Research into the relationship between the structure and function of proteins has been ongoing now for several decades. More recently, there has been an explosion in the investigation of the dynamic properties of proteins, and how their dynamic propensity relates to their function. This new direction in protein research requires new techniques to analyse protein dynamics, since most traditional techniques are biased towards a fixed tertiary structure. Mass spectrometry (MS) is emerging as a powerful tool to probe protein dynamics since it can provide information on interconverting conformations and has no preference towards the folded state. Furthermore, its low sample consumption, rapid data acquisition and low data processing positions MS as an attractive tool in protein structure research. The hybrid technique of ion mobility-mass spectrometry provides further insight into the range of conformations adopted by proteins and protein complexes, by providing information on the size in terms of rotationally averaged collision cross section. The work presented in this thesis considers proteins with a range of structural characteristics. We use ion mobility mass spectrometry to investigate proteins of different extents of disorder, protein complexes with dynamic entities and a system that undergoes structural rearrangement upon ligand binding. First, a framework of mass spectrometry experiments is described which allows identification of the extent of structure and disorder within proteins. This framework is tested on a range of different systems throughout the thesis. Differences in the gas-phase properties of two conformationally dynamic proteins which behave similarly in solution are investigated and from this research we postulate a new ionisation mechanism for partially folded proteins. The dynamic propensity of C-terminal p27 is investigated and compared to two permutants which allows us to delineate how the location of charged residues in a primary sequence affects the structure of a protein. We monitor the 'folding-upon-binding' behaviour of p27 upon association with its binding partners, and how this differs with the order of charged residues in the linear sequence. Finally, we describe the structural rearrangement of Fdc1 upon the binding of its cofactor; a prenylated FMN molecule. This thesis demonstrates the suitability of ion mobility-mass spectrometry for the investigation of dynamic properties of proteins and protein complexes.

Calcineurin (CaN) is a highly regulated Ser/Thr protein phosphatase that plays critical roles in learning and memory, cardiac development and function, and immune system activation. Alterations in CaN regulation contribute to multiple disease states such as Down syndrome, cardiac hypertrophy, Alzheimer’s disease, and autoimmune disease. In addition, CaN is the target of the immunosuppressant drugs FK506 and cyclosporin A. Despite its importance, CaN regulation is not well understood on a molecular level. Full CaN activation requires binding of calcium-loaded calmodulin (CaM), however little is known about how CaM binding releases CaN’s autoinhibitory domain from the active site. Previous work has demonstrated that the regulatory domain of CaN (RD) is disordered. The binding of CaM to CaN results in RD folding. Folding of the RD in turn causes the autoinhibitory domain (AID) located C-terminal to the RD to be ejected from CaN’s active site. This binding-induced disorder-to-order transition is responsible for the activation of CaN by CaM. In this work, we explore the nature of the disorder in the RD and its transition to an ordered state, demonstrating that the RD exists in a compact disordered state that undergoes further compaction upon CaM binding. We also demonstrate that a single CaM molecule is responsible for binding to and activating CaN. Finally, we determine that the CaM binding to CaN induces an amphipathic helix (the distal helix) C-terminal to the CaM binding region. The distal helix undergoes a hairpin-like chain reversal in order to interact with the surface of CaM, resulting in the removal of the AID from CaN’s active site. We employ site-directed mutagenesis, size-exclusion chromatography, protein crystallography, circular dichroism spectroscopy, fluorescence anisotropy and correlation spectroscopy, and phosphatase activity assays to investigate the ordering of CaN’s regulatory domain, the stoichiometry of CaN:CaM binding, and the impact of the distal helix on CaM activation of CaN.

