Conformational Transitions of 18.5-Kilodaltons Myelin Basic Protein Studied by Fluorescence Spectroscopy and Förster Resonance Energy Transfer

Conformational Transitions of 18.5-Kilodaltons Myelin Basic Protein studied by Fluorescence Spectroscopy and Förster Resonance Energy Transfer

by Andrew Daniel Jenkins

A Thesis presented to The University of Guelph

In partial fulfilment of requirements for the degree of Master of Science in Molecular and Cellular Biology

Guelph, Ontario, Canada © Andrew D. Jenkins, April, 2015

ABSTRACT

Conformational Transitions of 18.5-Kilodaltons Myelin Basic Protein studied by Fluorescence Spectroscopy and Förster Resonance Energy Transfer

Andrew Daniel Jenkins Advisor: University of Guelph, 2015 Professor G. Harauz

The intrinsically-disordered, 18.5-kiloDalton (kDa) isoform of myelin basic protein (MBP) is a peripheral membrane protein that is essential to proper myelin formation in the central nervous system. MBP multifunctionality arises from its high conformational plasticity and its ability to undergo reversible disorder-to-order transitions. One such transition is the disorder-to-α-helical conformational change which is induced upon MBP-membrane binding. We have investigated the disorder-to-α-helical transition of MBP-derived α-peptides as well as of the full-length 18.5-kDa protein. The data suggest that the disorder-to-α-helical transition of MBP follows a three-state model: disordered↔intermediate↔α-helical, with each of the identified equilibrium states likely representing a conformational ensemble. The disordered state is characterized by slight compaction, whereas the intermediate globally more compact. This study suggests that multifunctionality in MBP could arise from differences in the population of energetically distinct ensemble clusters in different conditions and also provides an example of an IDP that undergoes cooperative global conformation change.

ACKNOWLEDGEMENTS

This master's degree was really the collective doing of an extended group of individuals who supported me throughout the years.

First and foremost I would like to thank my graduate supervisor, Dr. George Harauz, who afforded me the opportunity to work, learn and grow as scientist in his lab. I am truly grateful for your patience and guidance.

I would also like to thank and my advisory committee members, Dr. Rod Merrill and Dr. Matthew Kimber, for their guidance and constructive input throughout the process that was my graduate education.

To the two post-doctoral fellows in our lab, Dr. Vladimir Bamm and Dr. Kenrick Vassall, who were responsible for bending my mind and wrapping it around many of the concepts that I touch on in this thesis, I would not have been near as successful or as well trained without your efforts. To past and present labbies (Aggie, Danielle, Miguel, Sergio), thank you for your advice and endless hilarity that dragged me through the days.

But most of all, I’d like to thank my family, especially my mom – thank you for all the help, love, and support. You have made this all possible.

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TABLE OF CONTENTS

ABSTRACT ...... ii

ACKNOWLEDGEMENTS ...... iii

TABLE OF CONTENTS ...... iv

LIST OF TABLES ...... vii

LIST OF FIGURES ...... viii

LIST OF ABBREVIATIONS ...... x

Chapter 1.0: Introduction ...... 1

1.1 Myelin architecture and Myelin Basic Protein (MBP) ...... 1 1.2 Multiple Sclerosis (MS) and Myelin Basic Protein...... 3 1.3 Myelin basic protein in health and disease...... 4 1.4 Post-translational modifications on MBP in MS pathology ...... 5 1.5 The Intrinsically-Disordered Protein (IDPs) Family ...... 6 1.6 MBP is an IDP and has alpha-helical propensity ...... 7 1.7 The recombinant MBP 18.5-kDa isoform ...... 8 1.8 TFE-Induced secondary structural changes ...... 14 1.9 Fluorescence and Fluorescence Anisotropy ...... 15 1.10 Fluorescence Resonance Energy Transfer (FRET) and its use in IDPs ...... 17 1.11 Measuring the ‘disorder-to-order’ transition(s) of MBP in TFE in order to probe the ‘major thermodynamic ensemble populations’ ...... 18

Chapter 2.0: Experimental Plan ...... 20

2.1 Hypotheses...... 20 2.2 Objectives ...... 20 2.2.1 Develop a labelling protocol ...... 20 2.2.2 Compare using CD and anisotropy ...... 21 2.2.3 Map intramolecular distances ...... 21 2.2.4 Combined analysis ...... 21 2.2.5 Mapping intramolecular distances in order to identify the ensemble populations ...... 21 iv

2.2.6 Structural and thermodynamic comparison ...... 22 2.3 Significance ...... 22 2.4 Statement of Publication ...... 22 Chapter 3.0: Experimental Methods ...... 23

3.1 Chemicals and Reagents ...... 23 3.2 Recombinant murine full-length untagged 18.5-kDa MBP-UTC1 and MBP-UTC8 ...... 23 3.3 Labelling of Cysteine-substituted MBP-UTC1 and MBP-UTC8 variants with IAEDANS ...... 26 3.4 Circular Dichroism Measurements...... 27 3.5 Fluorescence measurements ...... 28 3.6 Steady-state Fluorescence Anisotropy Measurements ...... 28 3.7 ANS fluorescence ...... 28 3.8 Fluorescence resonance energy transfer measurements ...... 29 3.9 TFE-titration curves monitored by tryptophan fluorescence quenching ...... 30 3.10 Quantitative analysis of TFE-titration curves ...... 30

Chapter 4.0: Results ...... 33

4.1 MBP Site-directed mutagenesis ...... 33 4.2 MBP purification ...... 33 4.3 MBP labelling with IAEDANS ...... 36 4.4 Determining MBP labelling efficiency ...... 37 4.5 Circular Dichroism Measurements of MBP ...... 37 4.6 ANS fluorescence in MBP ...... 42 4.7 Steady-state Fluorescence Anisotropy Measurements of MBP ...... 47 4.8 Fluorescence resonance energy transfer (FRET) measurements of MBP ...... 47 4.9 Measuring the ‘disorder-to-order’ conformational transition(s) of MBP in TFE ...... 52 4.10 The thermodynamic analysis of the ‘disorder-to-order’ transition of MBP in TFE in order to probe the ‘ensemble populations with different free energies’ ...... 52 4.11 Deconvolution of the major thermodynamic ensembles along the ‘disorder-to-order’ transition of MBP ...... 57 Chapter 5.0: Discussion ...... 63

5.1 MBP-UTC1 in solution ...... 63

5.2 Membrane mimetic solvents and their uses in IDPs ...... 65 5.3 Thermodynamic analysis of the disorder-to-α-helical transition in full-length 18.5-kDa MBP- UTC1 using 2,2,2-trifluoroethanol (TFE) ...... 67 5.4 Defining a physiological role for MBP-UTC8: A structural and thermodynamic comparison of the MS-MBP isoform ...... 68 Chapter 6.0: Conclusions, Significance and Future Directions ...... 70

References ...... 72

LIST OF TABLES

Table #1A and #1B. Steady-state fluorescence anisotropy (r) measurements of MBP-UTC1 (A) and MBP-UTC8 (B)...... 45

Tables #2A and 2B. Fluorescence resonance energy transfer (FRET) of MBP labelled with IAEDANS ...... 49

Table #3. FRET measurements in 6 M guanidinium chloride LIST OF TABLES (guanidine hydrochloride, GdmCl)...... 51

Table #4. Thermodynamic values were determined at 25 °C from global fitting of TFE titration curves monitored by changes in CD signal and Trp fluorescence quenching by IAEDANS...... 59

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LIST OF FIGURES

Figure Title Page

#1 The structure of myelin in the CNS and the primary function of 18.5-kDa 12 MBP

#2 Notable secondary structural elements of 18.5-kDa MBP 19-20

#3 Simple Jablonski diagram of three-state model of fluorescence 24

Amino acid sequence and secondary structural map of murine 18.5-kDa #4 42 MBP-UTC1 and MBP-UTC8 (168 residues)

#5 Purification of MBP-UTC1 and MBP-UTC8 43

Changes in circular dichroic (CD) spectra of 18.5-kDa MBP with #6 47 increasing trifluoroethanol (TFE) concentration

Comparison of TFE-titration curves of IEADANS-labelled MBP-UTC1- #7 48 S17C and S159C 18.5-kDa MBP with the unmodified protein

Circular dichroic (CD) spectra of unmodified 18.5-kDa MBP-UTC18 #8 49 and MBP-UTC IAEDANS-labelled cysteine variants.

Change in ANS fluorescence enhancement by 18.5-kDa MBP upon TFE #9 51 titration

Change in ANS fluorescence enhancement by 18.5-kDa MBP-UTC8 #10 52 upon TFE titration

#11 TFE-titration curves of full-length 18.5-kDa MBP 59

#12 Comparison of TFE-titration curves of IEADANS-labelled S17C and 60

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S159C 18.5-kDa MBP-UTC1 with the unmodified protein

#13 Preliminary TFE-titration curves of full-length 18.5-kDa MBP-UTC8 61

Fractional population of equilibrium conformers of MBP-UTC1 and #14 65 MBP-UTC8.

Thermodynamic mechanism of disorder-to-α-helical transition of MBP #15 66-67 and fractional population of equilibrium conformers

LIST OF ABBREVIATIONS

Abbreviation Term

α-helix-containing peptide fragments of murine 18.5-kDa MBP: α1, α2, α3 segments (A22-K56), (S72-S107), (S133-S159), respectively.

ANS anilinonaphthalene-1-sulfonic acid

AP action potential

AU arbitrary units

BCA bicinchoninic acid

CD circular dichroism

CNS central nervous system

DNA deoxyribonucleic acid

DPC dodecylphosphocholine

E. coli Escherichia coli

EAE experimental autoimmune encephalomyelitis

EPR electron paramagnetic resonance

FPLC fast protein liquid chromatography

FRET fluorescence (or Förster) resonance energy transfer

FTIR Fourier transform infrared golli gene in the oligodendrocyte lineage

GdmCl guanidinium chloride

HPLC high-performance liquid chromatography

IAEDANS 5-((((2-iodoacetyl) amino) ethyl) amino) naphthalene-1-sulfonic acid

IDP intrinsically-disordered protein

IPL intraperiod line

IPTG isopropyl β-D-thiogalactopyranoside kDa kilodaltons

LB Luria-Bertani

MAP mitogen-activated protein

MBP myelin basic protein (usually the 18.5-kDa size isoform)

MDL major dense line

MoRF molecular recognition fragment

MS Multiple Sclerosis

NaCl salt, sodium chloride

NMR nuclear magnetic resonance

PAD-2 peptidylarginine deiminase-2

PCR polymerase chain reaction

PLP proteolipid protein

PTM post-translational modifications

recombinant murine MBP (generic designation, usually with C-terminal rmMBP LEH6 tag yielding 176 residues)

SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis

TCEP Tris-2-carboxyethyl-phosphine

TEM transmission electron microscopy

TFA trifluoroacetic acid

TFE trifluoroethanol

UTC1 unmodified C1 charge component of MBP (untagged, 168 residues)

pseudo-deiminated C8 charge component of MBP (untagged, 168 UTC8 residues)

“untagged” recombinant variants of 18.5-kDa murine MBP, unmodified UTC1, UTC8 and pseudo-deiminated, respectively

UV ultraviolet

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Chapter 1.0: Introduction

1.1 Myelin architecture and Myelin Basic Protein (MBP)

The myelin sheath is a distinctive membrane complex formed when multiple spiral- wrapped lamellae surround neuronal axons. Glial cells known as oligodendrocytes produce myelin in the central nervous system (CNS), which includes the spinal cord and brain. The oligodendrocyte membrane extends towards and makes physical contact with the axon and initiates myelin sheath formation, concentrically winding itself onto an axon (1, 2).

The myelin sheath is important for proper neuronal function, as its integrity determines the speed at which action potential(s) (APs) can propagate (3, 4). Myelin itself has historically been viewed as an axonal insulator, working to limit the dissipation of ion current away from ion channels, and limiting depolarization events to the nodes of Ranvier (5). These nodes remain uninsulated and, due to the abundance of voltage-gated ion channels located within them, are capable of generating vast amounts of electrical activity. These nodes are spaced approximately 1 µm in distance away from each other, and thus AP conduction and propagation along the neuron “hops” or “jumps” from node to node: this mechanism of AP conduction and propagation along a myelinated nerve is known as saltatory conduction (6, 7). The process of myelination is thought to have facilitated fast and efficient communication in the brain, giving rise to animals with complex motor, sensory, and cognitive functions (8). Any disruption of the myelin sheath integrity compromises the ability of nerves to propagate APs effectively, and leads to impaired neurological function, where symptoms are highly correlated with the areas of the brain that are affected (5, 7, 9).

Figure #1 shows the structure of myelin sheath itself, composed of repeating lipid bilayers separated by 3 to 4 nm aqueous layers (10) and forms distinct regions: the intraperiod line (IPL) formed by the apposition of extracellular membrane layers, and the major dense line (MDL) formed by the apposition of the cytoplasmic membrane surfaces (11). The MDL contains a cytosolic, peripheral membrane protein, known as Myelin Basic Protein (MBP) which composes up to 30% of the dry weight of the total protein in myelin, making it the second most abundant myelin protein, following proteolipid protein (PLP) (3, 12, 13). 1

Figure #1. The structure of myelin in the CNS and the primary function of 18.5-kDa MBP. The myelin sheath is a distinctive membrane complex formed when multiple spiral-wrapped lamellae surround neuronal axons of nerve fibers. Glial cells known as oligodendrocytes produce myelin in the central nervous system (CNS), which includes the spinal cord and brain. The myelin sheath is composed of repeating lipid bilayers separated by 3 to 4 nm aqueous layers and forming distinct regions: the intraperiod line formed by the apposition of extracellular membrane layers and the major dense line formed by the apposition of the cytoplasmic membrane surfaces. A cytosolic, peripheral membrane protein, known as myelin basic protein (MBP), comprises up to 30% of the dry weight of protein and is present in the major dense line. The predominant 18.5-kDa isoform of MBP maintains myelin structure by conjoining the cytoplasmic leaflets of the oligodendrocyte membrane. MBP is the 'executive molecule' and the only structural protein essential for formation and maintenance of normal myelin. Adapted with permission from Ishiyama and colleagues (2001).

When discussing MBP, it must be clarified that it is not one single protein, but a large family of protein isoforms derived from alternative splicing events of the Golli (gene in the oligodendrocyte lineage) gene complex, with the common human MBP splice isoforms being 17.2, 18.5, 20.2, and 21.5 kDa in nominal molecular mass (3). These protein isoforms are developmentally-expressed, translocatable, and highly post-translationally modified, and show a diversity of binding partners (3). Historically, the 18.5-kDa MBP isoform’s role has been thought to maintain the structural integrity of the myelin sheath via adhesion to cytoplasmic leaflets of the oligodendrocyte membrane. But today, it is known that MBP is highly multi-functional, serving as a 'hub' protein within myelin. Thus, in addition to its main role of maintaining the structural integrity of myelin via interactions with bilayers and/or lipids (14), common roles for the 18.5-kDa MBP isoform include chemical signalling (15); cytoskeletal assembly (16); interactions with Cu2+ and Zn2+ (17, 18), with calcium-activated calmodulin (19), and with SH3- domain containing proteins (20-22). As the focus of this thesis and the experimental work has been performed on this classical 18.5-kDa MBP isoform, it will henceforth and throughout be referred to as MBP.

1.2 Multiple Sclerosis (MS) and Myelin Basic Protein

Multiple sclerosis (MS) is a debilitating disease directed against myelin and the myelin components in the CNS (3, 23). MS is characterized by inflammation, demyelination, lesion formation, and progressive neurodegeneration (24). Progressive deterioration of myelin causes impaired saltatory conduction resulting in increasing loss of neurological function with symptoms including cognitive dysfunction, depression, spasticity, muscle weakness, and paralysis (24).

