Development of Native Ion Mobility ? Mass Spectrometry Methods for the Analysis of Membrane Proteins from Various Membrane Mimetics

Abstract

Membrane proteins (MPs) are critical drug targets, making up 70%, and hold significant potential for future biomedical breakthroughs. Their structure and function are heavily influenced by the biophysical properties of the native membranes in which they reside. Key factors such as membrane curvature, fluidity, and lipid composition regulate MP functions, affecting oligomerization, ligand binding, and protein conformations. However, studying MPs is challenging due to their hydrophobic nature, which makes them unstable outside their native membranes and complicates purification and analysis. This thesis develops advanced native ion mobility-mass spectrometry (nIM-MS) and collision-induced unfolding (CIU) techniques to better capture complex interactions of MPs in different membrane environments. These tools enable direct measurement of MP stoichiometry, ligand binding, complex organization, and stability. In Chapter 2, we use nIM-MS and CIU to assess how different membrane mimetics affect MP structure, with their CIU pathways providing insights into protein stability, conformational interactions, and dynamics. By evaluating various MPs, including cytochrome P450 3A4, peripheral membrane proteins PMP22 and its L16P mutant, and the GDX transporter, we optimized MP reconstitution with detergent micelles, bicelles, and lipid-based nanodiscs. Our findings reveal that monotopic and transmembrane proteins exhibit over 26% RMSD differences in CIU pathways depending on the mimetic used. Bicelles uniquely reveal an additional fourth feature in transmembrane proteins absent when solubilized in detergents or nanodiscs. Moreover, for transmembrane proteins with available theoretical collision cross section (CCS) values, bicelle environments provided the closest match to experimental measurements, with deviations as small as 1.5 Ų. We demonstrate how different mimetics affect MP lipid-binding, oligomerization, and stability. We also address the challenges associated with using these mimetics, particularly due to unique chemical noise profiles, and emphasize the need for tailored guidelines in MP studies. Chapter 3 details the quantitative evaluation of detergent exchange procedures commonly used for nMS studies. Using high-resolution mass spectrometry (HR-MS), we developed a method to assess exchange efficiencies across commonly used nMS-compatible detergents, allowing detailed characterization of the detergent molecules solubilizing the MP. We found that in some cases, up to 99% of the measured detergent corresponds to the original pre-exchange detergent rather than the desired post-exchange detergent. Key factors affecting exchange rates include the primary detergent, critical micelle concentrations, and MP class. We further demonstrated that increasing the number of sequential BioSpin exchanges can improve detergent exchange efficiency by over 50%. In Chapter 4, we investigate how MP structure, lipid-binding specificity, and membrane properties interact. Our study of Vesicle-associated membrane protein 2 (VAMP2) in synaptic vesicle (SV) liposomes reveals that VAMP2 is stabilized by phosphatidylcholine (PC) in low-fluidity SV liposomes. This stabilization, supported by MD simulations and fusion assays, indicates that PC and cholesterol cluster in the membrane to facilitate VAMP2's functional conformation. Our findings shed light on the mechanisms of SV membrane fusion, showing that PC-mediated stabilization of VAMP2 regulates this process. Overall, nIM-MS and CIU methodologies have advanced our ability to characterize MP structure and function beyond traditional detergent-based approaches. These methodologies enable crucial insights into how different membrane mimetics influence key functional aspects of MPs, providing powerful tools for studying MPs in biologically relevant conditions. Chapter 5 concludes by discussing how this thesis bridges important gaps in MP research and lays the groundwork for future applications, such as studying complex MP interactions and improving the accessibility of MPs as therapeutic drug targets.