Investigation of Ec YbaK loop function by multisite mutagenesis and FRET analysis

CHED 869

Ian M. Taylor, taylorim@westminster.edu1, Byung Ran So, bran@chemistry.ohio-state.edu2, and Karin Musier-Forsyth, musier@chemistry.ohio-state.edu2. (1) Department of Chemistry, Westminster College/ The Ohio State University, 310 W. Lima St., Findlay, OH 45840, (2) Department of Chemistry, Ohio State University, 140 W. 18th Ave, Columbus, OH 43210
When considering the EcYbaK protein, it has been noted that the loop containing residues 26-30 (NQHFG) is not observed during x-ray structure analysis. It is hypothesized that the loop of interest is a free moving flap on the exterior of the protein that repositions too frequently to be observed by instrumentation. It was the intent of this investigation to complete FRET analysis to determine to loop function of the YbaK protein using the donor-acceptor pair of tryptophan and 5-(2-iodoacetylaminoethylamino) naphthalene-1-sulfonic acid (IAEDANS). These fluorophores were introduced to the YbaK protein by the F29W and the M86C mutations. Once mutagenesis reactions were completed, aminoacylation activity tests were completed with the produced protein in the presence of radioactive labeled cys-tRNApro. The results given by replicate tests showed that the presence of the F29W/M86C mutations in the EcYbaK species decreased activity to essentially inactive status.
 

Undergraduate Research Poster Session: Biochemistry
2:00 PM-4:00 PM, Monday, April 7, 2008 Morial Convention Center -- Hall A, Poster

Division of Chemical Education

The 235th ACS National Meeting, New Orleans, LA, April 6-10, 2008