Thermal denaturation of Mycobacterium tuberculosis protein Rv0045c monitored by intrinsic tryptophan fluorescence (770.1)

Abstract

Rv0045c is a novel esterase of Mycobacterium tuberculosis, the causative bacterium of the still prevalent and deadly disease tuberculosis. The six tryptophan residues in this enzyme were of interest to monitor the denaturation process by intrinsic fluorescence. To simplify the signals, the six tryptophans (W78, W98, W122, W246, W248, W263) were mutated to phenylalanine to preserve the relative size of the residue but remove the fluorescence capability; the goal being to create a quadruple mutant with only one pair of tryptophans interacting. Mutated proteins were synthesized through site directed mutagenesis, overexpressed in E.coli hosts, and purified using a Ni2+ affinity resin. Differential scanning fluorimetry and kinetic assays were performed to test the initial stability of the mutants. W78F and W98F were not as stable as the wild type and therefor were not used in further double and triple mutations. Significant changes in the thermal stability of Rv0045c triple and quadruple mutants will be analyzed via intrinsic fluorescence.Grant Funding Source: NSF TUES