Poster abstracts
Poster number 5 submitted by Connor Weyrick
Developing stability and functional screens and selections for the atypical helical bundle protein FruR
Connor Weyrick (Chemistry & Biochemistry Graduate Program), Thomas Magliery (Department of Chemistry & Biochemistry)
Abstract:
Decoding the relationship between protein sequence changes and their resulting impacts on structural stability and biological function is crucial for understanding how to better rationally design therapeutic proteins. The N-terminal DNA-binding domain of FruR is a 57 amino acid atypical helical bundle with a putative DNA-binding helix-turn-helix motif. FruR belongs to the superfamily of LacI transcriptional regulators and was originally identified by its transcriptional repression of phosphoenolpyruvate (PEP) fructose phosphotransferase system (PTS) enzymes in E. coli. FruR was later observed to interact with numerous operons involved in central carbon metabolism pathways, acting either as a transcriptional repressor or activator. Previous studies in the Magliery group have focused on developing screens and selections to identify stable and functional variants of an antiparallel four-helix bundle protein called Rop. We plan on extending these studies to the atypical helical bundle FruR to investigate the effects helical bundle orientation has on amino acid preferences in the core, on the surface, and in solvent-exposed loops. We have constructed DNA-binding domain and full length versions of wildtype FruR and a statistically-designed stabilized mutant named Algo FruR. Stability of the wildtype and mutant DNA-binding domains has been established by circular dichroism (CD) spectroscopy thermal denaturation and 1H-15N heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy. In addition, function of the full length versions has been assessed by electrophoretic mobility shift assays (EMSA). Finally, we have devised an assay exploiting E. coli growth differences on fructose minimal media when a missing fruR gene is rescued by FruR plasmid sequences. Active FruR sequences cause hindered growth while inactive sequences allow for normal growth rates. In the future, we plan on altering the assay format so that active FruR sequences instead result in faster growth. This way, we can use the assay to efficiently select for active sequences in a library of FruR variants.
Keywords: protein engineering