Determinants of membrane protein stability and folding investigated by solid state NMR

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Membrane proteins play many important roles in biology of the cell, from regulation of membrane transport to signal transduction. These essential biomolecules have evolved to perform their function in cell membranes. The overarching long-term goal of our research program is to build an understanding of the interplay between membrane protein structure, dynamics and function and the cell membrane environment. One of the essential questions is how membrane proteins achieve their native functional structures. Folding of membrane proteins (a process by which a polypeptide chain adopts its native structure) is substantially more complex than that of their soluble counterparts, as in addition to intraprotein interactions, the presence of amphipathic lipid bilayer creates physical boundaries that limit the number of conformations accessible to membrane proteins. Furthermore, there is a growing body of evidence that specific and non-specific protein-lipid interactions, oligomerization, as well as interactions with ligands and cofactors play major roles in defining folding pathways and in stabilizing membrane protein structures. Understanding these interactions is the key to understanding of one of the central questions in membrane protein folding - how protein structure is established within membranes. We have previously focused on the development of solid-state nuclear magnetic resonance (SSNMR) methodologies that can provide the atomic level detail on membrane protein conformations in lipids and in cell membranes. We used these methods to characterize structure and dynamics of a seven-transmembrane helical retinal-binding photoreceptor Anabaena Sensory Rhodopsin (ASR). Our previous studies strongly suggest that the oligomeric structure of ASR is stabilized by interactions with tightly bound lipids, and these interactions modulate its folding landscape. Using this well-characterized model protein, we will study the relationship between ASR interactions and its folding. Our aims are (i) to determine the structural roles of lipids and of protein-protein interactions in the stability of ASR; (ii) to determine structures of the thermally induced unfolding intermediates and of the misfolded state, and to delineate the roles of cofactors and lipids in modulating the unfolding pathways.  Unfolding pathways will be detected by SSNMR and complementary techniques (e.g., Electron Paramagnetic Resonance), using either methods established by us earlier, or by trapping intermediate states by flash freezing, and by detecting chemical shift perturbations and key interatomic distances as structural reporters. By investigating structures and revealing stabilizing interactions of partially unfolded and misfolded states, we will determine the key factors governing proper folding of membrane proteins, and gain more detailed understanding of the membrane protein folding mechanisms.