Analysis of Viroporins and Membrane Systems With Native Mass Spectrometry

Abstract

Viroporins are a class of viral membrane proteins that play diverse roles in the viral infection cycle. Viroporins are known to play roles in ion transport, inducing membrane curvature, as well as participating in a variety of protein-protein interactions. However, there is little known about the oligomeric state of viroporins. The lack of quantitative data on the oligomeric state of viroporins is due to key analytical challenges associated viroporins, including that they are 1) hydrophobic, and require a membrane mimetic for analysis, 2) small, making viroporins unamenable to some structural biology techniques such as cryo-EM, and 3) contain intrinsically disordered regions, which also poses challenges for structural characterization. To overcome these analytical challenges, this dissertation outlines the application of the unique combination of native mass spectrometry (MS) and nanodiscs for the oligomeric state determination of viroporins. The unique combination of native MS and nanodiscs can be used to determine the oligomeric state and lipid specificities of membrane proteins (such as viroporins) while embedded within a lipid bilayer using mass defect analysis. This dissertation reviews how to perform mass defect analysis, provides examples of how mass defect analysis has been applied in the past, as well as providing tips for overcoming some of the technical limitations associated with mass defect analysis. This dissertation also outlines a structure-activity relationship study performed to identify novel charge reducing agents for native MS. Charge reducing agents can be useful for the preservation of fragile complexes, such as viroporins, during native MS. Here, we found improved charge reducing agents that were effective on a wide range of analytes. We also uncovered the chemical principles governing charge reduction, which may provide a basis for the development of even better novel charge reagents in the future. The first viroporin characterized with native MS was M2 from influenza A. Here, native MS revealed that M2 assembled into a range of previously undetected oligomers, ranging from monomer through hexamer. Native MS also revealed that M2 oligomerization is highly sensitive to the local environment, being influenced by a range of factors including detergent type, solution pH, and the surrounding lipids. These results suggest that the behavior of M2 may be far more dynamic than initially thought. The methods developed to characterize M2 were then extended to characterize a broader range of viroporins from other clinically relevant viroporins, including viroporins from HIV, SARS-CoV-2, and polio. Native MS revealed that this broader range of viroporins also had complex patterns of oligomerization and could be highly influenced by the local environment. Overall, the methods outlined in this thesis detail the methods for determining the oligomeric state of viroporins in a wide range of chemical environments. Native MS revealed that the behavior of viroporins is more dynamic and complex than initially thought. The mass spectrometry approaches outlined in this dissertation provide directions for determining the oligomeric state of membrane proteins while embedded in a lipid bilayer, as well as may enable the development of novel therapeutics targeting viroporin complexes.