Authors: Kristine F. Parson
Source: Deep Blue (University of Michigan) (article, 2022-01-01)
Field: Chemistry — Spectroscopy

Development of Ion Mobility-Mass Spectrometry Methods for Membrane Proteins Incorporated into Nanodiscs Membrane proteins (MPs) are vital therapeutic targets which play important roles in a multitude of cellular functions. MPs interact intimately with the cellular membranes in which they embed. Interactions between proteins and ligands such as membranous lipids and other small molecules can affect MP structure and function. Ion mobility-mass spectrometry (IM-MS) has recently emerged as a valuable tool for interrogating the interactions between proteins and ligands, offering direct measurements of protein-complex stoichiometry, ligand binding strengths, and stabilities. This dissertation seeks to extend IM-MS technologies to study the relationships between MPs and their lipid environment, probing directly long-standing questions surrounding the functional role of local lipid environments on MP structure and function. In chapter 2 we develop a workflow for studying different MP classes using various solubilization techniques and discuss the implications such membrane mimetics carry in the context of the embedded protein structures. We utilize four different MPs that vary in both the way that they span the membrane and in terms of their native oligomeric states. Specifically, we utilize a small multidrug resistance transporter (GDX), a transmembrane protein that has a unique antiparallel orientation, WT and the L16P disease-associated mutant form of peripheral myelin protein (PMP22), a transmembrane protein which occupies both a monomeric and dimeric state, and Cytochrome P450 (CYP), a monotopic membrane-bound enzyme. Each MP system was studied within at least two different mimetics, including: detergent-based micelles, lipid-bicelles, or lipid-nanodiscs (NDs). In general, we find evidence of differences in MP structure, oligomeric state, and ligand binding that appears to depend strongly on the membrane mimetic used. In chapters 3, 4 and 5 we focus on CYP and deploy IM-MS and collision induced unfolding (CIU) to study how this centrally important enzyme interacts with binding partners, ligand and its membrane environment in order to carry out essential functions. In chapter 3, we use NDs of carefully designed compositions to study the role of different lipid environments and ND scaffolding proteins on CYP structure. We find that CYP CIU, and by extension its structure, strongly depends on its local environment, and that more native membrane environments can result in more compact and more destabilized CYP forms. In chapter 4 we focus on CYP ligand binding and develop CIU classifiers capable of differentiating CYP binders based on their mode of attachment to the protein and their hydrophilicities. The ability of our CIU assays to differentiate CYP-ligand complexes to discern hydrophobic from hydrophilic binders relates directly to the proximity of the CYP active site to the biological membrane and supports the conclusion that lipids are significantly involved in structure of the CYP active site. In chapter 5 we study the interactions between full length CYP, cytochrome b5 (cytb5), and P450 oxioreductase (POR) within NDs. When we co-incubated these proteins with NDs we observed no direct evidence of stable complexes, but significant alterations in CYP CIU, suggesting changes in CYP structure when present within the same local membrane environment to cytb5 or POR. We observe evidence of additional lipid binding events within POR when reduced by NADPH, suggesting deeper membrane engagement when the protein is in its reduced state. We conclude in Chapter 6 by discussing the future of MP structural biology and how this dissertation works has emphasized the impact that a membrane environment has on the membrane protein structure Ion-mobility spectrometry ; Mass spectrometry ; Chemistry ; Membrane ; Ion ; Chromatography ; Nanotechnology ; Materials science ; Biochemistry ; Organic chemistry Mass Spectrometry Techniques and Applications ; Ion-surface interactions and analysis ; Analytical Chemistry and Sensors