The work presented in this thesis has contributed with knowledge to several aspects of protein-protein interaction involving PDZ domains. A substantial amount of our proteome contains regions that are intrinsically disordered but fold upon ligand interaction. The mechanism by which disordered regions bind to their ligands is one important piece of the puzzle to understand why disorder is beneficial. A region in the PDZ domain of nNOS undergoes such a disorder-to-order transition to form a b-sheet in the binding pocket of its partner. By studying the kinetics of interaction, in combination with mutations that modulate the stability of the aforementioned region, we demonstrate that the binding mechanism consists of multiple steps in which the native binding interactions of the b-sheet are formed cooperatively after the rate-limiting transition state. These mechanistic aspects may be general for the binding reactions of intrinsically disordered protein regions, at least upon formation of β-sheets.               The second part of this thesis deals with the engineering of proteins for increasing affinity in protein-protein interaction. Infection by high-risk human papillomavirus (hrHPV) can lead to cancer, and the viral E6 protein is an attractive drug target. E6 from hrHPV natively interacts with the well-characterized PDZ2 domain in SAP97, which we used as a scaffold to develop a high affinity bivalent binder of hrHPV E6. We initially increased PDZ2's affinity for E6 6-fold, but at the cost of decreased specificity. Attaching a helix that binds E6 at a distant site, increasing the affinity another14-fold, completed the design.             The final work of this thesis investigates if binding studies conducted with isolated PDZ domains is representative of the full-length proteins they belong to. It has been suggested that ligand binding in PDZ domains can be influenced by factors such as adjacent domains and interactions outside of the binding pocket. We studied these aspects for the three PDZ domains of PSD-95 and found that they on the whole function in an independent manner with short peptides as ligands, but that interactions outside of the PDZ binding-pocket may be present. The representative length of the PDZ interaction partner should therefore be considered.

No description available.

Inhibition of the intrinsically disordered proteins (IDP) is a recognized issue in drug research. Standard approaches, based on key-lock model, cannot be used in the absence of rigid structure and defined active site. Here a basic helix-loop-helix leucine zipper (bHLHZip) domain of c-Myc was studied, which is intrinsically disordered and prone to aggregation. Chemical denaturation of proteins is a widely accepted technique to study protein folding, but here this methodology was applied to IDP, observing its effect on the structural ensemble of c-Myc by NMR spectroscopy. Nonlinear chemical shift changes indicated cooperative unfolding of the helical structure of the part of the leucine zipper domain in parallel with the melting of the N-terminal helix. Paramagnetic relaxation enhancement (PRE) was used to probe long-range structure and revealed presence of long-range contacts. The following search for inhibitors can be directed to the search for ligands, locking c-Myc in its more compact conformation. Protein self-association is a problem typical for IDPs and intrinsic process for all proteins at high concentrations. It leads to increased viscosity, gelation and possible precipitation, which cause problems in protein manufacturing, stability and delivery. If protein drugs require high dosing, special approaches are needed. At high concentrations proteins experience conditions close to the crystal state, therefore interactions in solution could potentially coincide with crystal lattice contacts. A range of diverse methods is used to study this process, but the complexity of the mechanism makes it hard to build a reliable model. Here, the self-association of streptococcal Protein G (PrtG) was studied using Nuclear Magnetic Resonance (NMR) spectroscopy in solution. The properties of protein-protein interactions at high concentration, up to ~ 160 mg/ml, were studied at residue-level resolution. Residue specific information on protein dynamics was obtained using 15N relaxation measurements. The experiments were carried out at multiple concentrations. Variation in the rotational correlation time over these concentrations showed changes in the protein dynamics, which indicated weak protein-protein interactions occurring in solution. Pulsed-field gradient NMR spectroscopy was used to monitor translational diffusion coefficients in order to estimate the degree of protein self-association. Oligomer formation was also monitored by looking at variations in 1H and 15N amide chemical shifts. Better understanding of protein self-association mechanisms under different conditions could assist in developing methods to reduce the level of reversible protein self-association in solution at high protein concentrations.

