(169e) Bioinformatics-Mediated Rational Approaches to Protein Engineering for Increased Stability

Efforts to understand protein function at the molecular level have yet to fully capture the general rules that govern protein design. One important aspect of protein design is the prediction and optimization of enzyme stability over long time periods and under various harsh industrial operating conditions. This work focuses on data-driven methods for design and improvement of functional protein properties, primarily protein stability. A sequence-based study of the Subtilase superfamily is presented, revealing a sequence motif responsible for thermodynamic stability in that family. We demonstrate that subsequent insertion of that motif into a mesophilic family member results in increased thermal stability in vitro. A structure-based protein redesign method is presented for the direct improvement of protein kinetic stability with minimal effects on protein activity. We hypothesize in this work that enzyme function is primarily conferred by intramolecular contacts between secondary structural units. By maintaining such interactions (and therefore the majority of residue-residue contacts) and intelligently changing the way in which those structural units are connected (i.e., changing the topology of the protein), we demonstrate that it is possible to change the rate of unfolding of a model protein, GFP, in vitro, directly effecting protein stability without effecting protein activity. This method may be universal and therefore applied to any protein with multiple structural units.