Grant: Discovery Grants Program - Individual

Proteins are the molecular machines of living systems, and like any machine, rely on motions to carry out their functions. This is also true for proteins that exist in the lipid membrane that defines cellular boundaries, known as membrane proteins. One class of membrane proteins that undergo motions that appear to be important for function is the rhomboid family of intramembrane proteases. Found across all kingdoms of life, rhomboids play a role in a wide range of biological processes, including parasite infections, cell growth and protein quality control. Consequently, there is interest in developing compounds that can inhibit or activate the rhomboid protease, making them targets for drug discovery. However, our ability to control rhomboid function relies on understanding how the rhomboid can be uniquely capable of cleaving target proteins that are normally embedded within the water-poor environment of the membrane in a state that would be inhibitory against a reaction that requires water. There is a body of evidence suggesting that both the rhomboid and its target must adopt more than one structure to allow this transition, and that this may be the slowest step in the catalytic cycle, thereby controlling the rate of rhomboid activity. However, the nature of this conformational change, and how these dynamics can be modulated by the lipid environment is not known, and represents a key gap in our understanding of rhomboid activity. Using a well-characterized rhomboid protease from bacteria as our model system, called GlpG, we will study the relationship between rhomboid function and its dynamics, and the role of the membrane environment in modulating populations and rates of exchange. We will take advantage of the ability of solution phase nuclear magnetic resonance (NMR) to resolve signals from individual atoms that report on structure, energetics and rates of conformational exchange. Since GlpG is a large integral membrane protein it is necessary to use special strategies normally reserved for NMR studies of large proteins. This will include the introduction of unique NMR-active labels by selective labeling schemes, site-specific chemical labeling, and genetic code expansion to introduce unnatural amino acids having the desired label. This latter strategy is particularly exciting since it will for the first time open the door to the study of GlpG dynamics in native bacterial membranes. We will isolate stable fragments of native membrane containing selectively labeled GlpG and study rhomboid dynamics in its native environment. This new method has the potential to be applied to the study of other dynamic membrane proteins in their native membrane environments. Taken together, the results from this program will improve our understanding of how dynamics regulates rhomboid protease function, how lipid membrane properties can modulate these dynamics, and how we might be able to ultimately control dynamics to alter function.