The myelin basic protein (MBP) family arises from different transcription start sites of the Golli (gene of oligodendrocyte lineage) gene, with further variety generated by differential splicing. The “classic” MBP isoforms are peripheral membrane proteins that facilitate compaction of the mature myelin sheath, but also have multiple protein interactions. As an intrinsically disordered protein, MBP has proven to have complex structural and functional relationships with proteins in vitro including actin, tubulin, Ca2+-calmodulin, and multiple protein kinases. The investigations reported in this thesis were to further examine the multifunctionality, and protein:protein interactions of MBP with cytoskeletal and SRC homology 3 domain (SH3) proteins in cells using an oligodendrocyte (OLG) model system to better understand MBP’s contributions to membrane structure, formation, and elaboration in the developing OLG. A new function of MBP has been described showing that classic MBPs can modulate voltage operated calcium channels (VOCCs) by direct or indirect protein-protein interactions at the OLG cytoplasmic leaflet. These interactions contribute to global calcium homeostasis, and may play a complex developmental and spatiotemporal role in the regulation of oligodendrocyte precursor cell (OPC) migration and OLG differentiation. The importance of MBPs SH3 ligand binding domain within its central amino acid region was investigated with the protein-tyrosine kinase Fyn. Co-expression of MBP with a constitutively-active form of Fyn in OLGs resulted in membrane process elaboration, a phenomenon that was abolished by amino acid substitutions within MBP’s SH3-ligand domain. These results suggest that MBP’s SH3-ligand domain plays a key role, and may be required for proper membrane elaboration of developing OLGs. Lastly, interactions of MBP with the OLG cytoskeleton were investigated in OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). MBP redistributes to distinct ‘membrane-ruffled’ regions of the plasma membrane where it had increased co-localization with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. The results presented here advance our understanding regarding protein:protein interactions of MBP, and its multifunctionality in OLGs with regards to membrane formation and elaboration. / This work was supported by the Canadian Institutes of Health Research (MOP #86483, J.M.B. and G.H.), and Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC, G.H., RG121541). G.S.T.S. was a recipient of a Doctoral Studentship from the Multiple Sclerosis Society of Canada

Alpha-synuclein (AS) is a natively unfolded protein whose structure is extremely sensitive to its environment. The hallmark of Parkinson’s disease (PD) is aggregation and deposition of AS in inclusion bodies. Formation of misfolded AS monomers which are partially folded is the first and critical stage in fibrillation of AS and is a good target for designing therapeutic strategies. Characterization the biochemical properties of partially folded intermediates induced by fibrillization and anti- fibrillization agents will help to design drugs as new inhibitors of AS misfolding and aggregation. Nanopore analysis is an emerging technique for studying the molecular mechanism of protein misfolding. This technique was used to characterize the conformational change of AS in the presence of two groups of chemicals; anti-parkinsonian small molecules (dopamine and nicotine) and Parkinson’s developing toxin (Cu(II) and methamphetamine). Other biophysical techniques such as NMR spectroscopy and isothermal titration calorimentry (ITC) were able to confirm the nanopore analysis results and also to study other biophysical properties of the partially folded intermediates such as the binding constant of the interaction and the secondary structure content. The results from nanopore analysis showed that both groups of ligands shifted the blockade current peak of AS (centered at -86 pA) to lower blockade currents but in a different manner. Anti-parkinsonian drugs shifted the blockade current of AS to intermediate peaks between -40 to -80 pA but Parkinson developing toxins shifted the peak to a lower blockade current centered at -25 pA which suggests a more compact conformation. Thus nanopore analysis distinguished the different conformation induced by different ligands. Furthermore nanopore analysis with AS fragments showed that these ligands bind to different regions of AS. NMR spectroscopy of AS in the presence of dopamine and nicotine isomers was in agreement with the nanopore analysis and showed conformational changes of AS in a concentration dependent manner. CD spectroscopy results showed that the secondary structure of AS alone and in the presence of ligands was mostly random coil and suggests a loop formation model for the interaction of ligands with AS. The results of this thesis showed the application of nanopore analysis as a real-time and label-free technique to screen a library of ligands for designing misfolding inhibitors for PD treatment. The result of a synergic experiment with nicotine and caffeine showed that combination of these anti-parkinsonian small molecules would be a promising new drug for treatment of PD.

