Thermal stability and activity of fluorinated single-isoleucine mutants of chloramphenicol acetyltransferase

MEDI 65

Natalya Voloshchuk, nvoloshc@poly.edu1, Man Xia Lee1, Wan Wen Zhu1, Ismet Caglar Tanrikulu2, and Jin K. Montclare, jmontcla@poly.edu1. (1) Department of Chemical and Biological Sciences, Polytechnic University, 6 Metrotech Center, Brooklyn, NY 11201, (2) Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Boulevard, Pasadena, CA 91125
Residue-specific incorporation of 5',5',5'-trifluoroleucine (TFL) into chloramphenicol acetyltransferase (CAT) results in reduction in the thermal stability of the protein. To deconvolute which TFL residues participate in the loss of thermal stability, a highly sensitive microplate-based cell lysate thermostability and activity screen was developed. Thirteen single-isoleucine mutants were created and screened. From the assay, fluorinated mutants L82I T and L208I T exhibited large losses in thermal stability while the fluorinated mutant L158I T was determined to be more active than the parent CAT T at elevated temperatures. Additional secondary structure characterization of L158I T confirmed the enhanced thermal stability suggesting that TFL at position 158 contributes to some of the loss in thermostability upon fluorination.
 

Poster Session
7:00 PM-9:00 PM, Sunday, August 19, 2007 BCEC -- Exhibit Hall - B2, Poster

Division of Medicinal Chemistry

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007