Membrane proteins (MPs) are important but arguably the most challenging biological molecules to study in solution. One major concern with studies of MPs is the requirement for extraction from their native environment, the membrane. Over several decades the chemistry and MP communities have contributed diverse membrane mimetics that help to solubilize and stabilize MPs. Despite their broad applications, these membrane mimetics do not fully replicate the native bilayer setting of MPs which contains hundreds of diverse lipids. This is a fundamental issue because lipid bilayers and specific lipid interactions are crucial to the structural assembly and function of many MPs. Notwithstanding, key challenges remain as to how to isolate and stabilize fragile MPs and MP complexes with little disturbance to their conformation, oligomer assembly, and function, tasks that are nevertheless crucial to structural, functional and drug interaction studies. To overcome these important challenges in MP research, we propose to develop a strategy to directly enrich or purify MPs within small patches of cell membranes. Our proposal exploits the favorable properties of two types of popular membrane reagents, i.e. small molecule detergents and amphiphilic polymers, and meanwhile avoid their shortcomings in that 1) detergent is effective and inevitably used to disperse cell membranes in most MP pruritions, but the detergent- solubilized membrane patches are highly dynamic and unstable structures; and 2) an amphiphilic polymer scaffold may entrap and stabilize a central bilayer patch, but most polymers used to stabilize MPs are ineffective to disperse cell membranes. To resolve the dilemma, we propose an ex novo polymer design to encircle small membrane domains by crosslinking detergents in situ, upon the dispersion of cell membranes. To establish the feasibility of this approach, we will explore chemistry for detergent crosslinking to solubilize and stabilize small patches of cell membranes (Aim 1) and validate chemical tools in the purification of diverse MPs (Aim 2). If successful, our development will provide a completely new and versatile approach to preparing MPs in nearly native environment. Achieving this goal together with new tool development will have a far-reaching impact on many areas of MP research in which the isolation of MPs is needed.
Membrane proteins account for about one third of human genes, perform a variety of essential biological functions, and comprise more than 50% of human drug targets. Despite their biological and biomedical significance, membrane proteins remain a class of the most challenging biological molecules to study. We propose to develop a novel technology to address the central challenges limiting the preparation of stable, active membrane proteins so as to facilitate their structural and functional studies and drug discovery.