Schools of thought on the origin of MS typically involve the degeneration (or failure to regenerate) of the insulating layer around neuronal axons, and generation of auto-antigens against components of the CNS myelin, specifically MBP which is a well-known candidate auto- antigen in MS (25-27). Although MS is synonymous with auto-immune disease, the exact causative mechanism remains elusive (3, 27-29). Increasing experimental evidence suggests that a genetic contribution manifested by the up-regulation of enzymes that govern specific post- translational modifications (PTMs) of MBP may be involved (30-33). The mechanisms through 3

which post-translational modifications and interacting partners alter the structure of MBP require further investigation (3).

1.3 Myelin basic protein in health and disease

MBP maintains myelin structure by conjoining the cytoplasmic leaflets of the oligodendrocyte membrane and linking the membrane to the underlying cytoskeleton, including the cytoskeletal proteins actin and tubulin (34), whose assembly MBP strongly enhances (16). Deletion or mutation of the mbp gene causes the loss of compact CNS myelin as was shown in the shiverer mice (35), and the shaker rat (36). As the 'executive molecule' and only structural protein essential for formation and maintenance of normal myelin, the loss of MBP, or change in MBP’s interaction with the bilayer, can have dramatic physiological and/or neurological effects (3, 37).

The association between MBP and oligodendrocyte membranes is thought to be dictated by hydrophobic and electrostatic forces working in cooperation to maintain the interactions between MBP residues and anionic lipids of the myelin bilayer (38). Alterations made to these hydrophobic or electrostatic interactions between MBP and myelin layers results in reduced adhesion and sheath instability (39).

Some post-translational modifications of MBP, specifically deimination (conversion of arginine to citrulline, resulting in a decrease in the net positive charge) is highly correlated with MS pathology (32, 40, 41) with the degree of arginine lost by deimination being around 45% in chronic MS, and 20% in normal healthy adult brain (42). But more relevantly, highly deiminated MBP is thought to be ‘less structurally ordered’ and also shows less affinity to maintain binding membrane lipids (40, 43). Yet, there is still a poor understanding of the role that deimination plays in MS pathogenesis, and the mechanisms through which PTMs and interacting partners alter the structure of MBP require further investigation (3).

1.4 Post-translational modifications on MBP in MS pathology

Recently, bioinformatics analysis has suggested that proteins with post-translational target sites that are great in number and variation, such as MBP, are more prone to be involved in disease (44). PTMs have also long been known to alter the nature and extent of secondary structure in proteins, permitting them to adopt various conformations required for binding and signalling events, as is seen in proteins such as tau and α-synuclein which are very similar to MBP in such properties (45, 46). The PTMs of MBP include methylation, deimination, deamidation, and phosphorylation (3), and give rise to a multitude of charge components and potentially a vast number of conformers (31, 47-49).

More specifically, the enzyme peptidylarginine deiminase type 2 (PAD2) is known to cause a highly relevant PTM of MBP that is associated with MS (31, 32, 50). Citrullination causes the irreversible conversion of the amino acid arginine into citrulline through replacement of the terminal ketamine group (=NH) on arginine with a ketone group (=O) by PAD. Deimination has been shown to alter the electrostatic and hydrophobic interactions between citrullinated residues and negatively-charged lipid head groups (3, 52) work to decrease MBP’s ability to interact with and organize lipid bilayers into compacted myelin layers (53). Deimination of MBP is thought to represent the primary defect that precedes demyelination and progressive neurodegeneration that is seen in MS pathology (3, 12). In MS research in our laboratory, we distinguish between two major charge components: MBP-UTC1 which is the least modified and most cationic variant of MBP with a net charge of +19 at neutral pH; and MBP- UTC8 which represents a more highly modified form and differs by deimination of 6 to 11 arginyl residues, resulting in a decrease of net-positive charge to +13 at neutral pH (32, 51). The MBP-UTC8 variant is associated with MS pathology as well as with myelin development (12).

Electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) studies suggest that deimination results in a loss of structural integrity of an intrinsic α-helix formed by MBP (discussed below). This PTM causes the α-helix to become shorter in length, and more surface-exposed when bound to the cytoplasmic leaflet of the myelin membrane, and is thus more susceptible to proteolysis as compared to MBP-UTC1 (52, 54). Other EPR studies on MBP-UTC1 and MBP-UTC8 determined that MBP-UTC8 formed a shorter, more surface- 5

exposed α-helix in MBP-UTC8 as compared to MBP-UTC1 (52), which nicely complemented studies using myelin-associated proteases such as cathepsin D, which showed an increase in the ability to digest MBP-UTC8 (3, 43). All in all, the specific effect of deimination of MBP-UTC1 to yield MBP-UTC8 results in a conformational change that leads to the cytoplasmic exposure of a Phe86-Phe87 pair making the α-helix more vulnerable to proteolytic attack and digestion (52). Therefore, the effect(s) of deimination on the structure of MBP, and the resultant conformational changes that lead to the exposure of this 'molecular switch' may define a new pathological role for MBP in MS (55-58). Furthermore, this model both complements and challenges the historical dogma of MS research, suggesting that MS may be in fact be a post-translational disease and that PTMs may represent the primary defect that leads to MS pathology (33, 59). But as discussed below, characterizing MBP’s structure, or the effects of PTMs and the resultant conformational changes, have historically been somewhat challenging.

1.5 The Intrinsically-Disordered Protein (IDPs) Family

MBP’s structure continues to elude researchers largely because MBP is not any one single protein. A central tenet of biochemistry, the so-called structure-function paradigm, states that having a well-defined 3D structure is a prerequisite for the proper function of proteins (60). Traditionally, proteins are characterized by a rigid, well-folded, three-dimensional structure, defined by a single set of backbone side-chain atom positions and dihedral angles, represented by an energetically-minimal assembly where limited fluctuations of conformation occur (61, 62). This interpretation of protein structure-function relationships revolves around two main principles. The first suggests that the network of enzymatic, protein-protein, and/or protein- nucleic acid interactions inside the cell would not occur unless proteins act in a highly "specific" manner (63-66). Secondly, the "lock-and-key" concept, used to understand specificity of ligand action, states that without a well-defined geometry and the implied rigidity, particularly in regions of molecular binding, specificity of interactions could not be assured (63-66).

Today, it is understood that proteins can exist in a trinity of structures (namely the ordered, molten globule, and random coil states (60)), that native protein structure and function can correspond to any of the three states, and finally, that intrinsically-disordered proteins (IDPs) are distinguished by their incapacity to adopt a well-defined structure under near-physiological 6

conditions (65). Most IDPs typically show low hydrophobicity, high net charge, and exist as dynamic, conformational ensembles where dihedral angles and atom arrangements vary significantly over-time, therefore attaining no equilibrium (66). Thus, IDPs lack the specific three-dimensional structure required for crystallography (67, 68). It was not until the late 1990's that this central tenet of biochemistry was challenged as more advanced methodologies for analysis allowed the discovery of functional proteins which were either completely or partially disordered.

In 2013, the database of protein disorder (Disprot) listed over 694 proteins, with 1539 disordered regions (69). Amino acid sequence analysis has suggested disorder is an encoded property and software packages predict that 45-50% of eukaryotic proteins contain partially, or completely, disordered regions in solution (70). Disordered proteins are now known to be functionally important, partaking in cellular processes such as signalling, transcription, replication, cell-cycle control, chaperoning, and more importantly, have been associated with various human pathologies, including multiple neurological disorders (71, 72).

Intrinsic disorder is advantageous as it can allow for rapid association with one or multiple binding partners, in response to alterations in environmental or chemical signals (73). Consequently, IDPs can form a large number of interactions, acting as network hubs, which allow protein-protein, protein-ligand, and protein-membrane interactions that facilitate the transmission of information across subcellular systems (49, 66, 73). These associations remain fairly specific as the energy of secondary and/or tertiary folding can act as a barrier to interaction(s) (60).

1.6 MBP is an IDP and has alpha-helical propensity

All variants of MBP are IDPs (3). Previous Fourier-transform infrared (FTIR) (74) and NMR (75) studies of recombinant 18.5-kDa MBP have shown that MBP does not adopt a significant proportion of regular secondary structure elements in aqueous solution (76). But whereas the behaviour of MBP is consistent with a random-coil conformation, the central region is known to have some degree of ordered secondary structure (77). MBP has demonstrated the potential to adopt long-range folding interactions, thought to be critical for compact assembly of

myelin (3). Hydrophobic moment analysis of the amino-acid sequence of MBP (78), and solution/solid state nuclear magnetic resonance (NMR) spectroscopy, demonstrate the existence of three amphipathic α-helices located in the N- and C-termini and in the central regions of the protein (78, 79). Regions with high α-helical propensity in MBP include: Thr33-Asp46, Val83- Thr92, and Try142-Leu154, designated as α1, α2, and α3, respectively (79-81) (Figure #2A and #2B). Depth measurements of unmodified MBP (MBP-UTC1) in simple lipid systems, via spin labelling and electron paramagnetic resonance (EPR) spectroscopy, have confirmed that these regions are embedded in the lipid membrane and have α-helical properties (82).

The α-helical regions, and more specifically the central region, which contains α2 followed by a poly-proline sequence, is present in almost all vertebrates, with residues Asp81- Gln118 being highly conserved in mammals (78). This region also contains mitogen-activated protein (MAP)-kinase sites located at T92 and T95 (murine 18.5-kDa sequence numbering), and includes the immunodominant epitope (murine N81-PVVHFFKNIVTPRTPPP-S99) used to induce EAE (experimental autoimmune encephalomyelitis) in model animals, and so may also act as a possible "molecular switch" (83). Characterizations of α2 and the molecular switch regions have been performed in aqueous buffer, in membrane-mimetic environments such as 2,2,2-trifluoroethanol (TFE) (83), and in dodecylphosphocholine (DPC) micelles (84-86). Generally, PTMs (with net positive charge reduction) have the potential to modify MBP electrostatic interactions with its binding partners, which result in conformational changes in both the molecular switch and/or α-helical regions. These changes are, in turn, thought to cause the destabilization of myelin sheath, as seen in EAE and MS (85).

1.7 The recombinant MBP 18.5-kDa isoform

The recombinant murine MBP (rmMBP) 18.5-kDa protein has been demonstrated as a successful substitute to purifying the protein from natural sources (87). Recombinant proteins from a bacterial over-expression system can sometimes be misrepresentative of a protein from a natural source for two main reasons: (1) if the recombinant protein is not biochemically representative of its natural counter-part, and (2) the normal eukaryotic post-translational modifications are absent (87, 88). With respect to the first point, there is no known functional assay to determine the structural integrity of MBP (3, 89), and therefore the structure of 8

recombinant murine MBP variants has typically been characterized by spectroscopic techniques such as nuclear magnetic resonance (NMR) (78-80), FTIR (74), circular dichroism (CD) (90), and electron paramagnetic resonance (EPR) (19, 33, 91).

Figure #2A and #2B. Notable secondary structural elements of 18.5-kDa MBP. Three regions previously shown to have α-helical propensity (Thr33-Asp46, Val83-Thr92, and Tyr142- Leu154) are displayed as red helices. Hydrophobic moment plot of three regions previously shown to have α-helical propensity (Thr33-Asp46, Val83-Thr92, and Tyr142-Leu154; shown in part A as segments of red helix) reveals that these helical regions are strongly amphipathic. This figure was reproduced with permission from Harauz et al. (2009). (B) Probability of regular secondary structure types in His6-tagged murine recombinant 18.5-kDa MBP in 30% TFE (v/v). The plot was obtained by analyzing previously-derived Hα, HN, Cα, Cβ, and C′ solution NMR chemical shift assignments (BMRB 6100) using the δ2D software and the associated web server created by the Vendruscolo group (Camilloni et al., 2012). The regions with highest propensity for α-helical structure are at the center (encompassed here by the α2-peptide) and at the C- terminus (encompassed here by the α3-peptide). Significant propensity for α-helical structure is also seen near the N-terminus (~I35-F42, murine species numbering) (encompassed here by the α1-peptide). The regions with the highest probabilities for α-helical content in 30% TFE (v/v) correspond to regions predicted to form membrane-associated α-helices based on a hydrophobic moment plot (Harauz et al., 2009). The locations of the α-helical regions also agree with CD experiments of different MBP α-peptides, and with solution NMR experiments of the central MBP segments in a dodecylphosphocholine micelle environment. These regions were also shown in site-directed spin labelling electron paramagnetic resonance measurements to associate with the lipid bilayer in reconstituted myelin-mimetic systems.

Figure2A

Figure2B

Studies on 18.5-kDa rmMBP have determined that the recombinant protein is similar to the natural bovine 18.5-kDa isoform in terms of (1) immunoreactivity to antibodies, (2) the ability to cause experimental autoimmune encephalomyelitis (EAE) in animal model systems (87, 92), (3) the degree of secondary structure in aqueous solution via CD (90), and (4) adhesion to lipids (93). The lack of normal eukaryotic PTMs in bacterial systems is evident when looking at differences between natural bovine MBP (bMBP) and rmMBP. For instance, bMBP arginine residue 106 is either non-, mono-, or di-methylated, and the amino-terminal end of bMBP is acetylated (94, 95). Similar modifications are present in natural human MBP 18.5-kDa (3, 90, 94) but the fact that PTMs typical of eukaryotic species are absent in bacterial systems can actually be beneficial to researchers, acting as a 'tabula rasa'.

Recombinant DNA technology allows for the production of homogenous preparations of recombinant proteins (88) and pseudo-modification of amino acids by site-directed mutagenesis, which would otherwise be impossible (50, 54, 87, 88). The incorporation of probes to converted residues can then be used to study the effects of specific PTMs individually, or in a combinatorial manner, on structure or interaction with proteins, small ligands, and other interacting partners. In our studies, MBP-UTC8 refers to recombinant murine MBP modified at these 6 particular sites (the murine residues R25, R33, K119, R127, R157, and R168 correspond to primary arginine deimination sites in the C8 component of the human protein. The MBP post- translational sites were recombinantly modified to best mimic the size, as well as the charge reduction, associated with the particular PTM. The murine protein’s sites of pseudo-citrullination (5 Arg + 1 Lys, each with a net charge +1) were modified to glutamine (net charge 0). This strategy has been employed previously in our lab to study MBP PTMs, and has been cited to be effective in mimicking structural changes and modulating ligand associations seen with in vivo PTMs in other proteins (50). As these modifications are determinate for normal MBP structure and/or function, they are therefore critical for the understanding of MS pathology (3).

1.8 TFE-Induced secondary structural changes

Conformational analysis of peptides and small proteins is commonly carried out in structure-stabilizing solvents such as TFE. Experimentally, TFE induces and stabilizes α-helicity in peptides and proteins (96). TFE is a hydrophilic and hydrogen-bonding solvent (97) and can also strongly influence secondary structure and global folding of peptides and proteins. It has been demonstrated that TFE induces pre-existing helix-coil formation in proteins limited to when the effect is saturated at low concentration of TFE (<40% vol) (98), and solvation of the helix structure by TFE stabilizes the intramolecular hydrogen bonds (99, 100).

