Interactions of membrane proteins with lipids, water, and other proteins in biological membranes

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BACKGROUND Membrane proteins constitute approximately a third of all proteins. They are involved in many important physiological processes, and represent almost a half of known drug targets. Studying membrane proteins in their native lipid bilayer environment is particularly challenging, as the two common structural methods, X-ray crystallography and solution-state Nuclear Magnetic Resonance (NMR), rely on crystals or detergent micelles, which are different from that of lipids. Solid-state NMR (SSNMR) provides an alternative approach to protein structure determination, and offers ways to study membrane proteins in the bilayer environment. OBJECTIVES In this application, we will pursue both the methodological objectives aimed at improving SSNMR ability to characterize protein structures and protein interactions, and to answer the fundamental biophysical questions related to the response of membrane protein conformations to changes in the environment. Our goal is to undestand how interactions between membrane proteins and their environment shape protein structure. Specifically, we will focuss on the following three types of effects: (i) Protein-water interactions. Water molecules play important roles in defining the structure, stability, dynamics and function of proteins. Buried waters are extensively hydrogen-bonded with backbone and side chain atoms, both stabilizing the secondary and tertiary structures, and playing functional roles. As protein-water interactions are inherently dynamic, NMR is ideally suited to study these interactions. We are interested in determining the location of bound water molecules, and in characterizing their dynamics. (ii) Protein-lipid interactions. We are interested in characterizing the effects of both specific and non-specific interactions, e.g., how tightly bound non-annular lipids affect the protein structure, and how variation of the bilayer properties affect membrane proteins structure, oligomerization and dynamics. (iii) Structure of membrane proteins in cellular membranes. Studying biomolecules in the cellular environment is the ultimate goal of molecular biology. SSNMR provides an excellent tool to elucidate the structural and dynamic changes in membrane proteins induced by the complex environment of E. coli membranes with many additional endogenous molecular components present. The fundamental question we strive to answer is to what extent the idealized environment of a reconstituted bilayer resembles that of a real membrane. SIGNIFICANCE A growing number of SSNMR structures of lipid-embedded membrane proteins underscores the potential of solid-state NMR as a powerful method to investigate membrane proteins in the native-like bilayer environment. The present proposal aims to go beyond the structure and to study functionally and structurally important interactions between membrane proteins, and water and lipids. We will develop new SSNMR methodologies for obtaining comprehensive view of membrane protein structure and dynamics that include bound water and lipid molecules, and will contribute detailed structural information on the long-standing questions of the effects of environment on the membrane protein functional assembly, structure, and dynamics.