. The slope has a negative gradient which indicates that the process of unfolding is endothermic.

Instead, ΔG was calculated according to equation 15, and plotted against temperature as shown in Figure 14. This is known as a Gibbs-Helmholtz plot47. There is a clear non-linear dependance of ΔG on temperature, which indicates that ΔH and ΔS are proportional to temperature. This is related the heat capacity of the solution as shown in equations 19 and 20, where ΔCp is the heat capacity change at constant pressure

In order to calculate ΔH, ΔS and ΔCp from the unfolding of FAP174, the Free energy change data were fitted to a modified version of the Gibbs-Helmholtz equation (equation 21) shown below, by non linear regression using Microsoft Excel48.

Where ΔGT is the free energy change at a given temperature, T is the temperature in Kelvin, ΔCp is the change in heat capacity, and ΔHr, ΔSr and Tr are the enthalpy change, entropy change and temperature respectively at an arbitrary reference temperature (298K was used here). The model was compared to the observed data and gave an excellent fit with an R2 value of 0.999. The fitting parameters are shown below in table 2.

Table 2. Thermodynamic data from the nonlinear regression of the Gibbs-Helmoltz equation with data from thermal

unfolding.

ΔHr/kJmol-1 ΔSr J K-1 mol-1 ΔCp/ kJ mol-1 K-1

13.81 33.7 2.12

�37

dΔHdT

= ΔCp

dΔSdT

= ΔCp

T

(19)

(20)

ΔGT = ΔΗr −TΔSr + ΔCp T −Tr −T ln TTr

⎛⎝⎜

⎞⎠⎟

⎡⎣⎢

⎤⎦⎥

(21)

By simple inspection of the Gibbs-Helmholtz plot (Figure 14), we can say that ΔCp is positive, since concave down plots always result from an increase in heat capacity47. The physical meaning of this is that the energy required to increase the temperature of the solution by a small amount is greater as the temperature increases and the protein unfolds. This is because of solvent reordering effects, which are larger at higher temperatures when solvent molecules get reorganised around the hydrophobic and negatively charged residues of the unfolded protein.

This non-linear relationship leads to the interesting result that ΔG= 0 at two points, one at high temperature and one at a low temperature. The consequence of this well known49 phenomenon is that proteins undergo both hot and cold unfolding/denaturation. The Gibbs-Helmholtz plot also shows temperature range where the protein exists mainly in its folded state, between 256K and 328K, and these temperatures represent the cold and hot denaturation points respectively. Cold denaturation is thought to occur when at low temperatures the interaction of buried apolar amino acid residues with H2O (as the solvent) becomes enthalpically favourable, contributing enough to the term for free energy to make the process of unfolding spontaneous.

This plot gives a value of ΔG = 3.77kJmol-1 for unfolding at 298K, which agrees moderately well with the value calculated from the chemical denaturation experiment. This low value of free energy of unfolding may explain the tendency of the protein to aggregate.

�38

220 240 260 280 300 320 340 360 380 400

-10.0

-7.5

-5.0

-2.5

0.0

2.5

5.0

7.5

dG T/K

Modified Gibbs-Helmholtz Model Experimental Data

∆G/ k

Jmol

-1

Figure 14. Extrapolated Gibbs-Helmholtz plot for WT FAP174. The solid line is the predicted value of ΔG according to the modified Gibbs-Helmholtz equation (21), and the circles are experimental data points. The graph shows the region of highest stability, and the 2 denaturing temperatures corresponding to hot and cold unfolding. The R2 value = 0.999 using this model

Since ΔG is low, at 298K there will be a large fraction of unfolded protein in solution. Unfolded proteins are known to aggregate more readily than folded ones2, 24, 27, 29,

and this may be the case with FAP174. The low free energy of unfolding may arise because of strong interactions with the solvent. At ∆Gmax,∆S is zero and unfolding is purely driven by the enthalpy. This is exothermic at because at pH 7.4 FAP174 is negatively charged and therefore highly solvated — unfolding will increase the exothermic interaction between the solvent and exposed carboxylate residues, which have high hydration affinities. The low value of ∆G does not say that the intramolecular forces are weak in FAP174, rather that there are strong solvent interactions which decreases the difference in G between folded and unfolded states.

It is interesting to consider that the most stable conditions are those under which life on earth evolved — the protein is optimised to fold correctly under the ambient and mild environment where C. reinhardtii evolved as a mesophile, and indeed the folded state only exists in a small temperature range. If we look at a large temperature range, protein folding is atypical behaviour!

This instability might also have implications for the role of the protein in vivo. The protein has a low stability with respect to unfolding, which indicates that it can easily undergo conformational changes. It is suggested that FAP14 undergoes a conformational change on binding to AKAP240 and RSP3. This may be important in the regulation of PKA activity.

