Acquire preliminary 3D NMR data G(alpha)i to support grant application
EMSL Project ID
38499
Abstract
We seek rapid access to the EMSL 800 MHz NMR spectrometer in order to obtain preliminary data to support a revised application to the NSF with a submission of January 12, 2010. The major aim of our proposal is to use heteronuclear NMR methods to assess the dynamic behavior of G(alpha)i, a 40.1 kDa regulatory protein that transduces signals generated by G Protein-coupled Receptors. G(alpha)i is a guanine nucleotide-binding "G protein", in which conversion of the GTP-bound active form of the protein is converted to the GDP-bound inactive form with a t(1/2) of ~ 30 s. The proposed studies will test the hypothesis that dynamics in the ps - ms range determine or influence the probability of conformational transitions that occur in the time domain of seconds to minutes. This project is highly relevant to the EMSL theme in Biological Interactions and Dynamics, specifically in exploring the dynamic behavior of proteins. Ultimately, it is our intent to use heteronuclear NMR experiments to probe the dynamics of G(alpha)i in both the GTP-bound ground state and in a GDP . AlFx . Mg2+ complex that mimics the excited, catalytically competent state. Experiments to characterize the dynamic behavior of G(alpha)i will involve analysis of the relaxation of proton-nitrogen resonances, obtained from Transverse Relaxation Optimized (TROSY) 1H,15N heteronuclear single quantum correlation (HSQC) spectra. Preliminary to such experiments, it is essential that the cross-peaks in the TROSY-HSQC spectra be assigned to the amino acid residues in G(alpha)i from which they originate. Because G(alpha)i is at the high end, with respect to molecular mass, of proteins amenable to high-resolution NMR analysis, the EMSL 800 MHz Varian spectrometer is requested to achieve the necessary sensitivity and resolution needed to obtain interpretable spectra. For this rapid access proposal, to demonstrate feasibility for assignment of backbone 1H, 15N, 13C(alpha) and 13CO chemical shifts in G(alpha)i . GDP complexes, we will prepare 2H/13C/15N-labeled protein samples and acquire TROSY-based 3D HN(CA)CB and HN(CO)CACB, triple resonance data from at 2H/13C/15N-labeled protein. Two weeks of access, at minimum, to the 800 MHz instrument are requested. The minimum block is one week, and both blocks are requested before January 5, 2010 at the latest. Resonance experiments should, in the most favorable case, demonstrate feasibility of assigning most of the main-chain resonances for G(alpha)i.
Project Details
Project type
Limited Scope
Start Date
2009-11-11
End Date
2010-01-11
Status
Closed
Released Data Link
Team
Principal Investigator