Developing Native Mass Spectrometry Methods to Study the Specificity of Membrane Protein-Lipid Interactions

Abstract

Membrane proteins play important physiological roles and are major drug targets. Lipids surrounding membrane proteins are critical modulators of the structure, function, and stability of membrane proteins. Due to the complexity and heterogeneity of lipid environments surrounding membrane proteins, it is challenging to study the affinity and energetics of these protein-lipid interactions. To overcome these challenges, we develop novel mass spectrometry (MS) techniques to investigate membrane protein-lipid interactions. This dissertation details native MS methods we have developed to explore three key aspects of membrane protein-lipid interactions.First, we explore the preferential associations of different lipids with membrane proteins using membrane mimetic nanodiscs. Chapter 2 presents a novel native MS approach for investigating membrane protein-lipid interactions using heterogeneous nanodiscs. Using both gas-phase ejection and solution-phase detergent extraction techniques, we demonstrated the effectiveness of this native MS approach in probing lipid preferences of membrane proteins. Our findings revealed that Aquaporin Z (AqpZ) prefers phosphatidylcholine (PC) and phosphatidylglycerol (PG) lipids over phosphatidylethanolamine (PE). This approach provides the ability to study membrane proteins in a near-native lipid environment and the capacity to analyze different lipid types simultaneously. Moreover, using different detergent types for the solution-phase extraction of membrane protein-lipid complexes highlights the importance of careful selection of detergents to preserve lipid interactions. Secondly, we identify specific binding sites of lipids on membrane protein structures. Chapter 3 develops a novel native MS approach using single mutants to measure the relative energetic contributions of specific residues to cardiolipin (CL) binding, a known specific lipid interaction of AqpZ. Here, we simultaneously resolved lipid binding to mutant and wild-type proteins in a single spectrum, allowing direct determination of relative affinities of CL binding. We identified W14 as contributing to the tightest binding CL site, which was also previously shown to interact strongly with AqpZ. We further identified R224 contributing to a lower affinity site on AqpZ. Our novel approach offers several advantages, such as the need for fewer titrations and reduced susceptibility to errors compared to traditional native MS techniques. This method provides a more direct and efficient way to rank lipid binding sites based on their thermodynamic trends. We further examined synergistic effects associated with CL binding to the two residues identified before. In Chapter 4, we used double mutant cycling to investigate the synergy between W14 and R224 sites in CL binding. We observed energetic coupling between the two amino acids in CL interactions. Building on these findings, we expanded our research to create affinity maps that highlight hotspots of lipid binding on membrane protein structures. To complement our native MS experiments, we used molecular dynamic (MD) simulations, enabling us to visualize key lipid binding sites. In Chapter 5, we expand our previous research by conducting a comprehensive screening of AqpZ mutants to identify specific lipid binding sites. We also explored the binding affinity of various lipid types to determine lipid selectivity at these sites. Our findings revealed that AqpZ binds more stably with CL at a specific orientation, with its headgroup facing the cytoplasmic-exposed region and acyl chains interacting with a hydrophobic pocket at the monomeric interface. This comprehensive analysis enables us to map the AqpZ protein structure based on lipid affinity, revealing the specificity and site-selectivity of diverse lipids on specific amino acid residues. Overall, these innovative native MS approaches provide detailed insights into the structure and thermodynamics of membrane protein-lipid interactions and have the potential to accelerate research in membrane protein biology and contribute to the development of more effective membrane protein-based therapeutics.