We applied single-molecule fluorescence (SMF) methods to probe the properties of individual fluorescent probes, and to characterize the proteins of interest to which these probes were attached. One remarkable advantage of SMF spectroscopy is the ability to investigate heterogeneous subpopulations of the ensemble, which are buried in ensemble averaging in other measurements. Other advantages include the ability to probe the entire dynamic sequences of a single molecule transitioning between different conformational states. For the purpose of having an extended observation of single molecules, while maintaining the native nanoscale surroundings, we developed an improved vesicle preparation method for encapsulating scarce biological samples. SMF investigations revealed that molecules trapped in vesicles exhibit nearly ideal single-emitter behavior, which therefore recommends the vesicle encapsulation for reproducible and reliable SMF studies. Hyperactive Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein contributes significantly to human cancers, such as leukemia and lymphoma. We have proposed a novel therapeutic strategy by designing a cholesterol-based protein membrane anchor (PMA), to tether STAT3 to the cell membrane and thus inhibit unwanted transcription at the cell nucleus. We designed in vitro proof-of-concept experiments by encapsulating STAT3 and PMAs in phospholipid vesicles. The efficiency and the stability of STAT3 anchoring in the lipid membrane were interrogated via quantitative fluorescence imaging and multiparameter SMF spectroscopy. Our in vitro data paved the way for the in vivo demonstration of STAT3 inhibition in live cells, thus demonstrating that PMA-induced protein localization is a conceptually viable therapeutic strategy. The recent discovery of intrinsically disordered proteins (IDPs) highlights important exceptions to the traditional structure-function paradigm. SMF methods are very suited for probing the properties of such highly heterogeneous systems. We studied in detail the effects of electrostatics on the conformational disorder of an IDP protein, Sic1 from yeast, and found that the electrostatic repulsion is a major factor controlling the dimensions of Sic1. Based on our data we also conclude that a rod-like shape seems a better candidate than a random Gaussian chain to describe and predict the behavior of Sic1.

We applied single-molecule fluorescence (SMF) methods to probe the properties of individual fluorescent probes, and to characterize the proteins of interest to which these probes were attached. One remarkable advantage of SMF spectroscopy is the ability to investigate heterogeneous subpopulations of the ensemble, which are buried in ensemble averaging in other measurements. Other advantages include the ability to probe the entire dynamic sequences of a single molecule transitioning between different conformational states. For the purpose of having an extended observation of single molecules, while maintaining the native nanoscale surroundings, we developed an improved vesicle preparation method for encapsulating scarce biological samples. SMF investigations revealed that molecules trapped in vesicles exhibit nearly ideal single-emitter behavior, which therefore recommends the vesicle encapsulation for reproducible and reliable SMF studies. Hyperactive Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein contributes significantly to human cancers, such as leukemia and lymphoma. We have proposed a novel therapeutic strategy by designing a cholesterol-based protein membrane anchor (PMA), to tether STAT3 to the cell membrane and thus inhibit unwanted transcription at the cell nucleus. We designed in vitro proof-of-concept experiments by encapsulating STAT3 and PMAs in phospholipid vesicles. The efficiency and the stability of STAT3 anchoring in the lipid membrane were interrogated via quantitative fluorescence imaging and multiparameter SMF spectroscopy. Our in vitro data paved the way for the in vivo demonstration of STAT3 inhibition in live cells, thus demonstrating that PMA-induced protein localization is a conceptually viable therapeutic strategy. The recent discovery of intrinsically disordered proteins (IDPs) highlights important exceptions to the traditional structure-function paradigm. SMF methods are very suited for probing the properties of such highly heterogeneous systems. We studied in detail the effects of electrostatics on the conformational disorder of an IDP protein, Sic1 from yeast, and found that the electrostatic repulsion is a major factor controlling the dimensions of Sic1. Based on our data we also conclude that a rod-like shape seems a better candidate than a random Gaussian chain to describe and predict the behavior of Sic1.

Eukaryotic initiation factor-4E (eIF4E) controls the rate of cap-dependent translation initiation and is in turn exquisitely regulated by 4E-BPs. 4E-BP2 binds eIF4E with the highest affinity and is implicated in cancer, and metabolic and neurological disorders. Herein we use NMR, ITC and fluorescence to characterize 4E-BP2 structural and binding properties. Isolated 4E-BP2 is intrinsically disordered, but possesses some transient secondary structural propensities. eIF4E, however, is folded but has a disordered N-terminus. The eIF4E:4E-BP2 interaction is tight (Kd = 10-9 nM) and involves 4E-BP2 C-terminal and canonical binding regions, and the disordered eIF4E N-terminus. 4E-BP2 remains largely disordered upon binding to eIF4E. Noteworthy, high affinity interactions are not necessarily mediated by static structures, and 4E-BP2 binding is not the simple “disorder-to-order” transition observed in many interactions involving disordered proteins. This study offers molecular insights into 4E-BP2 functionality, and lays a foundation for development of novel therapies for cancer and neurological disorders.