Probing the folding pathway of bacterial luciferase via mutagenesis

Due to the character of the original source materials and the nature of batch digitization, quality control issues may be present in this document. Please report any quality issues you encounter to digital@library.tamu.edu, referencing the URI of the item.Includes bibliographical references: p. 63-65.Issued also on microfiche from Lange Micrographics.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