Although we understand the key forces that drive protein folding, it remains difficult to predict the effects of mutations on thermodynamic stability.  Knowledge based potentials have improved such predictions, but it is unlikely that AI/machine based methods will be as useful as they have for predicting the structures of natural proteins, since not nearly as many well-characterized mutants are available.  Our lab has taken a library-based experimental approach to get a more expansive view of thermodynamic effects in model proteins.  In particular, we have used the four-helix bundle protein Rop, in combination with a cell-based screen and high-throughput implementation of a dye-binding screen for stability, to examine the effects of mutations in the core, turn and surface of Rop, with special attention to places with non-idealities in the core and heptad repeat.  We have also used selection to examine the fitness of all single point mutants of Rop.  Here I will review some of the most important and surprising lessons from these studies.  Several ideas we will touch on are evidence for “oil droplet” or “bucket of bolts” versus “jigsaw puzzle” like constraints on core packing, as well as the surprising volume plasticity of the core.  Outside of the core, it has proven easier to find mutations that afford substantial stabilization among active variants, but these positions also yield other changes in properties such as solubility, which highlight competing evolutionary forces that can be difficult to recapitulate in laboratory selection. These studies tend to reveal that average or consensus behavior of active variants matches with what we know to be key forces in protein folding, but the distributions of active variants show many surprising ways to achieve folded molecules.

Library-based engineering of a model four-helix bundle protein reveals diverse ways to stabilize and competing evolutionary forces

Fall 2022 abstracts, posters, and presentations will launch in mid-September.

ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.