Poster abstracts
Poster number 59 submitted by Connor Weyrick
Developing a Cell-based Selection for the DNA-binding Helical Bundle Domain of Cra
Connor T. Weyrick (Chemistry and Biochemistry Graduate Program), Thomas J. Magliery (Department of Chemistry and Biochemistry)
Abstract:
Understanding the relationship between protein sequence changes and their resulting impact on structural stability and biological function is leading to advances in therapeutic protein design. The N-terminal DNA-binding domain of the bacterial catabolite repressor/activator protein (Cra) contains a nonparallel three-helix bundle with the helix-turn-helix major structural motif. Despite recent efforts uncovering the sequence-fitness relationship of antiparallel helical bundles, considerably less is known about nonparallel helical bundles. Here, we have developed a cell-based selection strategy that leverages bacterial growth differences on gluconeogenic substrates to identify stable and functional variants of Cra. We validated the approach by applying a library of single point mutants previously characterized with circular dichroism spectroscopy thermal denaturation, electrophoretic mobility shift assays, and fluorescence anisotropy. The selection successfully enriched variants with wildtype or improved levels of stability and function while depleting variants with deleterious mutations. This technology can now be applied to site saturation mutagenesis libraries of Cra to establish the sequence determinants of stability and function for nonparallel helical bundle proteins. The knowledge gained can be extended to therapeutic proteins to identify mutations that will improve their biophysical and pharmacological properties.
Keywords: protein engineering