Analytical Characterization and Biological Impacts of Multi-Lipid Nanodiscs

Abstract

Membrane proteins participate in numerous biological processes such as signal transduction and metabolism. Consequently, they have become popular therapeutic targets, comprising about 60% of current drugs despite making up only 30% of the proteome. This necessitates their characterization, but membrane proteins only make up about 3% of structures deposited in the Protein Data Bank. This is due to their propensity to aggregate and misfold in aqueous solution which poses a significant challenge for in vitro analyses. To overcome this challenge, membrane mimetics have been developed to solubilize and stabilize membrane proteins in solution. One such mimetic is the Nanodisc which is a discoidal lipid bilayer encircled by an amphipathic helical belt protein termed membrane scaffold protein (MSP). Nanodiscs self-assemble from a mixture of lipids, MSP, and membrane protein(s) upon detergent removal into relatively monodisperse particles. Nanodiscs have become a popular membrane mimetic system offering a well-defined bilayer environment to stabilize membrane proteins for in vitro analyses; however, lipid compositions common in their deployment are simplistic and often fail to model native membrane complexity. Additionally, our understanding of how Nanodisc synthesis conditions impact the final lipid composition is still very limited, primarily resulting from the lack of rigorous analytical and biophysical characterization of Nanodiscs comprised of more than one lipid. We sought to address these challenges through the development of LC-MS/MS strategies to quantify and profile lipid incorporation into Nanodiscs. This thesis comprises the development and application of two LC-MS/MS methods to interrogate lipid incorporation into Nanodiscs. The first is a targeted LC-MS/MS approach to quantify Nanodisc lipid compositions of 2-6 lipids. The second is an untargeted LC-MS/MS assay to profile the lipid landscape in Nanodiscs synthesized with natural lipid extracts and varied detergent, temperature, MSP sizes, and synthetic lipid additives. We utilized the targeted lipidomics approach to determine that lipids do not always incorporate stoichiometrically into Nanodiscs depending on their physical properties like curvature and fluidity (Chapter 2). The untargeted lipidomics approach was used to discover that Nanodisc synthesis parameters can enrich or deplete the incorporation of specific lipid species into Nanodiscs (Chapter 3). We then sought to utilize this enhanced understanding of Nanodisc synthesis parameters to selectively enrich different membrane protein classes. Using a bottom-up proteomics approach, we found that similarly to how detergents and neighboring lipids could influence lipid incorporation, these conditions could be tuned to enrich different protein classes (Chapter 4). Overall, these studies were among the first to quantitatively confirm Nanodisc lipid compositions as well as to profile the Nanodisc lipid and membrane protein environment upon modulation of synthesis parameters. We hope these studies serve as a foundation to better understand how multi-lipid Nanodiscs are formed and to promote more robust analytical characterization for necessary quality control experiments. These studies are essential to enable the use of Nanodiscs in structural and/or functional characterization of membrane proteins in more native-like environments.