Using the parameters in table 2 and the equations below, ΔH and ΔS can be calculated for any temperature (making the assumption that ΔΔCp = 0)46

Where subscript r denotes the reference temperature.Generally both ΔH and ΔS increase with temperature. We can plot ΔH and TΔS on the

same graph, against temperature. Where TΔS becomes larger than ΔH, ΔG becomes negative (according to equation 24) with respect to unfolding, and the protein denatures. This is shown in Figure 15; the 2 lines meet at the hot and cold unfolding temperatures, when ΔH = TΔS, and therefore ΔG = 0.

It should also be noted that the temperature of maximum stability, i.e., where ΔG is at its maximum, is the temperature where ΔS=0, and ΔG = ΔH. This means that unfolding is entirely an enthalpically driven process.

�39

ΔΗ = ΔΗr + ΔCp(T −Tr)

ΔS = ΔSr + ΔCp ln TTr

⎛⎝⎜

⎞⎠⎟

(22)

(23)

ΔG = ΔH −TΔS (24)

�40

!100$

!50$

0$

50$

100$

150$

250$ 260$ 270$ 280$ 290$ 300$ 310$ 320$ 330$ 340$ 350$

TΔS$an

d$ΔH

$$/kJmol

/1$

T/K$Figure 15. ΔH (solid line) and TΔS (dashed line) vs temperature. Points A and B represent the hot and cold unfolding temperatures respectively. C is the Temperature (T∆Gmax) of maximum stability of the folded protein. Above T∆Gmax, the protein stability decreases because in spite of the increasingly endothermic enthalpic term, T∆S increases nonlinearly. This means hot unfolding is an entropic effect. Below T∆Gmax, FAP174 becomes less stable because in spite of the negative entropy of unfolding, the enthalpy is exothermic. Therefore cold unfolding is an enthalpic effect. Between A and C, the protein has enthalpic stability, and between B and C the protein has entropic stability. Below T∆Gmax T∆S is negative because as the protein unfolds the solvent becomes organised around the exposed negative residues, increasing the level of order.

B

A

∆H a

nd T∆S

/ kJm

ol-1

CT/K

Extrinsic Fluorescence shift

In both thermal and chemical denaturing experiments, there is a significant red shift from native to unfolded FAP174. This is due to the dominance of different decay routes for excited state ANS50 Figure 16 explains how the behaviour of ANS differs when bound to a protein.

Solvent relaxation (SR), Twisted Intramolecular Charge transfer (TICT) and Intersystem Crossing (IC) all play a role in changing the fluorescence behaviour of ANS. TICT occurs when there is an intramolecular transfer of charge from the electron donating amino aryl group of ANS to the Sulfonated Naphthalene system (see Figure 16b for ANS structure).

�41

Incr

easin

g En

ergy

Figure 16. a) Simplified Jablonski diagram for the relaxation of ANS. F is phosphorescence, F is Fluorescence, hv is the excitation energy. When bound to a protein, ANS decays mainly from excited state S1 by fluorescence, whereas when not bound it undergoes solvent relaxation (SR) and Twisted Intramolecular Charge Transfer (TICT) to a lower energy level, where its fluorescence quantum yield is much lower. From the S(TICT) state, ANS decays rapidly by non radiative processes such as internal conversion (IC) and vibrational relaxation (VR). Intersystem crossing (ISC) to the triplet state is also more rapid from the S(TICT) state. This accounts for the red shift and low florescence intensity as FAP 174 unfolds, because ANS looses its affinity for proteins at high temperatures as hydrophobic patches are disrupted. Solvent relation (SR) occurs when the dipole of ANS changes on excitation, causing polar solvent molecules to reorient

themselves, lowering the excited state energy and raising the ground state energy.b) Structure of ANS

a)

b)

Intrinsic Protein Fluorescence

The intrinsic fluorescence spectra of FAP174 give some qualitative information about the environment of the fluorescent residues in the protein51. On unfolding there is small but significant blue shift in the fluorescence signal (Figure 17).

This wavelength shift can be explained by considering microenvironment of the 4 Tyrosine fluorophores. The native protein has a broad fluorescence spectrum, which suggests each tyrosine exists in a different micro-environment. The signal is therefore broadened, as each residue fluoresces at a different wavelength. The unfolded spectrum is more narrow, suggesting smaller spread in tyrosine environment. This is expected, as on unfolding the tertiary structure of the protein is disrupted and Tyrosine residues no longer sit in specific sub molecular environments . It is interesting that there is a blue shift on unfolding. The phenolic side chain of Tyrosine is a hydrophobic , and is more stable in a hydrophobic environment. The blue shift indicates tyrosine residues are moving from a hydrophilic to a hydrophobic environment (Figure 18)

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280 300 320 340 360 380 400 420 440 460-10

0

10

20

30

40

50

60

70

80

90

Flu

ores

cem

ce In

tens

ity/a

.u.