Trifluoroethanol has been shown to induce α-helical structure mainly in regions which already have α-helix-forming propensity, and it has been shown to be an effective membrane- mimetic solvent for many proteins, including MBP (75, 99). In corroboration, analysis of previously published NMR data of MBP in 30% TFE (v/v) has demonstrated that the pattern of α-helical formation in TFE is consistent with what has been found in lipid (101). At low concentrations, it has been suggested that TFE can effectively simulate membrane surface conditions by lowering the dielectric constant of the solution, whereas at higher concentrations, there is the added effect of the preferential interaction of TFE with the protein, which can effectively simulate the effects of protein-membrane binding (99, 102). Thus, TFE is useful in analyzing the disorder-to-α-helical transition of MBP that occurs as it approaches the oligodendrocyte membrane surface and eventually interacts.

TFE is also compatible with a number of techniques including fluorescence and circular dichroism (CD). CD/TFE titration curves are particularly useful as they can be used to determine the average α-helicity of peptides and small proteins (83). This method can also allow for the calculation of the solvation free energy and free energy change associated with the coil-helix transition, and defining the thermodynamics for α-helical formation of IDPs (98). Previous CD- monitored TFE-titration curves of four α2-peptides (unmodified, phosphorylated T92 and/or T95) have been performed (83); however, the effects of other typical PTMs such as deimination in this respect have yet to be determined.

1.9 Fluorescence and Fluorescence Anisotropy

Over the last two decades, fluorescence has become a standard tool for deciphering complex biological systems. Fluorescence is the release or emission of radiation produced by some molecules (fluorophores) due to the absorption of incident radiation of a shorter wavelength (103). A three-stage process responsible for the fluorescence of fluorophores is illustrated by the simple electronic-state diagram shown in Figure #3. Excitation is the process of photon absorption creating an excited electronic singlet state (S1). The S1 state is then dissipated, yielding a relaxed state from which fluorescence emission originates (104), and finally by fluorescence emission, where a photon of energy is emitted, returning the fluorophore to its ground state S0 (103). Measurable parameters of a fluorophore, such as absorption wavelength (λ), emission λ, quantum yield, fluorescence quenching, fluorescence intensity and anisotropy, can provide a wealth of information pertinent to the structure of IDPs, and specifically MBP (103, 105-107).

For example, excitation of some molecules by polarized light in one dimension (x, y, or z) subsequently causes the emission of polarized light within the same dimension (103). A molecule’s anisotropy is defined as the difference between the intensity of polarized light in the vertical direction and the intensity of polarized light in the horizontal direction, normalized to the total intensity of light (103). Fluorophores with non-zero anisotropies display changes in the dimension of polarized light emission, and the extent of this emission is defined by its relative anisotropy (r), a unit-less measure (103). Proteins in aqueous solution, particularly IDPs which are highly mobile, tumble during the excited state. Consequently, the polarized light is emitted in various directions as compared to the incident light (103, 108). The displacement of polarized light is dependent upon the speed and freedom of rotational diffusion during the lifetime of a fluorophore’s excited state (108). Rotational diffusion of a protein depends on the solvent, and the protein’s size and intrinsic shape (103, 109). Macromolecules and protein-binding interactions with various binding partners can also be measured by anisotropy as a result of changes in rotational diffusion (108, 109). Experimentally, a sample is excited by polarized light which is oriented in the vertical or the z-axis (103). The intensity of the emission is measured through a polarizer, and if emission is oriented parallel (║) to the direction of the excitation light,

Figure #3. Simple Jablonski diagram of three-state model of fluorescence. (1) Excitation is the process whereby a photon is supplied by an external source and absorbed by the fluorophore, creating an excited electronic singlet state (S1'). (2) The excited state lifetime is finite, lasting 1–10 nanoseconds (in most cases), and during this time, a fluorophore is subjected to a multitude of possible interactions with its molecular environment. The energy of S1' is partially dissipated, yielding a relaxed singlet excited state (S1) from which fluorescence emission originates. (3) A photon is emitted, returning the fluorophore to its ground state S0. Due to energy dissipation during the excited-state lifetime, the energy of this photon is lower, and therefore of longer wavelength, than the excitation photon (Figure created by Author).

the observed intensity is called I (║). If the polarizer is oriented perpendicular (┴) to the excitation light, the intensity is called I (┴) (109). These values can be used to calculate anisotropy (r), in order to infer the relative environment of a fluorescent probe as well as to determine if the probe has free-rotation or is restricted. Data will be collected for both MBP-UTC1 and MBP-UTC8 in aqueous buffer and in TFE.

1.10 Fluorescence Resonance Energy Transfer (FRET) and its use in IDPs

The realization that MBP is an IDP shifted focus to understanding the ‘binding-induced folding’ or ‘disorder-to-order transitions’ influenced by interactions with its cellular partners (zinc, salt, etc.) as reviewed in (94), or/and in the presence of membrane-mimetic environments such as TFE or synthetic lipid(s) (39, 110). As mentioned previously, multifunctionality in IDPs such as MBP is related to their conformational plasticity and their ability to undergo disorder-to- order transitions. It is important to understand the mechanism of these structural conversions (47). As the organization of the protein conformation may already start forming “in solution” before it interacts fully with the two leaflets, MBP therefore has the potential to interact with various cellular partners before being inserted into the membrane (111). It is, therefore, important to obtain more information on the changes in ensemble conformation of MBP; as such, FRET will be performed in a series of environments.

Fluorescence (or Förster) resonance energy transfer (FRET) can be, and has been, employed for the determination of long-range interactions between amino acid residues in IDPs (46, 106, 112), often exploiting the intrinsic tryptophan residue(s) of proteins as the donor, and the selection of a suitable fluorescent probe as an acceptor (46, 113). The FRET approach is uniquely suited to the study of IDP structure, and by extension MBP’s structure, as the technique has high binding specificity, accuracy at angstrom (Å) level distances and/or molecular dimensions, and the ability to resolve distributions of intramolecular distances of fast fluctuating disordered protein(s) regions (106). Minimally, FRET requires that: (1) the absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor, (2) the donor and acceptor molecules must be in proximity (typically 10–100 Å), and (3) donor and acceptor transition dipole orientations must be roughly parallel (103, 113, 114). The FRET involves the distance-dependent irradiative transfer of energy from a donor molecule to an acceptor molecule 17

via a dipole-dipole coupling mechanism (113). The donor molecule is the chromophore that initially absorbs energy when excited by photons and which subsequently transfers energy to an acceptor chromophore (46, 113) Energy transfer efficiency is determined by the ratio between donor fluorescence intensity in the presence and absence of acceptor, which can then be used to determine the physical distance between the donor and acceptor (114). Previous studies have demonstrated that fluorescence techniques, including FRET, are well-suited to study the conformational ensemble of MBP caused by PTMs in aqueous buffers, in membrane-mimetic solvents such as TFE, in protein denaturants, and, finally, in the presence of small-interacting ligands such as zinc and NaCl (46, 103, 113, 115). For the reasons outlined above, it is clear that FRET is a natural complement to previous studies that have been conducted by the Harauz group on MBP using NMR, EPR, and CD spectroscopy.

1.11 Measuring the ‘disorder-to-order’ transition(s) of MBP in TFE in order to probe the ‘major thermodynamic ensemble populations’

As part of an integration of multiple sources of data, it is also possible to define the mechanism of the disordered-to-α-helical transition of MBP. This can be accomplished by thermodynamic analysis of the disordered-to-α-helical transition that is induced upon titration with TFE. It is important to monitor conformational changes using multiple probes specific for secondary and/or tertiary (or global) structural changes. In this study, this analysis will be performed by CD spectroscopy, which probes for changes in secondary structure, and will also be monitored by tryptophan fluorescence quenching by IAEDANS, which can be used to probe for concomitant changes in global organization. By utilizing combinations of techniques, including circular dichroism (CD) spectroscopy, tryptophan fluorescence quenching, and 1- anilinonaphthalene-8-sulfonic acid (ANS)-binding, it is possible to define the ‘major thermodynamic clusters of ensemble populations’ along the transition pathway, as previously done with other prominent IDPs, such as tau and α-synuclein (46, 115, 116),

MBP likely undergoes global conformational changes resulting in the population of various energetically distinct sets of conformational ensembles which are regulated by PTMs, as has been seen for a variety of IDPs (46, 117), each of which potentially having varying levels of compaction and secondary structure. A full thermodynamic description of the mechanism of

disorder-to-order transition is useful both for defining the protein’s conformational space which may be important in binding-induced folding mechanisms, and also for identifying populations of energetically distinct conformations along the transition pathway which may be instrumental in conformational selection.

Chapter 2.0: Experimental Plan

2.1 Hypotheses

First, we posit that the MBP-UTC1 variant adopts a hairpin or paperclip shape in the presence of membrane-mimetic solvents, as observed in previous studies of other IDPs similar to MBP, like tau (46, 115). This conformation is important for close apposition of oligodendrocyte membrane bilayers, yet MBP is conformationally adaptive and is subject to change upon interaction with its various binding partners, and in the presence of different solvent systems, which is a key property for its multifunctionality.

Second, we hypothesise that deimination of MBP associated with charge reduction will alter the global structure of the MBP-UTC8 variant. In particular, we believe that the extensive deimination of MBP-UTC1 (+19) to MBP-UTC8 (+13) will result in different long-range intramolecular folding interactions. Subsequently, this conformational change in MBP may lead to the destabilization of the compact myelin sheath and to the degeneration seen in MS.

Third, we suggest that the multifunctionality seen in MBP could arise from differences in the population of energetically distinct ensemble clusters in different conditions which can be measured as energetically distinct states.

Finally, we hold the expectation that if pseudo-deimination in MBP-UTC8 causes global structural changes as compared to MBP-UTC1, there will be also a measurable thermodynamic difference in the population of energetically distinct ensemble clusters between MBP-UTC1 and MBP-UTC8 (47).

2.2 Objectives

2.2.1 Develop a labelling protocol

Using single cysteine mutants of the MBP-UTC1 and MBP-UTC8 variants, we will develop a labelling protocol with the fluorescent probe 5-((((2-iodoacetyl) amino) ethyl) amino) naphthalene-1-sulfonic acid (1, 5-IAEDANS, henceforth just IAEDANS, for simplicity) to create FRET pairs with the intrinsic single tryptophan at position 113.

2.2.2 Compare using CD and anisotropy

We will compare IAEDANS-labelled MBP-UTC1 and MBP-UTC8 using CD spectroscopy and fluorescence anisotropy measurements to ensure that site-directed mutagenesis and label incorporation did not alter MBP-UTC1 and MBP-UTC8 variant secondary structural propensities.

2.2.3 Map intramolecular distances

We will map intramolecular distances using fluorescence resonance energy transfer (FRET) in full-length MBP-UTC1 and MBP-UTC8 variants, with the goal of determining the location, flexibility, and distribution of various MBP domains in aqueous buffer, TFE, NaCl, and 6 M guanidinium chloride, and in the presence of zinc. These distances will complement our previous EPR, NMR, FTIR, and CD studies of MBP-UTC1 and MBP-UTC8 variants, and will facilitate the understanding of MBP topology in the myelin sheath.

2.2.4 Combined analysis

Using a combined analysis of CD spectroscopy, ANS fluorescence, and FRET we aim to determine the minimal applicable mechanism of disorder-to-α-helical-transition of full-length 18.5-kDa MBP. This will be performed on both MBP-UTC1 and MBP-UTC8. We also hope to determine the free energy change associated with the coil-helix transition of MBP. This transition will be induced through titration of the membrane-mimetic solvent trifluoroethanol (TFE) into protein solutions. A full thermodynamic description of the mechanism of disorder-to- order transition is useful both for defining the protein’s conformational landscape, which may be important in binding-induced folding mechanisms.

2.2.5 Mapping intramolecular distances in order to identify the ensemble populations

We hope to identify populations of distinct conformations along the transition pathway which may be instrumental for understanding the conformational landscape of MBP.

2.2.6 Structural and thermodynamic comparison

We hope to draw a structural and thermodynamic comparison of MBP-UTC1 and MBP- UTC8 in order to better define a physiological role for these isoforms.

2.3 Significance

This work will allow us to define more precisely the role of MBP in the underlying molecular architecture of CNS myelin, including MBP polymorphism and modifications linked to multifunctionality. In addition, understanding the molecular basis of induction of demyelination, and failure to remyelinate in MS, will aid development of therapeutic strategies for MS.

2.4 Statement of Publication

Parts of this thesis work have been published and several figures are reproduced with permission as noted (K.A. Vassall, A.D. Jenkins, V.V. Bamm, G. Harauz. “Thermodynamic analysis of the disorder-to-α-helical transition of 18.5-kDa myelin basic protein reveals equilibrium intermediate representing the most compact conformation”. Journal of Molecular Biology, volume 427, pp 1977-1992, 2015, doi: 10.1016/j.jmb.2015.03.011).

Chapter 3.0: Experimental Methods

3.1 Chemicals and Reagents

The reagents 5-((2-[(iodoacetyl)amino]ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) and 1-anilinonaphthalene-8-sulfonic acid (ANS) were obtained from Invitrogen Life Technology (Burlington, ON, Canada). All other chemicals were reagent grade and acquired from either Fisher Scientific (Unionville, ON, Canada), or Sigma-Aldrich (Oakville, ON, Canada).

3.2 Recombinant murine full-length untagged 18.5-kDa MBP-UTC1 and MBP-UTC8

Seven MBP variants containing single cysteine substitutions (S17C, S44C, S67C, H85C, S99C, S129C, and S159C) were produced for each MBP charge variant. Targets for mutagenesis were chosen as in previous EPR studies (118), and were constructed by the polymerase chain reaction (PCR) with site-directed mutagenesis using the QuickChange® protocol (Roche- Stratgene, La Jolla, CA). Previously described expression vector(s) pET22b (+) MBP-UTC1 and MBP-UTC8 were used as the template DNA (87). Primers were supplied by the Guelph Molecular Supercenter (University of Guelph, ON, Canada) and were supplied at a minimum concentration ~0.400 nmole/µL. Mutant strand synthesis was performed using a BioRad (ON, Canada) thermal cycler PCR system. The PCR mutant plasmid products were subjected to digestion by the restriction enzyme FastDigest DpnI (Fermentas, Ottawa, ON) to remove parental DNA. A volume of 1 µL of DpnI (10 units) was added to 20 µL of each of the PCR products, and was placed in a 37°C water bath for 2 hours. Following digestion, plasmid DNA was stored at -20°C.

Mutant plasmids were transformed via heat shock into competent E. coli-DH5α cells. An amount of 5 µL of the digested PCR product was added to competent E. coli-DH5 cells and incubated on ice for 5 minutes. The cells were then transferred to a 42°C water bath for 45 seconds and then placed back on ice for another 2 minutes. Then, 300 μL of LB media was added and the mixture was incubated at 37°C with shaking for 1 hour. The entire volume of each mutant culture was plated on agar with 50 µg/mL ampicillin. Plates were incubated overnight at 23

37°C. Both positive and negative control plates were used. Selected E. coli-DH5α colonies from the ampicillin agar plates for each mutant were inoculated in 10 mL of LB media (+50 µg/ mL ampicillin) and were incubated with shaking for ~17 hours at 37°C. A miniprep plasmid extraction kit (Roche Diagnostics, Laval, Quebec) was used according to the provided protocol for the small-scale extraction of plasmid DNA. The concentration of plasmid DNA was determined using a POLARstar Omega® (BMG Labtech, NC, U.S.A.) micro-plate reader. The amount of DNA was quantified by absorbance at 260 nm (A260) using the following equation: [DNA] = A260 / (ε x l), where (ε) is the extinction coefficient (mL mg-1 cm-1) and (l) is the path length (cm). The ε coefficient used was 50 mL mg-1 cm-1 and the path length was 1 cm. A volume of 10 µL of each mutant plasmid was collected and sent for sequencing (Guelph Molecular Supercenter, University of Guelph, ON).

