The parent grant supports our work to determine the mechanism of rhomboid intramembrane protease GlpG. Intramembrane proteases are implicated in many important biological processes. The prototypical Drosophila rhomboid-1, by controlling growth factor release, is essential for EGF receptor signaling. In P. falciparum, the parasite that causes human malaria, rhomboid proteases play a key role in the invasion of host cells. We previously determined the X-ray structures of E. coli rhomboid GlpG and its complexes with mechanism- specific inhibitors, which have provided us with a structural framework to investigate further the mechanistic basis of intramembrane proteolysis. While our work on the parent grant continues to pursue crystallographic, site-directed mutagenesis, cross-linking, cysteine accessibility, optical tweezer and chemical synthesis experiments, we realize that our recent progress has opened up a novel opportunity to gain additional insight into two key questions in the mechanism of intramembrane proteolysis: 1) the conformational change in GlpG that is induced by the binding of substrate?s TM domain and 2) the unfolding of substrate?s TM domain, a prerequisite for the intramembrane cleavage reaction. Our ability to address both questions rely on quantitative assessment of the conformation of the membrane protein (and its substrate), and the extent that the protein is buried within the lipid bilayer, which are difficult to achieve using conventional biophysical approaches because the conformational differences could be subtle. This request is for supplemental funds with which we will purchase, together several other PIs within the pharmacology department, a SYNAPT G2-Si mass spectrometer to upgrade our system for hydrogen deuterium exchange mass spectrometry that has already proven itself as a powerful tool in studying protein conformational dynamics. The requested instrument will also allow significant advances in other NIGMS-funded grants whose PIs are submitting cross-referenced Administrative Supplement applications: Titus Boggon and Benjamin Turk (MPI: R01-GM102262), Karen Anderson (R01-GM049551), and Mark Lemmon (R35-GM122485), supported with funds from the Yale Cancer Biology Institute.
Rhomboid represents an ancient intramembrane protease family. The proteases have acquired a variety of biological functions during the course of evolution, many relevant to human health. Biochemical and biophysical experiments are planned in this application to study how rhomboid protease GlpG interacts with transmembrane protein substrates and how they change conformation inside membrane bilayers.
