Poster abstracts

Poster number 4 submitted by Elihu Ihms

Identification of regulatory complexes from polydisperse solutions

Elihu C. Ihms (Biophysics, The Ohio State University), Mark P. Foster (Biochemistry, The Ohio State University), Paul Gollnick (Biological Sciences, SUNY Buffalo)

Abstract:
In many Bacilli, the undecameric (11-mer) toroidal protein TRAP acts as a tryptophan sensor, becoming allosterically activated by tryptophan to bind to specific mRNAs. TRAP binding to these mRNAs favors the formation of alternate secondary structure elements, which abort the production of nascent transcripts or block translation of completed mRNAs. A second protein, the trimeric Anti-TRAP (AT), is expressed under tryptophan starvation conditions and prevents the TRAP/RNA interaction by occluding the RNA binding site of TRAP.

Solution-state studies of TRAP-AT complexes via analytical ultracentrifugation (AUC) and Small-Angle X-Ray Scattering (SAXS) reveal that complexes assembled at low AT:TRAP ratios (i.e., 1-2) are oligomeric and heterogeneous, likely consisting of multiple AT trimers and TRAP 11-mers. Interestingly, as the ratio of AT to TRAP is increased, dimensions of these complexes dramatically decrease.

To determine the structural configurations adopted under various conditions by TRAP-AT complexes, we have developed software that aims to identify a minimal ensemble of structures consistent with a range of structural data, including SAXS, AUC, and FRET. The software utilizes a genetic algorithm capable of determining the best-fitting ensemble from thousands of candidate structural conformers. Using this approach, we analyzed SAXS solution-state titration data from 4 to 8 AT₃:TRAP₁₁ ratios and observe that the experimental data are reliably fit when two complex configurations are present: TRAP in which all five binding sites are occupied by AT trimers, and TRAP bound to only three AT trimers. This result is consistent with isothermal titration calorimetry data that reveal 2-3 high affinity, and 2-3 low affinity binding modes.

This global analysis method represents a powerful approach by which to study the highly variable structural configurations, and has already provided important insight into the TRAP-AT interaction.

Keywords: SAXS, Regulation, TRAP