Talk abstracts

Talk on Monday 04:00-04:15pm submitted by Ian Kleckner

Mechanisms of Allosteric Gene Regulation by NMR Quantification of μs-ms Protein Dynamics

Ian R. Kleckner (Biophysics Program, The Ohio State University), Paul Gollnick (Biological Sciences, University at Buffalo, the State University of New York), Mark P. Foster (Biochemistry Dept., The Ohio State aUniversity)

Abstract:
The trp RNA-binding attenuation protein (TRAP) is a paradigmatic allosteric protein that regulates the tryptophan biosynthetic genes associated with the trp operon in Bacilli. The ring-shaped 11-mer TRAP is activated for recognition of a specific trp-mRNA target by binding up to 11 tryptophan molecules. To characterize the mechanisms of tryptophan-induced TRAP activation we have performed methyl relaxation dispersion (MRD) nuclear magnetic resonance (NMR) experiments that probe the time-dependent structure of TRAP in the microsecond to millisecond “chemical exchange” time window. We find significant side chain flexibility localized to the RNA and tryptophan binding sites of the apo protein, and that these dynamics are dramatically reduced upon ligand binding. Analysis of the MRD NMR data provides detailed insights into the structure and thermodynamics of transiently populated conformations sampled in solution by apo TRAP. Contrary to commonly assumed global two-state behavior, rigorous χ² mapping of fitted parameters reveals that conformational sampling in apo TRAP is asynchronous, reflecting a temporally heterogenous population of structures that are incompatible with RNA binding. Moreover, we find structure- and residue-specific partitioning of μs-ms side chain flexibility, which may prove to be prevalent in sidechain dynamics of other proteins. This substantiates the study of TRAP as a paradigm for probing and understanding essential dynamics in allosteric, regulatory proteins.

References:
1. Heddle JG et al. (2007) Dynamic allostery in the ring protein TRAP. Journal of molecular biology 371:154-67.
2. McElroy C a, Manfredo A, Gollnick P, Foster MP (2006) Thermodynamics of tryptophan-mediated activation of the trp RNA-binding attenuation protein. Biochemistry 45:7844-53.

Keywords: global analysis, stopped flow, itc