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Mammalian respiratory complex I (NADH:ubiquinone oxidoreductase) is an intricate multi-subunit, energy-transducing membrane protein that is essential for aerobic energy metabolism and NADH/NAD⁺ homeostasis. It couples the energy released from NADH oxidation and ubiquinone (Q) reduction to pump four protons across the inner mitochondrial membrane, contributing to the proton motive force used to synthesise ATP. Despite recent advances in structural knowledge and decades of biochemical investigations, the mechanism of redox-coupled proton translocation by complex I is still unknown. In the work presented in this thesis, electron cryomicroscopy (cryo-EM) was used as a primary tool to provide fundamental insight on this mechanism by generating three-dimensional reconstructions of complex I bound to substrates, ligands or inhibitors. Analyses of the structural data and further interrogation by complementary biochemical, biophysical, and computational approaches were used to develop an integrated understanding of substrate and inhibitor binding to complex I. First, to investigate the mechanism of action of a drug in phase 1 clinical trials against cancers reliant on oxidative phosphorylation (IACS-010759), the structure of mouse complex I inhibited by IACS-2858 - a tighter binding derivative - was resolved to a global resolution of 3.0 Å. The inhibitor, which bears little resemblance to ubiquinone-10 (Q₁₀), occupies the entrance to the Q-binding channel in a 'cork-in-bottle' binding mode not previously observed for complex I. Key inhibitor-enzyme interactions were identified, providing a molecular basis for understanding cross-species differences in binding affinities. Modelling of kinetic data showed that IACS-2858 is a simple one-site competitive inhibitor, and the structural motif of a 'chain' of aromatic rings was proposed as a characteristic that promotes complex I inhibition. Next, a strategy for reconstituting bovine complex I into lipid nanodiscs supplemented with exogenous Q₁₀ was devised to probe how the native substrate Q₁₀ binds to the 'reactive site' of the Q-binding channel. Five structurally and biochemically distinct conformational classes were identified at global resolutions up to 2.3 Å. These structures fall into three major states: an 'active' ready-to-catalyse state, a 'deactive' pronounced resting state, and a 'slack' state that appears partially disrupted and is of uncertain physiological and biochemical relevance. Comparisons of the deactive structures suggested how substrate/ligand binding restructures the Q-binding site and why both Q and NADH are required for reactivation. Importantly, a Q₁₀ molecule spanning the entirety of the Q-binding site was observed with the Q-headgroup close to its proposed ligating partners NDUFS2-His59 and NDUFS2-Tyr108. Combined with results from molecular dynamics simulations, these structures reveal how the charge states of key active-site residues influence the Q₁₀ binding pose. The bound Q₁₀ species is attributed to a quinone paused in a 'pre-reactive' conformation.

Membrane proteins perform various important and wide-ranging functions. They exist within a lipid bilayer and their interactions with the components of this bilayer are of great scientific and pharmacological interest due to the ability of these components to regulate protein structure and function. Computational approaches such as multiscale molecular dynamics simulations are well suited to the study of these systems. As such, the research presented here uses an array of these approaches to examine and characterise the interactions between membrane proteins and lipids in two environments: nanodiscs and lipid bilayers. There has been exponential growth in the use of nanodiscs for solubilisation of membrane proteins in a native-like environment, where the membrane protein is embedded in a patch of lipid bilayer constrained by helical proteins. However, the impact of constraining the nanodisc components in this manner is currently not well understood. In this research molecular dynamics simulations are used to investigate these effects; the results presented here describe the changes in lipid properties which occur as a consequence of constraining the nanodisc components and subsequent variations in protein lipid interactions. One family of membrane proteins, the G-protein-coupled receptors, was selected for further study. A simulation approach was used to examine the binding of a membrane lipid, cholesterol, to potential allosteric modulatory sites on these proteins, including the previously identified TM345 site. Interactions with cholesterol at this site were found to be common to several members of this family despite limited sequence conservation. The results presented here demonstrate the importance of understanding the environment in which membrane proteins exist and the impact of changes to this environment upon protein behaviour, properties and interactions.