Wavelength/nm

Denatured Intrinsic Fluorescence Native Intinsic Fluorescence

Figure 17. Intrinsic protein fluorescence spectra of FAP174 in the native (red) and unfolded (black) states. The Native spectra was recorded at 298 K in a pH 7.4 Sodium phosphate buffer, and the unfolded spectra at 298k in a 6M GuHCl pH7.7 Sodium phosphate buffer. The fluorescence signals are from the 4 tyrosine residues present in FAP174.

The tyrosine blue shift implies that in the native structure some or all of the tyrosine

residues are exposed at the protein surface, then as the protein is denatured move into a hydrophobic environment52. The ground state energy level decreases and the excited state energy increases as tyrosine moves into a more stable hydrophobic environment. This leads to a larger energy gap, ΔE = 0.053eV (equation 25 gives energy in Joules),

Where λF and λU are the wavelengths of maximum fluorescence for the folded and unfolded states respectively.

The 4 tyrosine residues are stabilised by approximately ΔE/2 (0.027eV) When FAP174 unfolds. However this number reflects calculations based on the sum of all tyrosine signals according to equation 1. Since each tyrosine exists in a different environment, ΔE will be different

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ΔE = hcλF

− hcλU

(25)

Figure 18. Schematic diagram of changing vibronic energy levels of tyrosine from FAP 174 on unfolding. VR is vibrational relaxation, E is energy, hv is the energy of radiation emitted. On unfolding, tyrosine residues are stabilised when they move to a hydrophobic environment. Transitions occur according to the Frank-Condon principle. For simplicity the diagram does not show all decay routes or anharmonic vibrational energy levels.

= 0.053eV

for each residue, probably larger than 0.027eV (the broad signal suggests some tyrosine residues are lower in energy than others).

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Recommended further work.

Fluorescence spectroscopy can give valuable information about biomolecules, but has its limitations. For instance, in order to detect different species in solution, they must have detectably different fluorescence properties. The aggregation of proteins may not give rise to a large enough change in signal to be detected. The following experiments are recommended to expand or clarify the work presented above. Dynamic light scattering (DLS)

This technique would be very useful to give both static and kinetic information about the aggregation of FAP17453. FAP174 monomers could be separated from a dynamic solution using FPLC. The monomer fraction could then be immediately transferred to a DLS instrument, and the distribution of particle sizes could be monitored over time as aggregates form. Kinetic parameters like rate constants could be calculated by this method. When the system reaches equilibrium, thermodynamic parameters could be calculated from the equilibrium constant. This experiment could be conducted at different temperatures to calculate ΔH and ΔS according to the van’t Hoff method, and under different solvent conditions to compliment the thermofluor experiment. A closely related technique (often bundled into the same instrument) is the measurement of Zeta potential. This gives information about the surface potential of particles in solution, and indicates the strength of interactions (a balance between attractive Van der Waals and repulsive electrostatic forces). It indicates the likelihood that a system will aggregate/flocculate.

Reducing Thermal Denaturing Experiment

TCEP is a strong temperature stable organic reducing agent that can reduce disulfide bond in proteins over a large temperature range. TCEP could be used as a reducing agent in a modified version of the thermal denaturation experiment presented above. This would remove ambiguity about signals coming from monomers or dimers, since the disulfide dimers would not form. Other reducing agents such as DTT could be used, but they are less temperature stable and need to be used in higher molar concentrations.

DOSY NMR

Diffusion ordered NMR spectroscopy can resolve macromolecules based on their coefficient of diffusion53. This gives information about the relative sizes of molecules in a solution, and would give complimentary information to DLS. 1D DOSY NMR is a convenient method as it can use 1H resonance signals, and does not require protein labelling with 13C or 15N, which can be both time consuming and inefficient.

Glutaraldehyde Cross-linking

Glutaraldehyde is a small di-aldehyde that can form strong intermolecular linkages between strongly and weakly interacting proteins alike54. This means dynamic aggregates can be ‘trapped’ and separated by SDS PAGE. This gives a snapshot of the species

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existent in solution. Glutaraldehyde must be used at very low concentrations as there are many side reactions that may give misleading results. For example 2 non-interacting species may get linked together.

X-ray crystallography

X-ray crystallography gives the exact spatial coordinates of atoms in a molecule based on electron density maps. It is a supremely powerful technique especially when investigating large biomolecules like proteins. The crystal structure of FAP174 would provide unique and enlightening information about the molecule. For example, by considering the residues at the surface of the molecule, aggregation mechanisms could be proposed. This would be an excellent way to validate the data collected in this experiment.