Mutant plasmids were transformed into E. coli-BL21-Codon Plus (DE3)-RP cells (see Cloning Techniques). Then 300 µL of LB media was added and cells were incubated at 37°C with shaking for 1 hour. The cell cultures were added to a 10 mL LB media with 50 µg/mL ampicillin and 34 µg/mL chloramphenicol, and incubated at 37°C with shaking for ~14 hours. Each culture was then added to a flask containing 150 mL of LB broth with 50 µg/mL ampicillin and 34 µg/mL chloramphenicol. The cells incubated at 37°C with shaking until the optical density at 600 nm (A600) was between 0.6 - 0.8 (arbitrary units, a.u.) as measured by an Ultraspec-2000® (Pharmacia-Biotech, QC, Canada) with LB broth as a reference. Cultures were transferred into centrifuge bottles and spun at 2000 rpm (Beckman-Coulter Rotor JA-10) for 15 minutes at 20°C. Supernatant was disposed. Cell pellets were re-suspended into 1.5 L of M9 media with additions of 3 mL of 1 M MgSO4, 150 µL of 1 M CaCl2, 1.5 mL of 50 µg/mL ampicillin, 1.5 mL of 34 µg/mL chloramphenicol, and 30 mL of 20% glucose solution.

The M9 cultures were incubated at 37°C with shaking until an A600 = 0.7 - 0.9 (A.U.) was achieved. A 1-mL negative-induction sample was taken for each variant. Protein induction was accomplished with the addition of 1.5 mL of 0.238 g/mL isopropyl β-D-1-thiogalactopyranoside (IPTG), and cultures were incubated for ~5 hours. Then 1-mL positive-induction samples were taken for each variant. The entire cell culture of 1.5 L for each variant was pelleted by centrifugation at 7000 rpm (Beckman-Coulter Rotor JA-10) for 15 minutes at 4°C. The pellet

was frozen at -20°C for storage. Negative and positive induction samples were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

Cell lysis was performed by re-suspension of each variant cell pellet into 60 mL of lysis buffer (8 M urea, 100 mM NaH2PO4, 500 mM NaCl, 10 mM Tris-base, 10 mM β- mercaptoethanol, 1% v/v Tween-20, pH 8.0) and homogenization. The lysate was frozen at - 80°C, thawed, and further stirred at 20°C for 1 hour. Lysate was then spun at 15,000 rpm using a JA-25.5 Beckman-Coulter rotor for 20 minutes at 4°C. Supernatant was collected and dialyzed for more than 5 hours against 2L IEX buffer (6 M urea, 80 mM glycine, pH 10.0) using 6000- 8000 Da molecular weight cut-off (MWCO) dialysis tubing.

Purification of MBP variants was achieved by fast protein liquid chromatography (FPLC) using a BioRad-DuoLogic® apparatus and a 1-mL Pall AcroSep® cation exchange column. The IEX buffer filtered through a 0.45-µm filter composed the mobile phase. Detection was performed at 280 nm, the flow rate was 1 mL/minute, and the column temperature was 4°C. A linear gradient of salt in IEX buffer (0-500 mM) was developed over the total volume of 50 mL with a flow rate of 2 mL/minute. The 1.5-mL fractions were collected automatically. Pure fractions were observed and confirmed by SDS-PAGE using a standard protein ladder. Isolated fractions then were transferred into 6000-8000 Da dialysis tubing and dialyzed against 2 x 2L of Buffer A (50 mM Tris-base, 250 mM NaCl, pH 7.4), 2 x 2L of Buffer B (100 mM NaCl), and 4 x 2L distilled water. Buffer changes were made every ~5 hours. Following dialysis, the variant proteins were collected and lyophilized.

After ion exchange chromatography, the MBP single cysteine-substituted variants were treated with a 10-fold molar excess of β-mercaptoethanol for 20 min at 25°C, in order to ensure that all cysteine residues were reduced. Finally, unmodified MBP as well as the cysteine variants were further purified by reversed-phase high-performance liquid chromatography (HPLC), using a Waters (Mississauga, ON) apparatus with a Symmetry 300, C18, 5 mm, 4.6 x 250 mm column. Acetonitrile constituted the mobile phase, and 0.1% trifluoroacetic acid was used as the ion- pairing agent. Detection was performed at 214 nm and 280 nm, the flow rate was 1 mL/minute, and the column temperature was 40°C. The elution gradient initiated at 75% Solvent A (sterile water and 0.1% TFA) and 25% Solvent B (acetonitrile and 0.1% TFA). The gradient was run at 25

1% Solvent B per minute for 15 minutes, and then increased to 100% Solvent B at 2 mL/minute for over a period of 2 minutes. Fractions representing the main peak were manually collected. The collected protein fractions were freeze-dried using a Labconco FreeZone freeze-dryer (Fisher Scientific, Markham, ON, Canada). Upon solubilising, protein purity was assessed by SDS-PAGE and HPLC. Reduced and non-reduced protein samples were analyzed by SDS- PAGE under non-reducing conditions. The final sample purity was analyzed by HPLC using the same chromatographic conditions as above, and was determined to be greater than 95% for all MBP variants.

3.3 Labelling of Cysteine-substituted MBP-UTC1 and MBP-UTC8 variants with IAEDANS

Freeze-dried MBP cysteine variants were re-solubilized in a solution containing 20 mM Hepes-NaOH, pH 7.4, to a final protein concentration of ~1 mg/mL (~54 μM). Next, IAEDANS was added to this protein solution to a final concentration that was 20-fold in excess of the protein (by moles). The labelling reaction occurred for 3 hours at 25° C, and was terminated using 12.5 mM β-mercaptoethanol. Unreacted IAEDANS and β-mercaptoethanol were removed by HPLC (utilizing the same method that was used for MBP purification), and the collected protein fraction was then freeze-dried. Protein concentrations were determined by measuring the absorbance of re-solubilized protein in water, using an extinction coefficient at 276 nm of 8421.4 M-1 cm-1 for MBP-UTC1 and 8432.9 M-1 cm-1 for MBP-UTC8, which was experimentally determined by resuspending a known mass of protein in a known volume of water, and measuring absorbance using the following equation:

푨 = 푪 풍 휺 (Eq. 1 )

To determine the concentration of protein and IAEDANS in the labelled protein samples, multifactorial analysis of UV absorption spectra in water was used and produced the following system of equations:

푰 푨ퟐퟕퟔ−([푰푨푬푫푨푵푺]×휺ퟐퟕퟔ [푴푩푷] = 푴푩푷 (Eq. 2 ) 휺ퟐퟕퟔ

푴푩푷 푨ퟐퟔퟎ−([푴푩푷]×휺ퟐퟔퟎ [푰푨푬푫푨푵푺] = 푰 (Eq. 3 ) 휺ퟐퟕퟔ

MBP-UTC1 -1 -1 MBP-UTC1 where ε 276 (extinction coefficient of MBP at 276 nm) is 8,421.4 M cm , ε 260 -1 -1 MBP-UTC8 (extinction coefficient of MBP at 260 nm) is 5,544.9 M cm , ε 276 (extinction coefficient -1 -1 MBP-UTC8 of MBP at 276 nm) is 8432.9 M cm , ε 260 (extinction coefficient of MBP at 260 nm) is -1 -1 I -1 -1 5685.9 M cm , ε 276 (extinction coefficient of IAEDANS at 276 nm) is 1,633.5 M cm , and I -1 -1 ε 260 (extinction coefficient of IAEDANS at 260 nm) is 13,423.5 M cm . The solution to these systems of equations is:

[MBP-UTC1] = 129.1A276 − 15.7A260 (Eq. 4 )

[IAEDANS-MBP-UTC1] = 81.0A260 − 53.3A276 (Eq. 5 )

[MBP-UTC8] = 129.4A276 − 14.1A260 (Eq. 6 )

[IAEDANS-MBP-UTC8] = 79.0A260 − 51.2A276 (Eq. 7 )

In addition to Equation 2, the concentration of MBP was also determined by the bicinchoninic acid (BCA) assay (119). In the BCA assay, a protein standard was prepared by dissolving a known mass of freeze-dried MBP into a known volume of water. The calculated concentrations of MBP and IAEDANS were subsequently used in the determination of labelling efficiency (fA) using the following equation:

[푰푨푬푫푨푵푺] 풇 = (Eq. 8 ) 푨 [푴푩푷]

3.4 Circular Dichroism Measurements

All CD data were acquired using a Jasco J-815 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan) using a quartz cuvette with a 1-mm path length (Hellma, Concord, ON, Canada), with thermostatting at 25°C using a Jasco PTC-424S/15 Peltier temperature controller (Japan Spectroscopic, Tokyo, Japan). The CD spectra were collected with solutions containing 10 µM protein, 5 mM HEPES-NaOH, pH 7.4, and various concentrations of TFE (0-30%, v/v). The reagent tris(2-carboxyethyl)phosphine (TCEP) was added to a final concentration of 0.5 mM when the samples contained cysteine-substituted variants, in order to maintain a reducing environment. Each spectrum was collected at a scan rate of 50 nm min-1, and represents an

average of 4 scans. Corresponding buffer scans were subtracted from sample scans before analysis and presentation of the data. Experiments were typically repeated 2 or more times with independently-prepared samples.

3.5 Fluorescence measurements

3.6 Steady-state Fluorescence Anisotropy Measurements

Steady-state fluorescence anisotropy (r) measurements were performed using 2-nm excitation, and 8-nm emission, slit widths. Time-based polarization was measured for both tryptophan and IAEDANS using excitation at 295 nm and emission at 350 nm, and excitation at 336 nm and emission at 500 nm, respectively. The “T-format” was used for the simultaneous detection of intensities for vertically- (IVV) and horizontally- (IVH) polarized emitted light following sample excitation by vertically-polarized light. The “G” factor was measured as

IVH/IHH, where IVH and IHH are the intensities of the vertically- and horizontally-polarized emitted light, respectively, from horizontally-polarized excitation light. A signal integration time of 30 s with the rate of 10 points per second was used, and each anisotropy value is the average of three replicates. The final anisotropy (r) values for all labelled cysteine variants for both MBP-UTC1 and MBP-UTC8 were calculated as shown in the subsequent equation:

푰 − 푮푰 풓 = 푽푽 푽푯 (Eq. 9 ) 푰푽푽 + ퟐ푮푰푽푯 3.7 ANS fluorescence

The ANS fluorescence in the absence and presence of full-length unmodified MBP was measured at different concentrations of TFE. In these experiments, stock solutions of ANS were prepared by dissolving ANS in water to a final concentration of ~1.5 mM. This solution was centrifuged at 18,000 x g for 10 minutes to remove undissolved ANS, and the final stock concentration of ANS was determined by measuring the absorbance at 350 nm using an extinction coefficient of 5,000 M-1 cm-1 (120-122). From these stock solutions, sets of samples 28

were prepared containing 50 µM ANS, 20 mM HEPES-NaOH, pH 7.4, concentrations of TFE ranging from 0-55% (v/v), and either 0 or 7.5 µM MBP. Measurements were made using a quartz cuvette (Hellma, Concord, ON, Canada) with a 3-mm path-length with the excitation wavelength set to 350 nm (2-nm slit width), and emission was measured from 400-600 nm (6-nm slit width). In addition to collection of full spectra, emission was also measured at the single wavelength of 480 nm and presented as an average of 30 readings taken at a rate of 1 s-1. All experiments were repeated a minimum of 3 times on independently-prepared samples.

3.8 Fluorescence resonance energy transfer measurements

All fluorescence data were obtained with a Fluoromax Spectrophotometer (Photon Technology International, Edison, NJ, USA). Temperature was maintained at 25° C with a circulating water bath in all experiments. In FRET experiments involving MBP, the lone intrinsic tryptophan (W113) was used as the donor, and an IAEDANS-labelled cysteine served as the acceptor. Prepared samples contained 2-4 μM unlabelled or IAEDANS-labelled MBP, 20 mM HEPES-NaOH, 0.5 mM TCEP, at pH 7.4, and were measured in various environmental conditions. Samples were prepared under various environmental conditions such as; in presence of 6 M GdmCl, 100 mM NaCl, Zn2+ (10x excess Zn to protein in moles), and finally at varying concentrations of TFE (0%, 10%, 15%, or 30% TFE (v/v)). Measurements were performed using a 5-mm path-length quartz cuvette (Hellma, Concord, ON, Canada). Tryptophan excitation was at 295 nm, and emission was measured from 305 nm to 550 nm; slit widths were each set to 4 nm. Each spectrum was recorded as an average of 3 scans and manually blank-corrected by subtracting buffer scans. The FRET efficiency (EFRET) was calculated based on the extent of tryptophan fluorescence quenching by IAEDANS using the following equation:

푫푨 ퟏ 푬푭푹푬푻 = (ퟏ − ) ( ) (Eq. 10 ) 푫 풇푨 where DA and D are the tryptophan fluorescence intensities at 355 nm of labelled and unlabelled

MBP, respectively, and ƒA is the labelling efficiency (fraction of labelled protein). All FRET efficiency measurements were repeated using a minimum of 3 independently-made samples, using protein derived from at least 2 independent IAEDANS labelling reactions.

After obtaining FRET efficiencies, the distance (r) between the donor and acceptor was then calculated using the following equation:

ퟏ/ퟔ ퟏ−푬푭푹푬푻 풓 = 푹풐 ( ) (Eq. 11 ) 푬푭푹푬푻 where Ro is the Förster distance, which was taken as 22.0 Å for the tryptophan-IAEDANS pair (46). Measurements in the various environments were then compared to the Flory random-coil model for denatured proteins in 6 M guanidinium chloride (123).

3.9 TFE-titration curves monitored by tryptophan fluorescence quenching

The MBP variants S17C-MBP and S159C-MBP were further analyzed by obtaining equilibrium TFE-titration curves monitored by the change in tryptophan fluorescence quenching by IAEDANS. In these quenching experiments, separate samples of unlabelled and IAEDANS- labelled MBP were excited at 295 nm, and emission was measured at 355 nm. A 3-mm quartz cuvette was used (Hellma, Concord, ON, Canada), and slit widths were 2 nm for excitation and 8 nm for emission. A total of 30 readings were acquired over 30 s, which were subsequently manually averaged. The labelled fluorescence signal was then subtracted from the unlabelled signal to determine quenched fluorescence. Samples were prepared identically to those used for FRET measurements (see above) with the exception that the range of TFE concentrations used was broader, ranging from 0-35% (v/v), and many more TFE concentrations were considered. Samples were incubated at 25 °C for a minimum of 2 h before measuring. Similarly to the CD- monitored curves, reversibility was confirmed by collecting reverse-titration curves in which samples were incubated at high concentration of TFE (~40%) for ~1 h, and then diluted to lower concentrations before measuring.

