The main objective of this proposal is to better understand how Ras proteins function as molecular switches that are controlled by a regulated GDP/GTP cycle. Oncogenic forms of Ras are defective in this cycling and exist chronically in the active GTP-bound form, and consequently, trigger the uncontrolled growth of the cancer cell. Although a wealth of information from structural, genetic, and biochemical studies has clearly established that Ras protein function is controlled by GDP/GTP cycling, we remain ignorant of the mechanism of GDP/GTP conversion and how this regulation is accomplished by proteins that stimulate Ras GTP hydrolysis and GDP/GTP exchange to control Ras biological activity. The experiments in this proposal have been designed to complement structural and functional information currently available on this system and to test mechanistic and structural models. A multi-disciplinary approach, combining tools of biophysics, biochemistry, molecular biology and cell biology is presented. Structural and dynamic information on wild type and mutant Ras proteins in their biologically active and inactive forms will be derived from high field NMR spectroscopy. NMR studies conducted in solution should supplement X-ray structural information obtained in the crystal state. The NMR studies will be employed in parallel with in vitro biochemical analyses of wild type and mutant Ras protein ligand binding, dissociation and GTPase rates. To establish structure/function relationships, biological characterization of Ras mutants will be inducted in collaboration with Der and Hwang laboratories. These studies will provide important information for understanding the fundamental structural features and dynamic properties critical for Ras GTP/GDP cycling and its importance in Ras modulator proteins recognition and activation.
The specific aims of this proposal are l) to investigate the biologically active Ras.GTP state and examine the structural and dynamic basis for GTP <=> GDP interconversion 2) to analyze the properties of a G60A Ras mutant and investigate its affect on Ras (GTP/GDP cycling and 3) to evaluate the role of Thr-35 in the Mg.GTP binding site and examine the properties of a position 35 mutation that disrupts signal transmission to downstream effectors. We anticipate that the approaches outlined in this proposal should generate a better understanding of the unique structural and biochemical features of Ras proteins important for Ras-mediated cell signaling. Ras proteins are members of a larger superfamily of GDP/GTP- regulated proteins. Therefore, determining how Ras protein function is controlled provides the foundation for understanding the functional role of other GTP-binding proteins in controlling such diverse cellular process as signal transduction, intracellular transport, and cytoskeletal organization and cell motility. These studies will also lay the ground work for our longer term goals, which are to investigate the binding modes of agents that modulate Ras function. Such studies will provide information helpful for therapeutic intervention in cancer.
