Directed Evolution Designed to Optimize the in vivo Protein Folding Environment.

Abstract

Protein folding is assisted by molecular chaperones and folding catalysts in vivo. Understanding how chaperones are regulated and how they function in vivo may provide new avenues for developing protein folding modulators. We used directed evolution which combines DNA manipulation and powerful selection procedures for beneficial mutations in proteins to specifically address these questions. My work focused on two distinct, though closely related problems, both of which have to do with the directed evolution of the periplasmic folding environment of bacteria. My first set of experiments concerned the relationship between the CXXC active site and the functional properties of thiol-disulfide oxidoreductases. Thiol-disulfide oxidoreductases are involved in catalyzing disulfide bond formation, isomerization and reduction during protein folding. We selected for mutants in the CXXC motif of a reducing oxidoreductase, thioredoxin, that complement null mutants in the very oxidizing oxidoreductase, DsbA. We found that altering the CXXC motif affects not only the reduction potential of thioredoxin, but also the ability of the protein to interact with folding protein substrates and reoxidants. Furthermore, the CXXC motif also impacts the ability of thioredoxin to function as a disulfide isomerase. Our results indicate that the CXXC motif has the remarkable ability to confer a large number of very specific properties on thioredoxin related proteins, in addition to their usual roles of regulating redox potentials. The second phase of my work sought to optimize the in vivo folding of proteins by linking folding to antibiotic resistance, thereby forcing bacteria to either effectively fold the selected proteins or perish. Here we were able to show that when Escherichia coli is challenged to fold a very unstable protein, it responds by overproducing a protein called Spy, which increases the steady state level of unstable proteins up to nearly 700-fold. In vitro studies demonstrate that Spy functions as a very effective ATP-independent chaperone that suppresses protein aggregation and aids protein refolding. Our strategy opens up new routes for chaperone discovery and the custom tailoring of the in vivo folding environment. Spy forms thin flexible cradle-shape dimers with an apolar concave surface, unlike the structure of any previously solved chaperone.