3.10 Quantitative analysis of TFE-titration curves

All quantitative analysis was performed using OriginPro 8 (OriginLab Corporation, Northampton, MA, USA). In the analysis of TFE-titration curves the curves were fit to a 3-state model (disordered↔intermediate↔α-helical). In this analysis, we assume a linear dependence of ΔG (change in free energy) with TFE concentration for each protein variant (124-126), in which the equilibrium constant (KTFE) at any given TFE concentration can be defined as:

푻푭푬 푯ퟐ푶 −휟푮 풎[푻푭푬]−휟푮 풎[푻푭푬]−풎[푻푭푬]풎풊풅 푲푻푭푬 = 풆 푹푻 = 풆 푹푻 = 풆 푹푻 (Eq. 12 ) where, ΔGTFE is the free energy change at a given concentration of TFE, ΔGH2O is the free energy change in the absence of TFE (i.e., in 20 mM Hepes-NaOH, pH 7.4), m is the dependence of TFE ΔG on [TFE], [TFE]mid is the concentration of TFE where the transition is half completed, R is the universal gas constant, and T is the temperature in degrees Kelvin. For both a 2-state and 3- state equilibrium model, the conformational transitions can be mathematically related to the measured optical signal (127). The 2-state transition was defined by:

(풀 +푺 [푻푭푬])+(풀 +푺 [푻푭푬])푲푻푭푬 풀 = 푫 푫 푯 푯 (Eq. 13 ) 풐풃풔 ퟏ+푲푻푭푬 where Yobs is the measured optical signal, YD and YH are the intercepts of the disordered conformation and helical conformation baselines, respectively, and SD and SH are the slopes of the disordered conformation and α-helical conformation baselines, respectively. The values of

YD, YH, SD, and SH were determined by linear regression analysis and subsequently fixed when fitting to Equation 9, while m and [TFE]mid were floating parameters. The 3-state transition was defined by:

푻푭푬 푻푭푬 푻푭푬 (풀푫+푺푫[푻푭푬])+(풀푰+푺푰[푻푭푬])푲ퟏ +(풀푯+푺푯[푻푭푬])푲ퟏ 푲ퟐ 풀풐풃풔 = 푻푭푬 푻푭푬 푻푭푬 (Eq. 14 ) ퟏ+푲ퟏ +푲ퟏ 푲ퟐ

TFE TFE where YI and SI are the slopes and intercept of the intermediate, respectively, and K1 and K2 are the equilibrium constants of the first (disordered↔intermediate) and second (intermediate↔α-helical) transitions, respectively. In 3-state fitting, all the CD and fluorescence H2O quenching curves for both S17C-MBP and S159C-MBP were globally fit with ΔG1 and H2O st nd ΔG2 (free energy change for the 1 and 2 transitions, respectively), and m1 and m2 (the dependence of ΔG on TFE for the 1st and 2nd transitions, respectively) as shared floating parameters. For the CD data, the parameters defining the disordered and α-helical conformation baselines were determined by linear regression, and subsequently fixed during global fitting. It was assumed that the CD signal of the intermediate conformation is indistinguishable from the disordered conformation, and hence the baseline values of the intermediate conformation were set to be equal to those of the disordered conformation during fitting. For the fluorescence

quenching data, it was assumed that all slopes were zero; however, the intercepts of all three baselines were left as floating parameters.

Chapter 4.0: Results

*Statement of Work Performed

The work described in this chapter was performed entirely by the author, except for the TFE titration curves monitored by CD spectroscopy, ANS-binding, and tryptophan fluorescence quenching, along with subsequent data fitting to equilibrium thermodynamic models.

4.1 MBP Site-directed mutagenesis

Using site-directed mutagenesis via PCR, seven MBP-UTC1 and seven MBP-UTC8 protein constructs were produced, each containing single-cysteine substitution at positions: S17C, S44C, S67C, H85C, S99C, S129C, and S159C (Figure #4). Primers for mutagenesis were designed according to the QuikChange® protocol, ordered, and supplied by the Guelph Molecular Supercenter. The PCR conditions for mutation were performed as per the protocol described in the previous chapter. Transformation by heat-shock into E. coli-DH5α cells was confirmed using agar plates containing ampicillin as indicated by positive and negative controls. Plasmid DNA was purified using a mini-prep kit for all fourteen protein constructs, and their concentrations were determined via A260 measurements. Sequences of the isolated plasmids were confirmed by the Guelph Molecular Supercenter, Guelph, ON.

4.2 MBP purification

Cysteine-containing MBP variants were successfully transformed and expressed in E. coli BL-21(DE3)-Codon Plus-RP cells. Induction by IPTG produced significant quantities of variant protein(s), as confirmed by the comparison of the SDS-PAGE profile of induced and uninduced controls (Figure #5A). Extraction and purification of MBP from the bacterial cells were performed under denaturing conditions as described by (87) with minor changes to the protocol for purification. According to the chromatogram, MBP was eluted from the column at an approximate salt concentration of 160 mM. As the buffer used had a pH of 10.0, we would expect that all bacterial proteins should have been removed by FPLC, and therefore, it is likely

Figure #4. Amino acid sequence and secondary structural map of murine 18.5-kDa MBP-UTC1 and MBP-UTC8 (168 residues). MBP-UTC1 and MBP-UTC8 are intrinsically-disordered in aqueous solution but have three distinct segments which have strong α-helical propensity α1, α2, α3 (top to bottom). These regions consist of residues which can form a amphipathic α-helices when associated with lipids or in the presence of membrane-mimetic solvents. For purposes of this study, recombinant murine MBP-UTC1 and MBP-UTC8 constructs containing a series of point mutations at positions S17C, S44C, S67C, H85C, S99C, S129C, and S159C were generated. Cysteinyl-containing proteins were utilized for labelling with the thiol-reactive fluorescent probe 1,5-IAEDANS, in order to create a FRET donor-acceptor pair with an intrinsic tryptophan at position 113. Adapted and modified with permission from Vassall et al. (2013). 34

A B C

1 2 3 1 2 3 4 5 6 7 8 9 10 11 1 2 3 12

Figure #5. Purification of MBP-UTC1 and MBP-UTC8. (A) 14% SDS-PAGE gel for MBP- C1 cysteine-containing variants in pET22b(+) expression vector in E. coli BL21-CodonPlus (DE3)-RP cells induced with 1 mM IPTG. Samples were prepared by dissolving variant cell pellets in 5x reducing loading dye and boiled at >100°C for 2 minutes. Lane A-1 = Post-IPTG induction, Lane A-2 = Pre-IPTG induction, Lane A3 = Pink Plus Prestained Protein Ladder from BioRadTM. The gel was stained with Coomassie brilliant blue for 15 minutes, and destained with 2% methanol and 1% acetic acid. Results show overexpressed 18.5-kDa MBP as indicated in Lane #3 by the large band occurring at ~21 kDa. Data shown are for the MBP-C1-S17C variant. (B) 14% SDS-PAGE gel for FPLC-pure MBP-UTC1 cysteine-containing variant. Samples were prepared as above. Lanes B2-B12 are shown to contain overexpressed MBP-UTC1 protein variant (~18.50 kDa). Data shown are for the MBP-UTC1-S17C variant. (C) 14% SDS-PAGE gel for MBP-UTC1 cysteine containing variant reduction by 125 mM β-mercaptoethanol. Fractions were analyzed by SDS-PAGE. Samples of each fraction were prepared by addition of 5x reducing loading dye to make 20 µL samples, and were then boiled at >100°C for 2 minutes. Lane 1 = Reducing loading buffer. Lane 2 = Non-reducing loading buffer. Lane 3 = Post- reduction with non-reducing loading buffer. Lane 4 =Pink Plus Prestained Protein Ladder from BioRad. The reduction of dimers is shown be successful as indicated by the absence of bands occurring between 42 and 29 kDa in Lane 3. Data shown are for the MBP-C1-S17C variant.

that the observed low molecular mass impurities are truncations of full-length 18.5-kDa MBP. High molecular mass impurities were observed on the SDS-PAGE gel following FPLC purification (Figure #5B). Regarding these species, one possible explanation is the result of cross-linking of free cysteine residues, resulting in dimerization or oligomerization of MBP monomers. Analytical samples for each variant confirmed the presence of minor contaminants from dialyzed fractions of FPLC-pure variant protein. Purification of samples was successful as indicated by the HPLC chromatogram (data not shown). According to the chromatogram, MBP was eluted at approximately 37% acetonitrile, and exhibited a double-peak pattern. This pattern is indicative of MBP monomers and dimers. The nature of MBP dimers are likely the result of disulfide bonds between the introduced cysteine residues. In order to achieve high efficiency labelling, these dimers must be reduced. Reduction of variant protein dimers was achieved by incubation with 125 mM β-mercaptoethanol and subsequent analysis by SDS-PAGE (Figure #5C) revealed only monomeric MBP. In order to remove excess β-mercaptoethanol the samples were purified by HPLC under the same chromatographic conditons as mentioned previously. The final purity was assessed by HPLC and was determined to be greater than 95% for all MBP variants (data not shown).

4.3 MBP labelling with IAEDANS

Protein labelling was achieved by incubation of variant protein with 15-20x molar excess IAEDANS fluorescent label in 20 mM Hepes Buffer at pH 7.4. The reaction was terminated using 0.125 mM β-mercaptoethanol after a 4-hour period. Excess label, and the β- mercaptoethanol used to terminate the reaction, were successfully removed by HPLC under the same chromatographic conditons as stated above, though the absorbance channels that were monitored were altered to 214 nm and 336 nm, with 336 nm representing the IAEDANS label (data not shown). The fractions representing the main, labelled protein peak were manually collected and lyophilized.

4.4 Determining MBP labelling efficiency

Absorbance wavelength spectra were collected from 220 nm to 700 nm for both unlabelled and labelled samples (data not shown). A characteristic 3-peak shape was observed for all labelled MBP-UTC1 and MBP-UTC8, suggesting the successful linking of label to protein. In order to resolve the peaks and determine the amount of bound label, a system of equations was developed to subtract the IAEDANS contribution at A276 to determine protein concentration, as well as to subtract the protein contribution at A260 to determine the concentration of the amount of bound IAEDANS label (Section 3.3). A series of extinction coefficients were determined for both MBP-UTC1 and MBP-UTC8, as well as IAEDANS label, which were determined by solubilizing known weights in water. The final values calculated are MBP-UTC1 -1 -1 MBP- listed: ε 276 (extinction coefficient of MBP-UTC1 at 276 nm) is 8,421.4 M cm , ε UTC1 -1 -1 MBP-UTC8 260 (extinction coefficient of MBP-UTC1 at 260 nm) is 5,544.9 M cm , ε 276 -1 -1 MBP-UTC8 (extinction coefficient of MBP-UTC8 at 276 nm) is 8432.2.1 M cm , ε 260 (extinction -1 -1 I coefficient of MBP-UTC8 at 260 nm) is 5685.9 M cm . The values for IAEDANS are ε 276 -1 -1 I (extinction coefficient of IAEDANS at 276 nm) is 1,633.5 M cm , and ε 260 (extinction coefficient of IAEDANS at 260 nm) is 13,423.5 M-1 cm-1. Both unlabelled stock variant protein and labelled variant protein concentrations were successfully determined via this multifactorial analysis, which was confirmed by performing the bicinchoninic acid protein assay (BCA) for protein concentration. The BCA Protein Assay Kit (Thermo-Scienftic, ON, Canada) was performed as per the provided protocol, with the standard being modified to be either pure MBP- UTC1 or pure MBP-UTC8 protein (128). The standard concentration range used was from 200 µg/mL to 0.5 µg/mL, and was developed by serial dilution of pure MPB-UTC1 or MBP-UTC8 with known concentration, which were determined by solubilizing known weights in water. The labelling event was successful providing a labelling efficiency of >60%.

4.5 Circular Dichroism Measurements of MBP

In order to reaffirm that neither the incorporation of the cysteine residue nor the labelling with IAEDANS fluorescent probe resulted in alteration of MBP-UTC1 and MBP-UTC8 protein structure, we performed CD spectroscopy. Previously, NMR (16, 84, 129) and CD (130) spectroscopic studies have demonstrated that MBP is a disordered, monomeric protein in

solution (59, 77). However, in the presence of membrane-mimetic solvents such as TFE, MBP has been shown to adopt significant α-helical secondary structure (131). Previous studies using site-directed spin labelling and EPR spectroscopy (57, 82, 132), solid-state NMR spectroscopy in a lipid milieu (80, 84), as well as solution NMR spectroscopy in 30% TFE (v/v) (133, 134), have all demonstrated that α-helical formation is localized predominantly in three regions (approximately T33-F43, P82-V91, and Y141-K153). The disorder-to-α-helical transition of MBP can be directly monitored by measuring the change in far-UV CD spectra with increasing concentrations of TFE. This was done originally for natural protein purified from brain representing a heterogeneous population of modified forms, then again previously by us for a hexa-histidine tagged construct of MBP (135), and has been repeated here for the MBP-UTC1 (Figure #6) and MBP-UTC8 (data not shown) cysteine variants. Far-UV CD spectra of untagged MBP-UTC1 demonstrate here that there is pronounced α-helical content at TFE concentrations >25% (v/v) and conversely, there is relatively little ɑ-helical content between 0-10%. This disorder-to-α-helical transition of MBP in TFE is protein concentration independent, suggesting that α-helical formation is not accompanied by oligomerization of the protein (Figure #7). Significantly, there is an isodichroic point at 203 nm, which is usually suggestive of a 2-state transition (136, 137). However, we were cautious in analyzing the data via a 2-state model before analyzing the disorder-to-α-helical transition using additional probes (see below).

The CD spectra collected at 0% TFE of the labelled UTC1 and UTC8 MBP variants displayed the typical minimum of ∼200 nm, indicating a random coil structure. Conversely, CD spectra of MBP-UTC1 and MBP-UTC8 protein samples measured in 30% TFE displayed a α- helical rich secondary structure profile. It should be noted that far-UV CD spectra, of the hexa- histidine tagged construct of MBP, revealed that a second transition also occurs at ~90% TFE (101). We have identified a similar, well-separated, second transition in the untagged protein (data not shown), but have not considered it in our analysis in light of prior experimental evidence that α-helical formation above 40% TFE may not be physiologically relevant (122). Overall, control CD spectra collected for all the variants at 0% and 30% TFE suggest that the presence of the cysteine-substitutions and label do not significantly perturb the protein’s native secondary structure composition (Figure #8).

Figure #6. Changes in circular dichroic (CD) spectra of 18.5-kDa MBP-UTC1 with increasing trifluoroethanol (TFE) concentration. Samples contained 10 µM MBP, 5 mM Hepes-NaOH, pH 7.4, and measurements were made at 25 °C. Blank-subtracted representative data are shown and data were normalized to mean residue ellipticity. The α-helical content increases with increasing TFE concentrations (0% to 35%, v/v), and there is an isodichroic point at ~203 nm.

Figure #7. Comparison of TFE-titration curves of IEADANS-labelled MBP-UTC1-S17C and S159C 18.5-kDa MBP with the unmodified protein. The representative titration curves were monitored by change in circular dichroic signal at 222 nm and normalized to facilitate comparison. Unmodified MBP samples contained 4 μM, 7.7 μM, and 25 μM protein concentration, as indicated, whereas S17C and S159C MBP samples contained 4 μM protein. All solutions were buffered with 20 mM Hepes-NaOH, pH 7.4, and measurements were performed at 25 °C.

UT-MBP S17C S44C S67C H85C S99C S129C S159C

20 A B

)

-1 -10

dmol -20 2

20 degcm C

( D

-3 10

*10

]  [ 0

-10

-20

200 225 250 200 225 250

Wavelength (nm)

Figure #8. Circular dichroic (CD) spectra of unmodified 18.5-kDa MBP-UTC18 and MBP- UTC IAEDANS-labelled cysteine variants. The labeling efficiency of the MBP cysteine- substituted variants was >60% in all cases. All samples contained 10 µM MBP, 5 mM Hepes- NaOH, pH 7.4, and measurements were made at 25 °C. Blank-subtracted data are shown, and data were normalized to mean residue ellipticity. Panels (A) and (B) show far-UV CD spectra collected in 0% and 30% TFE for MBP-UTC1 variants (v/v), respectively. Panels (C) and (D) show far-UV CD spectra collected in 0% and 30% TFE for MBP-UTC8 variants (v/v), respectively. All variants displayed consistent levels of secondary structure in both 0% and 30% TFE.

