Abstract
Bacterial luciferase from Vibrio harveyi is a heterodimeric protein consisting of homologous [ ] and [ ] subunits. It has been shown to undergo reversible denaturation in urea with spectrally distinct species along the folding pathway. In the absence of [ ], the [ ] subunit can form a kinetically trapped homodimer which exhibits hysteresis during equilibrium unfolding due to the extremely slow rate of dissociation. Comparison of the interfaces demonstrated the presenceof a solvent channel in the heterodimer which is occluded in the homodimer. Site-directed mutagenesis was used to generate the following mutations atthe interface of the [ ]subunit: A8 1H, D89E, Tl 19R, A8 1H/D89E, A81H/T119R. D89E/Tll9R and A81H/D89E/Tll9R. The circular dichroism spectra of the three single mutants showed changes in the environment of the aromatic chromophores. Unfolding transitions for the mutants were monitored spectroscopically for urea-induced denaturation at fixed temperatures. These measurements demonstrate the amino acid changes had no effect on the overall stabilities of the native species when compared to wild-type. Activity recovery studies demonstrated a decrease in the final yield of active enzyme with 40, 22, and 39 percentrecovery for [ ]D89E, [ ]Tll9R and [ ]A8lHrespectively.
Corley, Lori Denee (1997). Probing the folding pathway of bacterial luciferase via mutagenesis. Master's thesis, Texas A&M University. Available electronically from
https : / /hdl .handle .net /1969 .1 /ETD -TAMU -1997 -THESIS -C676.