Membrane proteins play fundamental roles in the regulation of biological processes in cells and organisms. They have many responsibilities, such as signalling and transport of substances across lipid bilayers. Additionally, they provide targets for around 70 % of pharmaceutical drugs. Although membrane proteins are known for their importance, there is a distinct deficit in their structural and functional characterisation. This bottleneck of information is caused by a number of reasons, such as their fragility and ability to degrade and mis-fold. In 2009, a new method was developed to extract these proteins from membranes, utilising the copolymer poly (styrene-co-maleic acid) (SMA), creating nanodiscs to support membrane protein extraction. In the past decade, more copolymers have been developed to counteract limitations of the original SMA; many membrane proteins have also been studied using this technique, and overall structural characterisation of the nanodisc formation has been further investigated. Yet, there is little information on the interactions occurring within the nanodisc, with limited studies exploring membrane protein activity in nanodiscs. The initial purpose behind this research was to investigate the protein-polymer interactions within the nanodisc, by examining protein functionality using two different proteins. This in turn would provide a platform to screen newly synthesised copolymers, to determine their success in extracting membrane proteins, which maintain their natural functionality. However, the findings spurred on additional lines of enquiry, focussing on particular physical aspects of membrane proteins documented to be successfully extracted in literature, and comparing them to those deemed less successful. Further study using mass spectroscopy (MS) proteomics has provided a global insight into potential preferential extraction when comparing SMA to control extracted samples. Lastly, further study into structural characterisation of membrane proteins that have been incorporated into nanodiscs has been investigated. This study used a combination of different techniques; circular dichroism (CD), dynamic light scattering (DLS), small angle neutron scattering (SANS) and ab initio modelling constructs to gather structural information on the protein and nanodisc formation, comparing these methods to others, such as protein crystallography. These findings highlighted the differences between various techniques and the importance of using complementary methods to fully determine nanodisc structures. Overall, this thesis emphasises the complexity of the protein-nanodisc system and introduces potential factors to consider when using this technique, such as protein-polymer interactions.

The cytochrome P450 enzyme system is a multicomponent electron-transfer chain composed of a haem-containing monooxygenase cytochrome P450 (CYP) and one or more redox partners. Eukaryotic CYPs and their redox partner NADPH-dependent cytochrome P450 oxidoreductase (CPR) are involved in many biological processes. Each protein has one N- terminal membrane anchor domain for location within the endoplasmic reticulum (ER). In mammals, CYPs and CPR are especially abundant in liver cells, where they play important roles in the metabolism of steroids, fatty acids, and xenobiotic compounds including numerous drugs of pharmaceutical importance. Incorporation into lipid membranes is an important aspect of CYP and CPR function, influencing their kinetic properties and interactions. In this thesis, soluble nanometer-scale phospholipid bilayer membrane discs, "nanodiscs", were used as a reconstitution system to study the influence of lipid membrane composition on the activities of the abundant human CYP3A4 and human CPR. Both enzymes were expressed and purified from bacteria, and assembled into functionally active membrane-bound complexes in nanodiscs. Nanodisc assembly was assessed by a combination of native and denaturing gel electrophoresis, and a fluorimetric assay was developed to study CYP3A4 reaction kinetics using 7-benzyloxyquinoline as substrate. Kinetic properties were investigated with respect to different lipid membrane compositions: phosphatidyl choline; a synthetic lipid mixture resembling the ER; and natural lipids extracted from liver microsomes. Full activity of the CYP3A4 system, with electron transfer from NADPH via CPR, could only be reconstituted when both CYP3A4 and CPR were membrane-bound within the same nanodiscs. No activity was observed when CPR and CYP3A4 were each incorporated seperately into naodiscs then mixed together, or when soluble forms of CPR were mixed with pre-assembled CYP3A4-nanodiscs. Thus, assembly of the two proteins within the same membrane was shown to be essential for the function of the CPR-CYP3A4 electron transfer system. Comparison of the reaction kinetics in different membrane compositions revealed liver microsomal lipid to have an enhancing effect both on the activity of the assembled CPR-CYP3A4 nanodisc complex, and on the activity of CPR alone incorporated in nanodiscs, when compared either to the synthetic lipid mixture or to phosphatidyl choline alone. Thus, natural lipids appear to possess properties or include components important for the catalytic function of the CYP system, which are absent from synthetic lipid. Input of electrons, measured by NADPH consumption, exceeded product formation rate by the CPR-CYP3A4 complex in nanodiscs, indicating "leakage" in the electron flow, possibly due to uncoupling of the two enzymes. Uncoupling was shown to occur by developing a novel fluorimetric method using the dye MitSOX to detect superoxide production. The significance of this, and to what extent control of coupling could be a natural means of regulation of the CPR-CYP system, remains to be determined. Thus, phospholipid bilayer nanodiscs prove a powerful tool to enable detailed analysis of the reaction kinetics of membrane-reconstituted CPR-CYP systems, and to allow pertinent questions to be addressed concerning the integral significance of the membrane environment.

