Poster abstracts
Poster number 13 submitted by Sidharth Mohan
Consensus, Correlations and Combinatorics in Engineering and Exploring stability in Triosephosphate Isomerases
Sidharth Mohan (Biophysics Graduate Program), Nicholas W. Callahan (Chemistry and Biochemistry), Kimberly R. Stephany (Chemistry and Biochemistry)
Abstract:
Nature exploits similar protein folds in a plethora of evolutionary niches, which suggests that a common structural scaffold can be encoded by a variety of amino-acid sequences. With increasing access to protein sequences comes the incumbent need to exploit such information in a useful way; and while attempts to engineer thermodynamic stability using multiple sequence alignments have proven useful in the past, we posit a more reliable and simplistic approach to do so. Information-theory based metrics of conservation and co-variation across positions in protein sequence alignments have revealed some patterns of stability and enzymatic activity in the Triosephosphate Isomerases (TIMs). We have used these metrics to delineate an algorithm that targets specific positions in a protein to mutate to the consensus residue, and we are able to generate stabilized and active proteins over their wild-type counterparts by this method. Our results show a ~90% probability in identifying stabilizing mutations, with successful application to proteins with different folds. Previously, a fully consensus variant of TIM (cTIM) suffered overall destabilization and impaired activity. However, a curated database of sequences yielded a variant with a native-like fold and activity (ccTIM). Since the variants differed minimally at the sequence level, we suspected the scrambling of statistical correlations in cTIM and developed combinatorial libraries to interrogate the exact nature of the differences between cTIM and ccTIM. Our data suggest global stabilization of the protein fold as a basis for the rescue of cTIM. Data from rational mutations in ccTIM to understand stability from the angle of destabilization of the fold suggest that the effects on stability are distributed. Our latest data from multi-mutants in the binomial pool of mutational differences between these two variants also points to this underlying reason for the biophysical differences.
Keywords: Consensus & Correlation, Protein Stability, Sequence Design