Poster abstracts
Poster number 77 submitted by Katie Lichtenthal
Determining the mechanism of cooperative binding of trp to TRAP using Bacillus halodurans
Katie Lichtenthal (Biological Sciences, University at Buffalo), Weicheng Li (Biochemistry, The Ohio State University), Mark Foster (Biochemistry, The Ohio State University), Paul Gollnick (Biological Sciences, University at Buffalo)
Abstract:
TRAP (trp RNA-binding attenuation protein) is an RNA-binding protein that regulates expression of tryptophan (trp) biosynthetic genes in several Baccilli. In response to excess trp levels, the amino acid binds to the 11 or 12-subunit TRAP ring. When activated by trp, TRAP binds to the mRNA of trp genes and down-regulates expression. Prior studies have focused mainly on B. subtilis (11 subunit TRAP), but recently we have been investigating the biophysical properties of TRAP (12 subunits) from the thermostable bacterium, B. halodurans. Trp binding is positively cooperative in both species, but the mechanism remains unclear. To further examine the mechanism of cooperative binding, we constructed B. halodurans TRAP 12mers from covalently linked dimers. The dimers were composed of all WT subunits or substitutions that abolish trp binding to the first or second subunit of each dimer. In addition, we created a TRAP protein composed of 1 WT subunit and 11 subunits containing substitutions that also abolish trp binding. Kds and Hill coefficients (n) for the WT dimer were determined to be 2uM and 0.6, respectively, which agree with the Kds value and n of 2uM and 1.7 for B. halodurans WT monomeric TRAP, respectively. In contrast, heteromeric TRAP (1WT:11Mut) gave an undetectable Kds value. Further approaches to study trp binding to the substituted versions of the TRAP dimer and heteromer are being developed.
References:
E. C. Ihms, I. R. Kleckner, P. Gollnick, M. P. Foster. Mechanistic Models Fit to Variable Temperature Calorimetric Data Provide Insights into Cooperativity. Biophys J. 112(7):1328-1338 (2017).
Keywords: Tryptophan, TRAP, Cooperativity