In order to meet the growing demand for energy in our society and the environmental need to reduce carbon dioxide pollution in our atmosphere, it is imperative that we devise a scalable strategy to convert sunlight to electricity without the use of Earth-limited resources and rare elements. Biohybrid solar devices (BHSDs), described here, which incorporate photosynthetic proteins onto electrode surfaces to facilitate the capture and conversion of sunlight to electricity offer such a strategy, though the efficiencies of these devices remain very low at present. This work is focused on the innovation of retaining the native environment of photosystem I (PSI) within polymer bound nanodiscs, which demonstrate enhanced photochemistry in vitro, and the downstream implications for this innovation to be used in biohybrid solar devices. Here, we demonstrate that the efficiency of BHSDs is significantly reduced at elevated temperature (40 °C), and these effects can be partially mitigated by the introduction of osmolytes such as glycine-betaine and sucrose at 0.5 - 1 M. Further, we present data that suggest retaining the native thylakoid membrane environment of PSI within nanodiscs leads to enhanced photochemistry in vitro. In particular, PSI encapsulated within styrene maleic acid lipid particles (SMALPs) display a 1,000-fold faster conversion of photons to electrons compared to detergent solubilized PSI. To investigate what may be causing this discrepancy, we proceeded to characterize these PSI-SMALPs using a number of biochemical and biophysical analyses. Using small angle neutron and X-ray scattering techniques we determined the PSI-SMALP is approximately 30% larger than detergent solubilized PSI, surrounded by a tightly bound polymer belt with little protrusion from the particle. Mass spectrometry suggests that this increase in size is due to the retention of a lipid annulus surrounding the protein that is highly enriched in one particular lipid, sulfoquinovosyldiacylglycerol (SQDG), compared to the bulk thylakoid membrane, suggesting lateral heterogeneity in both the proteins and lipids in cyanobacterial thylakoid membranes. We also present neutron reflectivity data that suggests that these polymers are most disruptive to SQDG containing lipid monolayers. Lastly, we determined that the esterification of SMA copolymers leads to greatly enhanced solubilization of galactolipid membranes.

Diabetes mellitus affects approximately 350 million people worldwide, leading to the death of about 4.6 million people per year. As a complication of diabetes, 30-40 percent of patients age 50 and older develop peripheral artery disease (PAD). The current standard of care treatments for PAD includes surgical revascularization with bypass grafting or percutaneous interventions. However, these interventions cannot be performed in a significant portion of patients, and many do not respond to these therapies. An alternative approach for treating PAD is to use proteins to stimulate the body to create new vasculature, thus restoring blood flow through its own regenerative processes. Stem cell factor (SCF) is a cytokine that acts through the receptor tyrosine kinase c-Kit to regulate hematopoiesis and has been a candidate protein for treating PAD. Clinical use of soluble SCF would be highly beneficial but has been limited due to toxicity related to mast cell activation. SCF also exists in a transmembrane form that has differential activity from soluble SCF and has not been explored as a therapeutic agent. To explore the transmembrane SCF (tmSCF) as a therapeutic we created formulations of tmSCF embedded in proteoliposomes or in lipid nanodiscs. Mouse models of anaphylaxis and ischemia revealed the tmSCF-based therapies did not activate mast cells and were effective in improving the recovery from ischemia in both wild type and diabetic mice. We also found that the formulation of the lipid nanocarrier to deliver tmSCF altered the biological response and trophism of the tmSCF-based treatments. Proteoliposomal tmSCF preferentially acted on mature endothelial cells to induce angiogenesis while tmSCF nanodiscs had greater activity in inducing stem cell mobilization from the bone marrow and recruitment to ischemic sites. A mechanistic analysis of the effects of the treatments on mast cells, mature endothelial cells and endothelial progenitor cells, revealed that the nanocarriers altered the relative utilization of clathrin- versus caveolin-mediated uptake pathways of c-Kit in response to the treatments. Overall, our studies support tmSCF-based therapies can provide therapeutic benefits without off-target effects on mast cells and that lipid nanocarriers can be used to tailor the properties of membrane protein-based therapeutics.

When integral membrane proteins are visualized in detergents or other artificial systems, an important layer of information is lost regarding lipid interactions and their effects on protein structure. This is especially relevant to proteins for which lipids play both structural and regulatory roles. Here, we demonstrate the power of combining electron cryo-microscopy with lipid nanodisc technology to ascertain the structure of an integral membrane protein - the TRPV1 ion channel - in a native bilayer environment. Using this approach, we could ascertain the locations of annular and regulatory lipids, enabling us to show that specific phospholipid interactions enhance binding of a spider toxin to TRPV1 through formation of a tripartite complex. Furthermore, phosphatidylinositol lipids occupy the binding site for capsaicin and other vanilloid ligands, suggesting a mechanism whereby chemical or thermal stimuli elicit channel activation by promoting release of bioactive lipids from a critical allosteric regulatory site.

