The Complex Role of Sequence on the Stability and Function of a Thermophilic TIM Barrel Protein

Abstract

TIM barrel proteins are encoded by highly divergent sequences, but fold to highly conserved (βα)8 structures with varying stabilities and functions. The indole‐3‐glycerol phosphate synthase (IGPS) TIM barrel protein, required for tryptophan biosynthesis in yeast, was selected as a target for investigating how sequence influences structure, stability, and function. IGPS from the S. solfataricus (SsIGPS) thermophile was subjected to the EMPIRIC assay to assess the change in fitness accompanying saturating mutagenesis on the 8 β‐stands and the preceding loops, known to be important for protein stability. The strategy was designed to test the presumption that mutations in the β‐barrel would provide an in vivo estimate of protein stability by decreasing fitness. The majority of the mutations in the β‐strands and preceding loops decreased the fitness, as expected. Surprisingly, mutations that remove any of the four‐fold symmetric TIM barrel βα hairpin clamps provided a fitness advantage. In vitro studies on one such mutant protein, I107K, was found to be destabilized to urea denaturation. A functional assay revealed that the I107K mutation increases the Vmax as well as the substrate binding affinity, indicated by lower Km. We speculate that the decrease in stability from these mutations is accompanied by an increase in the dynamics of the platform and, therefore, an increase in the fitness of a thermophilic protein at a suboptimal temperature in a mesophilic host.Funding source: NIH GM23303