4.6 ANS fluorescence in MBP

Although it is clear that MBP forms amphipathic α-helices in at least three different segments in the presence of TFE, there is little known about the evolution of global structure that may accompany α-helix formation. Here, we obtained preliminary information on the evolution of tertiary structure for both MBP-UTC1 and MBP-UTC8 by utilizing the well-known hydrophobic fluorescent dye ANS. ANS is known to bind to solvent-exposed hydrophobic pockets in proteins, resulting in a blue shift in fluorescence maxima and an enhancement in intensity. Hence, this dye is commonly used to assess conformational changes within various proteins (120, 121), including IDPs (138, 139).

We found that MBP-UTC1 in the absence of TFE showed modest enhancement of ANS fluorescence. There is a sigmoidal increase in the wavelength of maximal ANS fluorescence enhancement from 0-45% TFE (Figure #9A), with maximal enhancement occurring close to 477 nm. Marked changes in the pattern of enhancement were seen when TFE was titrated into the system (Figure #9B). Additionally, there is a substantial increase in the intensity of enhancement between 5 and 10% TFE, compared to 0% TFE, and intensity is at a maximum at 10-17.5% TFE (Figure #9C). From 17.5-30% TFE, there is a steep decline in ANS fluorescence enhancement intensity, and from 30-45% TFE there is virtually no enhancement. Results for MBP-UTC8 were largely consistent with MBP-UTC1 (Figure #10) though maximal ANS enhancement was shown to occur at slightly lower percentages of TFE.

Overall, the pattern of ANS fluorescence enhancement between 0 and 45% TFE is bell- shaped, suggesting the presence of one or more intermediates along the disorder-to-α-helical pathway. This finding is in contrast to the CD results in which an isodichroic point at 203 nm is present when far-UV scans of MBP are conducted at different TFE concentrations, suggesting a simple 2-state transition. These results can be reconciled if we consider that the intermediate conformer may have a similar local secondary structure composition to the disordered conformer, but a different global structure. Thus, it may be difficult to differentiate between the disordered and intermediate conformations in the far-UV CD scans, as they are only sensitive to changes in secondary structure (124).

Figure #9. Change in ANS fluorescence enhancement by 18.5-kDa MBP upon TFE titration. Samples contained 20 mM Hepes-NaOH, pH 7.4, 50 µM ANS, and 7.5 µM MBP, and measurements were conducted at 25 °C with excitation at 350 nm. All data are presented after subtraction of corresponding data from samples that did not contain MBP. Panel (A) shows the effect of increasing TFE concentration on the enhancement of ANS fluorescence intensity by MBP (data are normalized) whereas panel (B) shows the corresponding wavelengths of maximal enhancement. Panel (C) shows the enhancement at 480 nm for each concentration of TFE relative to the enhancement in 0% TFE. Data points are an average of a minimum of 3-independently-made samples and error bars are standard deviations.

Relative Fluorescence Relative Enhancement.

[TFE] (% v/v)

Figure #10. Change in ANS fluorescence enhancement by 18.5-kDa MBP- UTC8-S17C upon TFE titration. Samples contained 20 mM Hepes-NaOH, pH 7.4, 50 µM ANS, and 7.5 µM MBP, and measurements were conducted at 25 °C with excitation at 350 nm. All data are presented after subtraction of corresponding data from samples that did not contain MBP. The enhancement at 480 nm for each concentration of TFE relative to the enhancement in 0% TFE. Data points are an average of a minimum of 3 independently-made samples and error bars are standard deviations.

Tables #1A and #1B. Steady-state fluorescence anisotropy (r) measurements of MBP-UTC1 (A) and MBP-UTC8 (B). Time-based polarization was measured for both tryptophan and IAEDANS using “T-format” was used for the simultaneous detection of intensities for vertically (IVV) and horizontally (IVH) polarized emitted light. “G” factor was measured as IVH/IHH, where IVH and IHH are the intensities of the vertically and horizontally polarized emitted light, respectively, from horizontally polarized excitation light. A signal integration time of 30 s with the rate of 10 points per second was used, and each anisotropy value is the average of three replicates. Final values for (r) were calculated via equation #9.

Table 1A

UTC1-S99C TFE concentration UTC1-S17C -Trp UTC1-S44C-Trp UTC1-S67C-Trp UTC1-H85C-Trp UTC1-S129C-Trp UTC1-S159C-Trp Trp

0% TFE 0.038 +/- 0.052 0.032 +/- 0.057 0.032 +/- 0.054 0.039 +/- 0.052 0.048 +/- 0.048 0.043 +/- 0.051 0.030 +/- 0.052

10% TFE 0.053 +/- 0.049 0.062 +/- 0.057 0.064 +/- 0.050 0.054 +/- 0.049 0.064 +/- 0.046 0.063 +/- 0.043 0.057 +/- 0.049

15% TFE 0.059 +/- 0.043 0.063 +/- 0.058 0.063 +/- 0.048 0.072 +/- 0.053 0.071 +/- 0.053 0.065 +/- 0.046 0.067 +/- 0.047

30% TFE 0.054 +/- 0.035 0.050 +/- 0.05 0.052 +/- 0.047 0.042 +/- 0.048 0.042 +/- 0.048 0.056 +/- 0.042 0.056 +/- 0.046

UTC1-S17C- UTC1-S159C- TFE concentration UTC1-S44C-IAEDANS UTC1-S67C-IAEDANS UTC1-H85C-IAEDANS UTC1-S99C-IAEDANS UTC1-S129C-IAEDANS IAEDANS IAEDANS

0% TFE 0.017 +/- 0.025 0.022 +/- 0.023 0.023 +/- 0.024 0.026 +/- 0.022 0.024 +/- 0.018 0.022 +/- 0.018 0.018 +/- 0.023

10% TFE 0.029 +/- 0.021 0.038 +/- 0.022 0.037 +/- 0.022 0.036 +/- 0.020 0.032 +/- 0.016 0.034 +/- 0.016 0.032 +/- 0.020

15% TFE 0.036 +/- 0.019 0.040 +/- 0.022 0.040 +/- 0.021 0.049 +/- 0.019 0.037 +/- 0.015 0.036 +/- 0.017 0.037 +/- 0.018

30% TFE 0.034 +/- 0.015 0.031 +/- 0.017 0.031 +/- 0.021 0.034 +/- 0.017 0.034 +/- 0.014 0.034 +/- 0.005 0.030 +/- 0.017

Table 1B

TFE UTC8-S44C- UTC8-S67C- UTC8-H85C- UTC8-S99C- UTC8-S17C -Trp UTC8-S129C-Trp UTC8-S159C-Trp concentration Trp Trp Trp Trp

0% TFE 0.031 +/- 0.082 0.035 +/- 0.034 0.029 +/- 0.085 0.039 +/- 0.069 0.059 +/- 0.073 0.116 +/- 0.074 0.035 +/- 0.073

10% TFE 0.035 +/- 0.074 0.062 +/- 0.05 0.023 +/- 0.071 0.048 +/- 0.074 0.030 +/- 0.073 0.037 +/- 0.077 0.084 +/- 0.070

15% TFE 0.042 +/- 0.074 0.046 +/- 0.048 0.027 +/- 0.072 0.065 +/- 0.073 0.078 +/- 0.067 0.026 +/- 0.071 0.143 +/- 0.074

30% TFE 0.031 +/- 0.075 0.033 +/- 0.045 0.051 +/- 0.077 0.028 +/- 0.073 0.029 +/- 0.074 0.107 +/- 0.074 0.046 +/- 0.074

TFE UTC8-S17C-IAEDANS UTC8-S44C-IAEDANS UTC8-S67C-IAEDANS UTC8-H85C-IAEDANS UTC8-S99C-IAEDANS UTC8-S129C-IAEDANS UTC8-S159C-IAEDANS concentration

0% TFE 0.057 +/- 0.059 0.037 +/- 0.059 0.031 +/- 0.063 0.047 +/- 0.052 0.055 +/- 0.053 0.051 +/- 0.049 0.42 +/- 0.47

10% TFE 0.041 +/- 0.047 0.046 +/- 0.033 0.014 +/- 0.050 0.047 +/- 0.048 0.029 +/- 0.052 0.049 +/- 0.048 0.081 +/- 0.061

15% TFE 0.046 +/- 0.048 0.051 +/- 0.045 0.026 +/- 0.045 0.065 +/- 0.056 0.065 +/- 0.048 0.039+/- 0.053 0.110 +/- 0.054

30% TFE 0.043 +/- 0.045 0.039 +/- 0.042 0.048 +/- 0.043 0.038 +/- 0.048 0.050 +/- 0.060 0.040 +/- 0.048 0.053 +/- 0.047

4.7 Steady-state Fluorescence Anisotropy Measurements of MBP

Tables #1A and #1B show the apparent fluorescence anisotropy values for MBP-UTC1 and MBP-UTC8 IAEDANS-labelled protein variants. Both tryptophan and cysteine-bound IAEDANS anisotropy values were measured for labelled proteins in various concentrations of TFE. All values that were determined showed relatively low fluorescence anisotropy values (<0.075), consistent with solvent-exposed sites and a high degree of probe mobility (109).

4.8 Fluorescence resonance energy transfer (FRET) measurements of MBP

The fluorescence spectra of unlabelled variants, with excitation at 295 nm, are consistent with the presence of a solvent-exposed tryptophan (residue Trp113) in both MBP-UTC1 and MBP-UTC8 (46), and show a fluorescence maximum at ~350 nm. The corresponding fluorescence spectra of the IAEDANS-labelled protein also have a fluorescence maximum at ~350 nm, but with diminished intensity, while there is another large peak in the spectra at ~500 nm, indicating resonance energy transfer between the donor tryptophan and the acceptor IAEDANS, even as the protein concentrations remained identical at 2.5 µM. The FRET efficiency between the lone intrinsic tryptophan at position 113 and the IAEDANS label at each of the seven positions was determined from the extent of tryptophan fluorescence quenching, and the distances between the label and Trp113 were also determined for both MBP-UTC1 and MBP-UTC8 (Table #2A and #2B). The FRET measurements were also successfully performed in 10x excess zinc (by moles) and again with 150 mM NaCl, and their results are compared to aqueous buffer (data not shown)

Comparisons were also made between the distances obtained in 0% TFE and theoretical random coil values (Table #3). All distances were found to be significantly less than would be expected for a random coil conformation indicating that MBP in aqueous buffer (0% TFE) retains a conformation which is somewhat globally compact. Additionally, denaturation was observed to occur upon addition of 6 M GdmCl (Table #3). This denaturation is demonstrated by the marked decrease in the transfer efficiencies, and the resulting increase in distances, as compared to native conditions. These results are consistent with recent small-angle X-ray

scattering (SAXS) experiments on MBP which also concluded that the protein is more compact than an ideal flexible polymer but, however, less compact than a globular protein (140).

Tables #2A and 2B. Fluorescence resonance energy transfer (FRET) of MBP labelled with IAEDANS. Samples contained 2.5 µM protein, 20 mM Hepes-KOH, pH 7.4, and 0.5 mM TCEP. Measurements were conducted at 25 °C and the excitation wavelength was 295 nm. Panel (A) shows the FRET efficiencies and distances calculated from donor (W113) quenching with IAEDANS at different positions within the primary sequence of 18.5-kDa MBP-UTC1 as indicated. Panel (B) shows the FRET efficiencies and distances calculated from donor (W113) quenching with IAEDANS at different positions within the primary sequence of 18.5-kDa MBP-UTC8 as indicated. Data are averages of 3-6 independent measurements, and the error bars represent the standard error. The general trend is for increased efficiency at 10% and/or 15% TFE, and lower efficiencies at 0% TFE and 30% TFE. This trend is most prominent at position S17, H85, and S159 indicating a global transition in MBP and the possible presence of an intermediate conformation.

Table 2A 0% TFE (v/v) 10% TFE (v/v) 15% TFE (v/v) 30% TFE (v/v)

Efficiency (%) Distance (Å) Efficiency (%) Distance (Å) Efficiency (%) Distance (Å) Efficiency (%) Distance (Å)

UTC1 -S17C 9.8±3.9 32.2±2.1 23.2±3.6 26.9±0.9 26.4±0.6 26.1±0.1 12.8±3.4 30.5±1.9 UTC1-S44C 21.5±2.8 27.4±0.8 24.9±3.0 26.5±0.7 30.2±4.8 25.3±1.0 14.8±4.0 30.0±1.8 UTC1-S67C 24.5±4.9 26.6±1.2 32.4±4.8 25.0±0.9 29.3±3.9 25.5±0.8 17.8±3.0 28.7±1.3 UTC1-H85C 35.1±4.4 24.4±0.8 51.1±7.9 21.9±1.2 46.0±7.8 22.6±1.2 29.5±5.0 25.4±0.9 UTC1-S99C 51.2±8.7 22.3±0.6 62.9±3.9 20.2±0.6 61.1±3.7 20.4±0.5 48.3±3.2 22.1±0.3 UTC1-S129C 44.0±8.4 23.2±0.9 56.9±11.7 21.1±1.7 51.6±8.0 21.8±1.2 40.4±3.1 23.5±0.5 UTC1-S159C 23.8±8.0 27.1±1.3 40.9±6.1 23.5±0.9 31.7±13.6 24.9±1.9 19.0±6.7 27.5±1.3

Table 2B

0% TFE (v/v) 10% TFE (v/v) 15% TFE (v/v) 30% TFE (v/v)

Efficiency (%) Distance (Å) Efficiency (%) Distance (Å) Efficiency (%) Distance (Å) Efficiency (%) Distance (Å)

UTC8 -S17C 12.1±4.0 30.9±2.1 33.6±11.8 26.5±3.7 29.8±6.5 25.5± 1.4 8.5±3.2 33.0±2.2

UTC8-S44C 34.1±.4.0 24.1±0.9 42.8±6.4 23.1±1.0 41.9±6.7 23.3±1.1 21.7±4.0 27.3±1.0 UTC8-S67C 35.9±9.7 24.4±2.0 41.9±4.3 23.2±0.7 47.3±6.3 22.4±0.9 34.9±9.7 24.5±1.8

UTC8-H85C 39.9±12.2 23.7±2.1 54.4±6.6 21.4±0.9 47.2±3.6 22.4±0.5 39.5±9.5 23.7±1.7 UTC8-S99C 57.3±6.6 21.0±1.0 59.9±8.5 20.6±1.2 52.6±9.2 21.6 ±2.2 48.7±6.1 22.2±0.9 UTC8-S129C 52.1±12.4 21.8±1.9 68.6±11.4 19.2±1.8 49.6±6.7 19.2±1.0 43.3±10.2 23.1±1.7 UTC8-S159C 49.9±7.7 22.0±1.1 54.5±3.1 21.3±0.4 51.0±4.1 21.3 ±0.6 36.4±11.2 24.3±2.1 0% TFE (v/v) 10% TFE (v/v) 15% TFE (v/v) 30% TFE (v/v)

Table #3. FRET measurements in 6 M guanidinium chloride (guanidine hydrochloride, GdmCl). Comparison of FRET-derived distances in IAEDANS-labelled 18.5-kDa MBP samples with theoretical random coil predictions. MBP was labelled with IAEDANS via engineered free cysteines at the positions indicated. The FRET was measured between IAEDANS and Trp113. Samples contained 20 mM Hepes-NaOH, pH 7.4.Theoretical distances are the mean end-to-end distances for a Gaussian coil and were calculated as 8.37√N, where N is the number of amino acids separating IAEDANS and Trp113.

