Our ultimate goal is to develop a rapid means to create non-antibody-based affinity reagents. Our strategy involves using directed molecular evolution and yeast surface display as a tool to generate panels of variant proteins that bind a defined ligand with improved affinity compared to the native protein. Through the analysis of these variants, we gain an understanding of the drivers that govern molecular recognition such that computational models for the rational design of new binding clefts can be developed. Our model affinity scaffold is based on calmodulin, a naturally occurring bivalent protein with considerable molecular plasticity and high structural stability. We have identified mutants with enhanced binding affinities to natural peptide targets that in all cases involve mutations far from the binding interface. Specifically, a range of mutations that either destabilize salt bridges in the unbound state or stabilize salt bridges in the bound state enhance binding. These results suggest that simple energetic calculations based on changes in the free energies of proteins provides a predictive means for rationale protein engineering.

Using directed molecular evolution to understand and improve molecular recognition in novel scaffold affinity reagents

243rd National Meeting (2012)

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