Electron density maps also provide a starting point for molecular simulations. However the crystals required for protein crystallography are difficult and time consuming to grow, and there may be problems if multiple species are present in solution. There are also always concerns that the X-ray structure does not represent the molecule as it exist in solution. For this information 2D protein NMR is invaluable, especially because of the dynamic information it can provide. The problems of structural NMR on FAP174 are mentioned earlier.

Differential Scanning Calorimetry (DSC)

DSC allows the direct measurement of ΔCp, and ΔH for unfolding55. This method is particularly convenient, and is generally gives precise and easily interpreted data. The technique works by measuring how much heat is absorbed by the sample as a function of temperature, and compares to a reference sample of known heat capacity. It can be used to calculate Tm, and would give interesting complimentary data to that provided here.

Modified Thermofluor

To test the hypothesis that Hofmeister effects play a role in determining the melting point of FAP174, the thermofluor experiment could be repeated using different salts from the Hofmeister series — we would expect to see different salting in and salting out behaviour based on the salts position in the series, and these results compared to DLS data on the same salts. We could also measure protein solubility56 under these conditions to determine whether solubility is related to melting temperature.

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Conclusion

The protein FAP174 has been shown to be unstable with respect to unfolding. Values for ∆G calculated by two different methods give similar results. These low values (3.77 kJmol-1 and 5.01kJmol-1) may explain why the protein has a tendency to aggregate. Unfolded proteins have been shown to have a higher aggregation propensity, and small aggregates can act as nucleation sites for further aggregation. A simple model of the aggregation of FAP174 is presented in Figure 19, which highlights a potential source of error in the experiments above. Indeed using a 2 state assumption can lead to an underestimation of �G and m values57 if there are undetected intermediates, and this may account for the low value of �G found in this report.

Using the Gibbs-Helmholtz model, cold unfolding is predicted to occur at 261K. Some early experiments on other proteins have observed cold unfolding by using novel conditions like high pressure or non aqueous solvents, and this could be a possible direction for future research.

The instability of FAP174 also indicates that it might undergo some conformational change on binding to AKAP240 and RSP3, as the free energy requirement for unfolding is very low. There may be a level of regulation related to the conformational change of FAP174, for instance its affinity for AKAPs may vary depending on the solution conditions in the cell.

Presumably FAP174 does not aggregate in vivo. There are numerous reasons for this. Firstly proteins tend to be relatively short lived in cells, so if the aggregation process is kinetically slow, there is not time for aggregation. Molecules called molecular chaperones can bind to unstable proteins and prevent their aggregation. These have been observed in C.reinhardtii, and may be involved in the regulation of FAP174 and the PKA control mechanism.

The three FAP174 mutants are not well folded. Thermofluor data suggests that all three have a disrupted tertiary structure, but CD spectra indicate the secondary structure is intact. Therefore the residues VLV21, VLV25 and C46 are all somehow involved in preserving the tertiary structure in WT FAP174. Importantly, because of the dissimilarity in the unfolding behaviour, it is recommended that these mutants not be used in binding studies with AKAPs. They may have a seriously compromised 3D structure, that could give misleading results in affinity experiments.

Intrinsic fluorescence suggests the 4 Tyrosine residues are exposed to a hydrophilic environment in FAP174. They probably exist in different micro-environments at the protein surface. This is surprising as Tyrosine is hydrophobic, and suggests that they may play a role in protein function — perhaps by acting as H-bond donors in a binding event at the protein surface.

The hexa-histidine tag was not removed during this project. However, further work should make every effort to produce high yield FAP174 and remove the His tag using the

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U

UaggFagg

F

Figure 19. This model of some of the aggregation pathways available to FAP174 highlights a potential source of error in these experiments. F and U are the folded and unfolded states respectively, and agg is an aggregated state Many of these species may be present in solution, but give similar fluorescence signals. This could lead to misleading or inaccurate data.

thrombin cleavage site. The His tag gives a constant level of ambiguity in results, since we do not know how it will affect the structure or function of the FAP17458. Further experiments have been recommended to remove as much ambiguity in the data as possible. Protein science is difficult because of the vast number of variables, and the difficulty in applying knowledge about one protein to another. However, carefully designed experiments can help clarify protein behaviour, and add to the growing pool of protein literature.

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Acknowledgements

I would like to thank the following people who's help and kindness made this project so enjoyable. Dr Jacinta D’Souza who kindly allowed me to work under her supervision, and helped get me on my feet in the lab. Dr Basir Ahmad who pointed me in the right direction. Dr Avinash Kale for his friendly and calm encouragement, who engaged in every detail of the project with true care. Special thanks to (the soon to be Dr) Venkatramanan G.Rao, who give up countless hours teaching me the skills I needed to undertake the project. I would like to thank Angelika Sebald and John Slattery at The University of York for their dedication to the exchange program, and for their amazing support over the year. And finally I thank CBS Mumbai, its staff and students, who extended to me such great hospitality. I wish you all success and happiness in the future.

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