Table 3

Under Native Theoretical Random MBP-UTC1 MBP-UTC8 conditions (Å) Coil (Å) in 6 M GdmCl (Å) in 6 M GdmCl (Å) UTC1 S17C 32.2±2.1 80.1 >34 >34

UTC1-S44C 27.4±0.8 68.0 n.d n.d

UTC1-S67C 26.6±1.2 55.7 >34 >34

UTC1-H85C 24.4±0.8 43.5 n.d n.d

UTC1-S99C 22.3±0.6 30.9 25.1±0.5 25.2±0.1

UTC1-S129C 23.2±0.9 33.0 n.d n.d

UTC1-S159C 27.1±1.3 55.7 >34 >34

4.9 Measuring the ‘disorder-to-order’ conformational transition(s) of MBP in TFE

Measurements were performed in various concentrations of TFE ranging from 0-30% TFE v/v for both MBP-UTC1 and MBP-UTC8. For both variants in the absence of TFE, the data are consistent with a disordered, flexible conformation, as the determined FRET efficiencies are qualitatively inversely proportional to the distance in primary sequence between the site of labelling and Trp113, and this general trend is also true in the presence of TFE. However, there is a clear trend for FRET efficiency to be higher at 10% and 15% TFE, and lower at 0% and 30% TFE (Table #2A and #2B), suggesting a tendency to populate more compact conformations at intermediate TFE concentrations. This general trend holds across all labelling positions, and is most prevalent with IAEDANS at positions S17C, H85C, and S159C. There are relatively larger changes in the FRET efficiencies between 0-30% for positions S17C and S159C compared to positions closer to Trp113. This observation suggests that both UTC1 and UTC8 MBP have relatively more flexible termini and a more rigid core, which is consist with what has been seen in previous studies (140). Overall, the FRET data suggest that both 18.5-kDa MBP-UTC1 and MBP-UTC8 structural ensembles are relatively extended at low and high TFE concentrations, but that more compact intermediate species are populated at TFE concentrations of 10-15% (v/v), in general agreement with the ANS-binding data for both variants (Figure #9A-C and Figure #10).

4.10 The thermodynamic analysis of the ‘disorder-to-order’ transition of MBP in TFE in order to probe the ‘ensemble populations with different free energies’

Previously, we had determined the free energy change (ΔG) associated with the disorder- to-α-helical transition of the MBP α2-peptide (S72-S107) by fitting CD-monitored TFE titration curves to a 2-state mechanism (83, 136), and this analysis and mechanism was recently extended to two other MBP α-peptides, A22-K56 and S133-S159 (Vassall et al., Journal of Molecular Biology, 427 (10), pp 1977-1992, 2015, doi: 10.1016/j.jmb.2015.03.011).

A combined analysis of the CD, ANS, and FRET data suggest that the minimal applicable mechanism of disorder-to-α-transition in full-length 18.5-kDa MBP is a 3-state

transition (disordered↔intermediate↔α-helical). The structural probes indicate that the intermediate conformation, when compared to the disordered conformation, has a different global structure but largely the same local α-helical content. We utilized the fact that the intermediate is more compact than both the disordered and α-helical conformations, and systematically monitored the TFE-titration curves of full-length 18.5-kDa MBP-UTC1 by measuring the change in tryptophan fluorescence quenching by IAEDANS bound at position S17C or S159C (Figure #11). In these TFE-titration curves, the intensity of tryptophan fluorescence was measured as TFE was titrated into different solutions containing either unlabelled or IAEDANS-labelled MBP. The extent of tryptophan fluorescence quenching was then determined by subtracting the fluorescence intensity of the labelled protein from the corresponding intensity of the unlabelled protein. Separate experiments using variants S17C and S159C for MBP-UTC1 were performed in order to assess whether conformational changes in 18.5-kDa MBP are global and if they propagate across the entire primary sequence. We also collected the corresponding CD-monitored titration curves for IAEDANS-labelled S17C and S159C for both variants, and found that these curves were highly congruent with the CD curves collected for unmodified MBP, suggesting again that the presence of the cysteine-substitution and label do not perturb the conformational transitions of 18.5-kDa MBP (Figure #12A and #12B). Preliminary experiments for MBP-UTC8 S17C have also been performed (Figure #13).

Reverse-titration curves were also collected which showed that, similar to all three peptides (83), the conformational transition of full-length 18.5-kDa MBP-UTC1 is reversible and can be analyzed thermodynamically (Figure #12C and #12D). In selecting the applicable thermodynamic model with which to analyze the data, we compared the titration curves monitored by far-UV CD spectroscopy, which follows α-helical formation, with curves monitored by fluorescence quenching, which follows the formation of new long-range contacts. In a two-state model, we would expect that new long-range contacts would occur concurrently with the formation of α-helical structure and hence, titration curves monitored by these two probes would be superimposable. However, when comparing the CD- and fluorescence quenching-monitored curves here, it is immediately apparent that the two cannot be superimposed, as the former is sigmoidal whereas the latter is bell shaped and bi-phasic. This

Figure #11. TFE-titration curves of full-length 18.5-kDa MBP-UTC1. Samples contained 4 µM MBP (labelled with IAEDANS), 20 mM Hepes-NaOH, pH 7.4, 0.5 mM TCEP. Experiments were conducted at 25 °C. Panels (A) and (B) are CD-monitored curves of S17C-MBP and S159C-MBP, respectively. Panels (C), (D), and (E) are curves of S17C-MBP, S159C-MBP, and a second independently-labelled S159C-MBP respectively, monitored by Trp113 fluorescence quenching by IAEDANS. Each data point is the mean of at least 2 independently-prepared samples, and error bars are the average deviation from the mean. Data were globally fit to a 3- state model: disordered↔intermediate↔α-helical (red lines) and fitted values are in Table #4. All data were normalized before plotting.

A B

C D

Figure #12. Comparison of TFE-titration curves of IEADANS-labelled S17C and S159C 18.5-kDa MBP-UTC1 with the unmodified protein. Forward (closed symbols) and reverse (open symbols) TFE-titration curves. Of IAEDANS-labelled MBP-UTC1-S17C and S159C variants. Panels (A and B) are representative titration curves monitored by the change in circular dichroic signal at 222 nm, whereas panels (C) and (D) show titration curves monitored by tryptophan fluorescence-quenching by IAEDANS. Samples contained 4 µM protein, 20 mM Hepes-NaOH, pH 7.4, and measurements were made at 25 °C after a minimum of 2 hours incubation.

1.0

0.8

0.6

0.4

Normalized CD (A.U.) NormalizedCD 0.2

0.0 0 5 10 15 20 25 30 35 [TFE] (% v/v) B

1.0

0.8

0.6

0.4 (A.U.)

0.2

0.0

Normalized Fluorescence 0 5 10 15 20 25 30 35 [TFE] (% v/v)

Figure #13. Preliminary TFE-titration curves of full-length 18.5-kDa MBP- UTC8. Samples contained 4 µM MBP (labelled with IAEDANS), 20 mM Hepes- NaOH, pH 7.4, 0.5 mM TCEP. Experiments were conducted at 25 °C. Panel (A) shows the CD-monitored curves of MBP-UTC8-S17C. Panel (B) shows MBP- UTC8-S17C monitored by Trp113 fluorescence quenching by IAEDANS. Each data point is the mean of at least 2 independently-prepared samples, and error bars are the average deviation from the mean. Data were globally fit to a 3-state model: disordered↔intermediate↔α-helical (red lines) and fitted values are in Table 4. All data were normalized before plotting 56

result provides further confirmation that the disordered-to-α-helical transition in MBP does not occur in a simple two-state process, but involves the presence of at least one intermediate (124). The data collected for both MBP-UTC1 S17C and S159C, as well as preliminary results for MBP UTC8 S17C across both probes (CD and fluorescence quenching), were globally fit to a 3- state model, comprising disordered ↔ intermediate ↔ α-helical), in which ΔG and m values were set as shared parameters, and there is good agreement between the data and the fits (Figure

#12, Table #4). From the fits, the ΔG1 (free energy change of disordered↔intermediate) and

ΔG2 (free energy change of intermediate↔α-helical) for MBP-UTC1 values are 1.3±0.5 and -1 2.5±0.6 kcal mol , respectively. The ΔGtotal (total free energy change of disordered↔α-helical -1 transition), which is calculated as the sum of ΔG1 and ΔG2, is 3.8±1.1 kcal mol (Table #4).

Preliminary data analysis for MBP-UTC8 S17C indicate a ΔG1 and ΔG2 of 1.0±1.2 and 4.8±0.9 -1 -1 kcal mol , respectively, whereas the ΔGtotal is 5.8±2.1 kcal mol (Figure #13)

4.11 Deconvolution of the major thermodynamic ensembles along the ‘disorder-to-order’ transition of MBP

The fitted thermodynamic values were also used to calculate the fractional population of each equilibrium species at different TFE concentrations (Figure #14) for both MBP-UTC1 and MBP-UTC8. The data concerning MBP-UTC1 show that the disordered conformation is present until ~17.5% TFE, whereas the population of intermediate conformation reaches a maximal fractional population of ~0.6 at 14% TFE. The α-helical conformation begins to become significantly populated at ~15% TFE, and by >30% TFE, this is virtually the only 18.5-kDa MBP conformation present at equilibrium. The variant MBP-UTC8 shows a large change in the behaviour of both the disordered and the intermediate states. The disordered conformation is only present until ~7% TFE, and the intermediate conformation begins to populate the ensemble at much lower concentrations of TFE, reaching a maximal fractional population of ~1.0 at 10% TFE. Similarly to MBP-UTC1, the α-helical conformation of MBP-UTC8 begins to become significantly populated at ~16% TFE, and by >30% TFE completely dominates the population.

Using the FRET efficiencies of both MBP-UTC1 and MBP UTC8 measured in different concentrations of TFE (Tables #2A and #2B), along with the fractional populations from thermodynamic analysis, we were able to deconvolve the individual contributions of each of the

equilibrium ensemble sets to the measured FRET efficiencies, and also the corresponding tryptophan-to-IAEDANS distances (Figures #15A, #15B, and #15C). We find that the distances between IAEDANS and Trp113 in the intermediate conformation are lower at all labelling positions, compared to the disordered and α-helical conformations, with the largest relative changes occurring at the S17C and S159C positions. This result is consistent with recent small- angle X-ray scattering data modelled by a coarse-grained structural ensemble, which suggested that the disordered 18.5-kDa MBP conformation has a relatively rigid core with flexible ends (140). When combining all the data, we propose that the mechanism of disorder-to-α-helical formation in 18.5-kDa MBP involves global compaction of the disordered conformation to an intermediate conformation, with little to no α-helical content, followed by re-expansion to an extended α-helical-rich conformation.

Table #4. Thermodynamic values were determined at 25 °C from global fitting of TFE titration curves monitored by changes in CD signal and Trp fluorescence quenching by IAEDANS. Errors (±) were estimated by the fitting program from the global fit. Thermodynamic values were determined at 25 °C from fitting CD-monitored TFE titration curves. All errors (±) are standard deviations from at least three independent experiments. Subscripts 1 and 2 refer to thermodynamic parameters corresponding to the first (D↔I) and second (I↔H) transition, respectively, for the three-state model. Also, m is the dependence of ∆G on [TFE] and Cmid is the [TFE] at the mid-point of the transition (i.e., [TFE] when half the peptide molecules contain a α- helical conformation). Both values were determined directly from fitting. e∆GH2O is the change in H2O free energy of the folding transition in the absence of TFE and was calculated as ∆G = Cmid f H2O H2O H2O •m. mtotal = m1 +m2; ΔGtotal = ΔG1 + ΔG2 .

Table 4

3-state model (D↔I↔H)

H2O H2O H2O m 1 ΔG1 m2 ΔG2 mtotal ΔGtotal

(kcal mol-1 (kcal (kcal mol-1 (kcal (kcal mol-1 (kcal mol-1)f M-1) mol-1) M-1) mol-1) M-1)f

MBP-UTC1 1.1±0.5 1.3±0.5 1.0±0.2 2.5±0.6 2.1±0.7 3.8±1.1

MBP-UTC8 3.0.0±2.0 1.0±1.2 1.7±0.3 4.8±0.9 4.7±2.3 5.8±2.1

1.2

Fi-C1 0.8 Fh-C1

0.6 Fd-C1

Fi-C8 0.4

Fractional Population Fractional Fh-C8 0.2 Fd-C8

0 0 10 20 30 40 [TFE] (%vol/vol)

Figure #14. Fractional population of equilibrium conformers of MBP-UTC1 and MBP- UTC8. The populations were determined from thermodynamic values (Table 1) obtained from global fitting of MBP TFE-titration curves to a 3-state model (disordered↔intermediate↔α- helical).

Figure 15A

∆G = 1.3 ∆G = 2.5 1-UTC1 2-UTC1

∆G = 1.0 ∆G = 5.8 1-UTC8 2-UTC8

Figure 15B

35 C

) from tryptophan ) from Å residue #113 residue 20

15 Distance ( Distance S17C S44C S67C H85C S99C S129C S159C S17C S44C S67C H85C S99C S129C S159C

MBP-UTC1 cysteine variant MBP-UTC8 cysteine variant 61

Figure #15A and #15B Thermodynamic mechanism of disorder-to-α-helical transition of MBP and fractional population of equilibrium conformers. Panel (A) shows the thermodynamic disorder-to-α-helical transition of MBP with the associated ΔGs for MBP-UTC1 and MBP-UTC8 which populations were obtained from global fitting of MBP TFE-titration curves to a 3-state model (disordered↔intermediate↔α-helical) (Table #4). The fractional populations of each equilibrium conformer of MBP used to deconvolute the contribution of each conformer to the measured FRET efficiencies (Table #2A and #2B), allowed the distances between the indicated positions and Trp113 within MBP to be calculated (Equation #11). The distances determined for MBP-UTC1 are shown in Panel (B), and Panel (C) shows the distances determined for MBP-UTC8.

Chapter 5.0: Discussion

5.1 MBP-UTC1 in solution

The earliest conformational studies showed that MBP is predominantly a “random coil” in aqueous solution (142) and that MBP’s lack of defined tertiary structure in solution prevents analysis by conventional techniques such as protein X-ray crystallography (67). Subsequent studies hinted at conformational transitions and some kind of “folded conformation” even in solution (e.g., (77, 143-145)). Although MBP is largely disordered in aqueous solution, many studies have revealed the presence of α-helical molecular recognition fragments (α-MoRFs) which are important for modulating the interaction of the protein with membranes and with protein partners (reviewed in (78, 131, 146)). As previously mentioned, the inherent intrinsic disorder of MBP is advantageous as it can allow for rapid association with one or multiple binding partners, in response to alterations in environmental or chemical signals (73), and it is well known that PTMs of MBP determine and regulate its conformation (55, 83, 147).

Fluorescence techniques such as FRET, when used in conjunction with other structural techniques, have been employed in IDPs similar to MBP such as tau and α-synuclein (46, 115). These techniques are well-suited to study the conformational ensembles of MBP, and structural changes caused by PTMs in aqueous buffers, in membrane-mimetic solvents such as TFE, in denaturants, and in the presence of small-interacting ligands such as zinc (115, 148, 149). In experiments presented here, seven MBP-UTC1 and seven MBP-UTC8 protein variants were used, each containing single-cysteine substitutions which were designed to span the entire primary sequence of the 168-residue protein (89, 150) in order to develop a ‘molecular ruler’. This strategy should give a sense of the ‘global’ conformational changes that occur in MBP during the disorder-to-order transitions of MBP isoforms. Additionally, by comparing the distances measured by FRET in solution with those predicted theoretically, we can gain insight into the nature of MBP conformational ensembles in solution. The FRET was measured between the center of the IAEDANS fluorescent probe and the center of Trp113.

