Role of Protein Dynamics in G Protein Signaling
EMSL Project ID
39934
Abstract
This is a multi-investigator project aimed at understanding the role of protein dynamics in controlling the catalytic properties of an important family of biological regulators: the G-alpha subunits of heterotrimeric G proteins. G-alpha subunits are guanine nucleotide-binding proteins with weak GTPase activity. When GTP is bound, G adopts an active signaling state with high affinity for effectors; in the GDP bound state, it is inactive, and has low affinity for effectors. Activation and deactivation of G-alpha are associated with GDP release/GTP binding and GTP hydrolysis, respectively. The kinetic barriers to both events are high, such that the intrinsic rate is on the order of 0.03 min-1 for the former, and 2-5 min-1 for the latter. The hypothesis to be tested is that ground state fluctuations in the picosecond to second time frame, are additive with respect to accessing high-energy states that are competent for nucleotide release and catalysis. A map of millisecond-to-second G-alpha fluctuations in the GTP-bound, transition-state and GDP-bound states, will be obtained by multidimensional nuclear magnetic resonance spectroscopy to characterize fluctuations in the microsecond-to-millisecond range at single-residue resolution. Molecular dymamics simulations, validated by NMR spectroscopy, will be used to determine the dynamic behavior of these complexes in the picosecond-nanosecond domain. To characterize fluctuations that are involved in catalysis, similar experiments will be conducted on all three nucleotide-bound states of G-alpha gain-of-function mutants, in which GTP hydrolytic activity or GDP release rates are individually enhanced. By this means, fluctuations over a 109 range in time scale that are specifically coupled to catalytic events will be identified. The results of this work will be of general significance to the mechanism of action of all members of the Ras superfamily, which includes hundreds of proteins engaged in virtually all of the regulatory and housekeeping activities that occur in eukaryotic cells. Importantly, the proposed studies will be the first to comprehensively explore the role of ps - s dynamic fluctuations in signaling molecules that undergo functional transitions at physiological time scales. Access to the EMSL cold-probe equipped 800 MHz NMR instrument is critical to this effort, which require high resolution and sensitivity for analysis of a high-molecular mass protein. No high field NMR instrument of comparable resolution and sensitivity is available at the University of Montana. The proposed molecular dynamics experiments involve long run times that require supercomputer facilities on the scale of Chinook. This application is also driven by the high convergence of the proposed research with the EMSL Biological Interactions and Dynamics theme.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2010-10-12
End Date
2011-10-16
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members