The Small GTPases are proteins involved in virtually all aspects of cellular function, from the control of cell proliferation, regulation of cell death, motility, transport of membrane vesicles containing molecular cargo within the cell and transport of molecules between the cell cytoplasm and nucleus. Their activity is regulated by conversion of a molecule (GTP) bound to their active site to GDP and inorganic phosphate. They belong to a group of over 150 proteins classified into 5 families: Ras, Rho, Rab, Arf and Ran. The goal of this research project is to determine how representative proteins in the Ras, Rho and Arf families, protein families that are found on the cell membrane, have evolved specific structural details associated with their distinct functions. The investigators will test how interactions with the cell membrane affect the function of these small GTPases. This will be done using a multidisciplinary approach based on complementary state of the art biophysical and biochemical experiments designed to elucidate the fundamental molecular mechanisms through which these proteins work. In addition to continuing to promote diverse laboratory and departmental environments in which this project will be conducted, there will be a new Future Faculty Fellowship mentoring program at the university level associated with the ADVANCE program at Northeastern.
Proteins in the Ras superfamily of GTPases (Small GTPases) are involved in vastly diverse functions in the cell, where they work through a tightly regulated mechanism of switching from an active GTP-bound state to an inactive GDP-bound state. There is now substantial evidence that interaction with the membrane is an integral part of the regulatory mechanism through allosteric modulation linking membrane-interacting hot spots to the active site. In this project, investigators aim to determine the nature of these interactions and to map the allosteric pathways in three representative members of the superfamily: HRas, RhoA and Arf1. The investigators hypothesize that specific membrane elements play a key role in regulating the function of G-proteins across most branches of the superfamily. They will will test this hypothesis by using a series of phospholipid head group mimics to determine the binding specificities of membrane components to HRas, RhoA and Arf1 and by studying the effects of strategically designed mutants on the conformational ensemble of states and associated hydrolysis rates. This will be done using a combination of X-ray crystallography, hydrolysis rate measurements, solution scattering experiments in the wide angle regime (WAXS) and accelerated MD (aMD) simulations, each method providing a perspective that together will lead to a mechanistic view of regulatory mechanisms that have thus far been unexplored within the superfamily.