As mentioned previously, MBP has been described as lacking ordered secondary structural elements in solution, yet it may be possible that MBP maintains some residual

compacted conformation in solution (77, 148). Recent studies have indicated that the Flory random-coil model can accurately predict dimensions of completely denatured (random coil) proteins successfully (148). This model may also be applicable to IDPs that have little to no long-range interactions. A comparison of FRET distances for MBP to the Flory model demonstrated that MBP is compacted relative to a random coil (Table #3). Using in accordance with the Flory random coil model, theoretical distances are the mean end-to-end distances for a Gaussian coil and were calculated as 8.37√N, where N is the number of amino acids separating IAEDANS and Trp113. Our experimental FRET distances of ~20-30 Å are much smaller than those predicted by the Flory model, suggesting that the assumption of a pure random coil is not applicable, and thus that MBP maintains some level of residual structure in both MBP-UTC1 and MBP-UTC8 whilst in solution.

The FRET measurements were also performed in 150 mM NaCl, as MBP can be viewed as a ‘polyampholyte’ which refers to a polymer composed of negatively- and positively-charged monomers interspersed along a protein chain. These chains are either neutral or have a net charge of one sign (151). If the net charge is high as is the case with MBP, the expectation is for the chain to behave like a conventional polyelectrolyte with all charges of the same sign, having a net repulsion and forming an ‘extended rod-like’ molecule, whereas the behaviour of neutral polyampholyte chains is entirely different as the net electrostatic forces are attractive, so that the chains have a tendency to collapse into a globule (151). When an ionic solution such as salt (NaCl) is added, a charge-screening effect alters the interactions and reduces the electrostatic attraction(s). The globule begins to increase in size when the concentration of salt becomes larger than the concentration of charge on the polymer itself. This screening effect occurs in ionic solutions and works by creating an "atmosphere" of charges of the opposite sign in order to screen the long-range interactions, and gives rise to a net gain in electrostatic energy (151). We measured the FRET for both MBP-UTC1 and MBP-UTC8 in the presence 150 mM NaCl in order to monitor the extent of change screening of the transfer efficiencies (data not shown). Preliminary results show no statistical change in FRET efficiency for either MBP-UTC1 or MBP-UTC8 in the presence of NaCl as compared to aqueous solution. More replicates must be performed in order to confirm this result.

MBP in CNS myelin is also known to interact with the small ligand zinc. Zinc is found in high concentration (~50 µM) in the myelin cytoplasm, and increases up to 3-fold in the erythrocytes during MS pathology (152). MBP is known to be a prominent zinc-binding protein via intrinsic histidyl-residues (74). Divalent cations, such as Zn2+, can induce a random coil to order transition in several IDPs, including MBP (153). Zinc is believed to stabilize the protein's association with the lipids in order to form compact myelin in the CNS (154), and thus is proposed to fulfill a similar role to that of zinc in the peripheral nervous system myelin (155). Previous studies by the Harauz group characterized the effect of zinc on MBP by FTIR, which spectroscopy revealed that there was a rearrangement of secondary structure components upon addition of zinc (74). Results for FRET distances and efficiencies in the presence of 10x excess Zn (by moles) suggest no significant change to either MBP-UTC1 or MBP-UTC8 as compared to aqueous solution. Previous studies, in true membrane- environments, showed changes in secondary structure upon increasing the amount of zinc, suggesting a synergistic effect of the myelin membrane and divalent cations on MBP structural rearrangement (156). Thus, it could be due to the lack of a lipid component that we see no significant change in conformation of MBP- UTC1 or MBP-UTC8. Furthermore, ANS binding (discussed below) performed in the presence of 10x molar excess Zn for both MBP-UTC1 and MBP-UTC8 showed no shift in the maximal enhancement (data not shown) as compared to aqueous solution.

5.2 Membrane mimetic solvents and their uses in IDPs

In this study, we sought to characterize the mechanism of 18.5-kDa MBP disorder-to-α- helical transition through a conformational and thermodynamic analysis of MBP-UTC1 and MBP-UTC8. From previous structural studies, we have produced a two-dimensional map of MBP (Figure #1, #2A, and #2B) revealing three regions with α-helical propensity (approximately T33-F43, P82-V91, Y141-K153) that we have shown are important for tethering

MBP to the oligodendrocyte membrane. Other studies have focused on the three MBP α1-, α2-, and α3-peptides (A22-K56, S72-S107, and S133-S159, respectively, murine 18.5-kDa species 2+ numbering), and found that both the α1 and α3 regions are important interaction sites for Ca - activated calmodulin, and that the α1-containing region may also interact with actin (158). The α2 region is part of a molecular switch, and may be conformationally linked to an adjacent proline- rich structure that is an SH3-ligand (20, 83, 141, 158). These three regions in 18.5-kDa MBP 65

may adopt the α-helical conformation upon interaction with its partners in a binding-induced folding mechanism, or alternatively, interaction may occur through conformational selection whereby a population of the α-helical conformer is present and available for interaction (66, 159, 160). However, the applications of such conventional ensemble spectroscopic techniques such as NMR, EPR, and CD have not determined all the necessary conformational information about MBP (distance restraints) in order to generate a structural model. Not all structured and unstructured domains of MBP, and/or the effect of PTMs on structure, have yet been resolved (131). There is also a lack of information regarding the mechanism of disorder-to-order transition which is useful both for defining the protein’s conformational space and may be important in binding-induced folding mechanisms. Furthermore, identifying the populations of energetically- distinct sets of ensembles along the transition pathway may be instrumental for determining the potential for conformational selection.

The propensity to form α-helical structure in the full-length MBP-UTC1 and MBP-UTC8 18.5-kDa protein variants was probed using TFE-titration curves which have long been used to determine the free energy of α-helix formation of peptides as well as of proteins, through fitting to thermodynamic folding models (124-126).

Trifluoroethanol has been shown to induce α-helical structure mainly in regions which already have α-helix-forming propensity, and it has been shown to be an effective membrane- mimetic solvent for many proteins, including MBP, when used judiciously (97, 158, 161-163). Consistent with this idea, analysis of previously published NMR data of MBP in 30% TFE has demonstrated that the pattern of α-helical formation in TFE corresponds to what has been found in reconstituted lipid bilayer environments (see Figure #2B and accompanying figure legend). At low concentrations, it has been suggested that TFE can effectively simulate membrane surface conditions by lowering the dielectric constant of the solution, whereas at higher concentrations, there is the added effect of the preferential interaction of TFE with the protein, which can effectively simulate the effects of protein-membrane binding (153). Thus, TFE is useful in analyzing the disorder-to-α-helical transition of MBP that occurs as it approaches the oligodendrocyte membrane surface and eventually interacts (164, 165). Of direct relevance to this work is an independent study by the group of Israelachvili et al., who demonstrated using a

surface forces apparatus and quartz crystal microbalance that MBP in solution first adsorbs to a solid surface such as mica or SiO2, then undergoes a conformational change (165). It can be predicted that the same phenomenon occurs with membrane surfaces (111).

5.3 Thermodynamic analysis of the disorder-to-α-helical transition in full- length 18.5-kDa MBP-UTC1 using 2,2,2-trifluoroethanol (TFE)

In this study, we performed a thermodynamic analysis of TFE-titration curves monitored by CD spectroscopy, which probed changes in secondary structure, and tryptophan fluorescence quenching by IAEDANS, which was used to probe for concomitant changes in global organization (Figure #11A-E, #13A and #13B and Table #4). The data are well described by a 3-state transition model (disordered↔intermediate↔α-helical), indicating that intermediate formation in MBP is a first-order process involving global cooperative interactions. The observed global cooperativity of the disordered↔α-helical transition of 18.5-kDa MBP suggests that the conformational space of each equilibrium species is somewhat limited. Nevertheless, it is important to note that each of the three identified equilibrium species will still have significant conformational freedom, and thus that their structural properties likely represent an average of ensemble conformations, with each ensemble arranged in distinct clusters that are separated by energy barriers (Figure #14).

The ΔGtotal of the disordered-to-α-helical transition in MBP-UTC1 is a fairly modest 3.8±1.1 kcal mol-1, and we found that the ΔG of the first transition (disordered↔intermediate) was more than 50% lower than the second transition (intermediate↔α-helical). Due to the relatively low ΔG1 value, there is a significant population of intermediate (fractional population of ~0.1) even in the absence of TFE (Figure #15). Surprisingly, upon transitioning from the intermediate to the α-helical conformation, we find that 18.5-kDa MBP globally re-expands. The α-helical conformation in MBP is highly populated above 25% TFE (v/v), and could be stabilized by preferential interaction with this membrane-mimetic solvent.

The observed collapse and subsequent re-expansion mechanism of the disorder-to-order transition in 18.5-kDa MBP helps to explain the observed pattern of ANS-enhancement at different TFE concentrations, which largely mirrors the fractional population curve of the intermediate (Figures #9, #10 and #15). These data thus suggest that it is predominantly the 67

intermediate that binds to ANS, whereas there is little ANS interaction with the more extended disordered or α-helical conformations. The ability to bind ANS is a commonly observed characteristic of collapsed protein conformations which have solvent-exposed hydrophobic pockets, and is a property associated with molten-globule and pre-molten globule conformations. Significantly, both the molten-globule and pre-molten globule conformations possess some ordered secondary structure which is notably absent from the MBP intermediate identified here. This phenomenon suggests that collapse in the intermediate state is not driven by cooperative interactions between the α-helical domains, as α-helical formation occurs concurrently with global re-expansion in a second transition. The presence of hydrophobic pockets in the intermediate suggests that compaction could be, at least in part, driven by the classical hydrophobic collapse model. However, since 18.5-kDa MBP, like most IDPs, has a low overall hydrophobicity, it is unlikely that this is the dominant mechanism for global collapse. Rather than being the driving force of collapse, the presence of hydrophobic patches may have functional significance; for example, they may be important for binding of the intermediate MBP conformer to a partner or surface (150, 164). It has been suggested that molecular recognition fragments in disordered proteins or regions may have evolved to have a high proportion of hydrophobic patches, which can serve as non-specific binding sites that mediate induced folding (166). The fact that the population of the intermediate is highest at ~14% TFE suggests that collapse is mediated by hydrogen bonding, consistent with the new models of protein collapse which argue that backbone interactions are the dominant force (167).

5.4 Defining a physiological role for MBP-UTC8: A structural and thermodynamic comparison of the MS-MBP isoform

Preliminary FRET results for MBP-UTC8 in solution (0% TFE) are largely similar to those seen for MBP-UTC1 showing the presence of a flexible but non-random coil state that completely lacks any secondary structure as monitored by CD spectroscopy. One discrepancy is that MBP-UTC8-S159C appears to remain much closer to W113 as compared to MBP-UTC1- S159C when in solution, and remains so over the entire range of TFE concentrations studied. This observation suggests a more compacted C-terminus, and possibly a more compact global structure.

From thermodynamic analysis and the resulting fractional populations (Figure #14 and #15 we see that the fractional population of the disordered ensemble is about ~0.8 at 0% TFE v/v as compared to ~0.9 for MBP-UTC1. Furthermore, in MBP-UTC8 the fractional population of the disordered species declines to only ~0.01 at 8% TFE. This result is in stark contrast to what is seen for MBP-UTC1, in which the disordered species remains populated over a much larger range of TFE concentrations, and decreases to ~0.01 at 20% TFE v/v. More importantly, the intermediate ensemble for MBP-UTC8 is maximally populated at much lower concentrations of TFE, totalling >0.95 of the ensemble population at as little as 8% TFE v/v, and reaching 1.0 at ~10% TFE v/v. The relatively higher population of the intermediate species when comparing

MBP-UTC1 and MBP-UTC8 mainly results from ΔG2 being significantly higher in the latter, suggesting that the intermediate is more stable compared to the α-helical conformation. In relating these preliminary thermodynamic data of MBP-UTC8 to a possible physiological role in MS, it is possible that a larger energy barrier between intermediate and α-helical ensembles may impede myelin stability, as formation of the α-helices is required for membrane association. While this is an interesting proposition, more data will have to be collected to verify this finding.

Chapter 6.0: Conclusions, Significance and Future Directions

Intrinsically-disordered proteins are highly flexible and dynamic, allowing them to sample a wide variety of conformations. They are usually envisioned as extended structures but are known to also sample collapsed conformations containing various degrees of ordered secondary structure. The conformational energy landscape of IDPs, in the absence of a binding partner, is often represented as either a shallow funnel, or as a totally random landscape in which all conformational states are readily accessible with little to no thermodynamic barriers (167- 168). Here, in our analysis of 18.5-kDa MBP, which is a stereotypical IDP, we find that the disorder-to-α-helical transition follows a biphasic mechanism at equilibrium in which three distinct states (or more likely, three ensemble clusters) are separated by measurable energy barriers (Figure #14A and #14B). The FRET results of MBP in solution are consistent with the presence of a relatively disordered conformation, yet more compacted than a random coil state, that completely lacks any ordered secondary structure as monitored by CD (Figure #8) and also by NMR spectroscopy (169). These kinds of disordered collapsed conformations have been observed in many IDPs including Sic1 (170) and osteopontin (171), and also in amyloidogenic IDPs such α-synuclein (172), where they may represent important aggregation precursors. It is, therefore, of significant interest to characterize these collapsed conformations within IDP ensembles both structurally and mechanistically.

Interestingly, when considering the thermodynamic data as well as the results from FRET measurements, we find that it is the intermediate conformational ensemble of 18.5-kDa MBP that is the most compact, and not the α-helical ensemble. A α-helical rich state that is relatively globally extended may, in fact, be functionally important, as it could potentially allow MBP to better bridge the two cytoplasmic leaflets in the major dense line of myelin, which is roughly 3 nm in size. Additionally, a more extended α-helical state may facilitate faster membrane binding by providing more surface area for interaction and by giving the α-helical segments greater flexibility to attain the correct orientation for insertion into the membrane. Furthermore, we suggest that differences observed here in both the structural conformation of MBP-UTC1 and MBP-UTC8 as well the differences that observed in the thermodynamic values for the

disordered↔intermediate↔α-helical transition provide evidence that further supports the suggestion that deimination of MBP may be an early event that precipitates MS pathogenesis.

Interesting avenues of future research would be to study MBP in a natural lipidic environment which is both physiologically and structurally pertinent. Even when membrane- associated, MBP is still largely mobile and able to interact with various cellular components (146, 173). Previously, DPC micelles have been used to induce α-helicity of MBP-UTC1 and its associated peptides by NMR and CD spectroscopy, but remains to be completed for the MBP- UTC8 variant. Furthermore, performing FRET studies in the presence of DPC would be well- suited for a structural comparison similar to the work completed here. The effects of other PTMs such as phosphorylation on the disorder-to-α-helical transition of MBP should also be measured. This modification, which reduces the overall net change of MBP and also alters the charge distribution on the protein (22, 83), could modulate the ensemble conformations sampled by the protein. Additionally, these PTMs could also directly affect α-helical formation. For example, using the MBP α2-peptide, the Harauz lab have shown previously that phosphorylation at mitogen-activated protein kinase sites (T92 and T95, murine 18.5-kDa numbering) alters the disorder-to-α-helical transition of the second α-helical segment (P82-I90) by destabilizing the C- terminal end of the α-helix (141). The methodology that we have illustrated here would allow us to quantitatively assess the effects of PTMs on the global disorder-to-α-helical transition of MBP, which could provide insights into the regulation of myelination, multiple sclerosis pathogenesis and a better theoretical framework of the factors that control the conformational energy landscape of IDPs.

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