Abstract: The interactions between water-soluble proteins and carbohydrates found on the surfaces of cells play important roles in many physiological and pathological cellular processes. Carbohydrates function as receptors for signaling, cellular recognition and adhesion, and pathogen infections. This thesis focuses on the development and application of electrospray ionization mass spectrometry (ESI-MS) methods to detect and quantify protein-carbohydrate interactions in vitro. In Chapter 2, the intrinsic affinities (per binding site) of the protruding domain dimer (P dimer, 69 kDa) of the human norovirus (NoV) strain VA387 to a panel of 47 soluble analogs of histo-blood group antigens (HBGAs) were quantified using the direct ESI-MS assay. Our results revealed that the P dimer exhibits a broad specificity for the HBGAs and bind, albeit weakly (intrinsic association constants (Ka,int) of 102 – 103 M-1), to all of the oligosaccharides tested. Overall, the A and B antigens exhibit stronger binding than the H and Lewis antigens. In addition, the affinities are also affected by the precursor chain type of HBGAs but not by the chain length. In Chapter 3, the applicability of the catch-and-release (CaR)-ESI-MS assay for screening carbohydrate libraries against large protein complexes was demonstrated for the first time. Libraries containing as many as 146 compounds were screened against NoV VA387 subviral P particle (24-mer, 865 kDa). Notably, the results of the screening experiments revealed NoV interactions with oligosaccharides with structures found in human milk and the cell wall of mycobacteria. The affinities of these newly discovered ligands are comparable to those of the HBGA receptors. In Chapters 4 and 6, the direct ESI-MS assay was combined with a competitive binding strategy in order to measure the affinities of protein-carbohydrate interactions that can’t be directly quantified by ESI-MS. In Chapter 4, the affinities of the NoV P particle and virus-like particle (VLP, 180-mer, 10.5 MDa) to HBGA ligands were quantified using the proxy protein ESI-MS method, which utilizes competitive protein binding. The results revealed that HBGA ligands exhibit similar affinities for the P particle and P dimer whereas the HBGA affinities for the VLP are consistently higher than those measured for the P dimer, but within a factor of three. In Chapter 6, the proxy ligand ESI-MS assay, which is on the basis of competitive ligand binding, combined with nanodisc technology to solubilize glycolipids was used to determine the interactions of cholera toxin B subunit homopentamer (CTB5) with GM1, and a family 51 carbohydrate-binding module (CBM) with B type 2 tetrasaccharide neoglycolipid. A notable finding of this study is that the affinities of the glycolipid ligands in the nanodisc are lower, by a factor of ≤5, than those of the corresponding oligosaccharides in solution. In Chapter 5, the screening using CaR-ESI-MS assay revealed the first evidence that human NoVs bind to gangliosides. Moreover, affinities measurements were reported for the NoV VA387 P dimer, P particle and VLP, and VA115 P dimer for a series of ganglioside oligosaccharides. Notably, the ganglioside affinities are similar in magnitude to those of HBGA receptors for NoVs. Additional confirmation of NoV-ganglioside interactions was provided by the binding measurements using the enzyme-linked immunosorbent assays. Finally, also in Chapter 6, a systematic ESI-MS investigation aimed at elucidating the processes that influence binding of water-soluble proteins to glycolipids incorporated into nanodiscs was described. The interactions of CTB5 to GM1 nanodiscs studied by ESI-MS indicated that proteins bind reversibly to nanodisc-associated glycolipids, and that proteins possessing multiple ligand binding sites are able to interact with glycolipids originating from different nanodiscs. Moreover, the nature of the protein-glycolipid complexes detected by ESI-MS is likely to be influenced by the diffusion of glycolipids between nanodiscs.

Abstract: This thesis describes the application of electrospray ionization mass spectrometry (ESI-MS) to investigate non-covalent This thesis describes the application of electrospray ionization mass spectrometry (ESI-MS) to investigate non-covalent interactions. The first research chapter focuses on the protein-carbohydrate interaction. The binding affinities (Ka) were measured for a library of thirty human oligosaccharides and recombinant sub-fragments of TcdA (TcdA-A2) and TcdB (TcdB-B3) using the direct ESI-MS assay. Affinity measurements were also carried on a series of Lewis X analogues that were previously identified as ligands for TcdA. The second research chapter focuses on the peptide-membrane interaction. ESI-MS was employed to investigate peptide-membrane interaction using antimicrobial peptide melittin and phospholipid nanodisc as the model system. The ESI-MS study revealed melittin interact with phospholipid bilayer specifically. This result opens up further investigation on the development of the optimal instrument conditions to study peptide-membrane interaction.