Mass spectrometry-based structural insights into protein assemblies

Abstract

Complications with protein homeostasis, genetic mutations or post-translational modifications can lead to deficits in the correct folding, and therefore function, of proteins. These misfolding events are often associated with protein aggregation via amorphous and fibrillar pathways. Protein fibrils have long been associated with a range of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases, through the formation of fibrillar plaques that are deposited in diseased regions of the brain. Current understanding of misfolding diseases point to low molecular weight oligomers en route to fibril formation, instead of fibrils themselves, as the pathological species. More specifically, the relationship between lipid membranes and misfolded oligomers plays a role fibril kinetics and may be a vital component of disease aetiology via membrane disruption or leakage by oligomeric pore formation. To date, there are no effective therapies associated with neurodegenerative misfolding diseases, despite great advances in our understandings. To treat diseases such as these we require a greater understand of the mechanisms at play. Traditional structure determination techniques such as X-Ray crystallography and nuclear magnetic resonance (NMR) spectroscopy are often not amenable to the study of misfolding proteins given their transient nature, therefore new structure determination techniques must be exploited. This thesis utilised and developed a combined biophysical approach, with a focus on mass spectrometry (MS), to better understand protein misfolding and aggregation. Protein quality control systems like chaperones combat misfolding but these can fail, leading to disease. The mechanisms of molecular chaperones depend in part on protein morphology and can interact differently with the same protein if it undergoes different structural stresses. In this work, ion-mobility mass spectrometry (IM-MS) was utilised to understand the protective mechanisms of a molecular chaperone, β-casein (β-CN), against the aggregation of α-lactalbumin (α-LA). α-LA is capable of both fibrillar and amorphous aggregation, both of which were inhibited by β-CN at substoichiometric concentrations. During amorphous aggregation, analytical size exclusion revealed α-LA formed stable, high molecular weight complex with β-CN. IM-MS coupled with collision-induced dissociation (CIU) described transient structural interactions wherein β-CN stabilised α-LA, as well as reduced conformational heterogeneity while in the gas-phase. Overall, this data demonstrates the practicality of biophysical techniques, particularly IM-MS with CIU, to explore the interactions between misfolding proteins and chaperones. α-LA is a powerful model for protein misfolding, but it is not associated with disease. Amyloid beta 40 (Aβ40) and α-synuclein (α-syn) proteins are implicated in Alzheimer’s and Parkinsons’s diseases, respectively. Here these intrinsically disordered proteins were examined to better understand their interaction with the cellular membrane and its effects on fibril inhibition by small molecules, EGCG and resveratrol. Large unilamellar vesicles (LUVs) were used to model the cellular membrane, and it was revealed that they both increased the rate of fibrillar aggregation and decreased the effectiveness of the known fibril inhibitors. Using oligomer specific immunoblotting and IM-MS it was shown that EGCG and resveratrol work through differing mechanisms, wherein resveratrol targeted the elongation phase of aggregation while EGCG targeted the nucleation phase. IM-MS showed EGCG to preference binding to more compact forms of monomeric Aβ40 and α-syn, while LUVs influenced conformational changes indicative of nucleation. These observations combined to form a more detailed mechanistic insight into the protein-lipid-inhibitor relationship and highlighted that current approaches toward drug design may be misguided if the effects of lipid membranes are not properly considered. The transient nature of misfolding proteins can make structural determination complicated using traditional high-resolution structure determination techniques like X-ray crystallography and NMR spectroscopy. While useful, native- and IM-MS cannot offer specific detail in terms of tertiary structure and subunit architecture. Cross-linking MS (XL-MS) has emerged as a valuable complementary tool for uncovering structural information from proteins. While cross-linkers are available commercially, the rapid emergence of new analytical strategies means specific features or combinations of features are not readily available, and the highly variable nature of proteins means the optimum cross-linking regent may be system specific. To remedy this and enable to a wide range of cross-linker chemistries, a modular synthetic protocol was designed to allow for the incorporation custom reactive groups, spacer-arms, enrichment motifs and other features. The protocol was used to develop a small library containing 8 unique cross-linkers, each containing different features. The reagents were optimised using model peptides to monitor efficiency, labile spacer-arm fragmentation and derivatisation with enrichment and dye motifs. The wide applicability and straightforward synthesis of the modular protocol aims to remedy the lacking diversity in available cross-linkers for broad uptake in XL-MS workflows. Overall, the work in this thesis aims to highlight and expand MS-based techniques for protein structure determination. The presented examples showcase that investigating transient proteins require adaptable techniques like native MS, IM-MS and XL-MS. The combination of these and other biophysical techniques provide a toolset capable of handling a great range of protein structures deemed too complicated for traditional structure determination techniques.