Generating quantitative binding landscapes through fractional binding selections combined with deep sequencing and data normalization

Quantifying the effects of various mutations on binding free energy is crucial for understanding the evolution of protein-protein interactions and would greatly facilitate protein engineering studies. Yet, measuring changes in binding free energy (ΔΔG(bind)) remains a tedious task that requires expression of each mutant, its purification, and affinity measurements. We developed an attractive approach that allows us to quantify ΔΔG(bind) for thousands of protein mutants in one experiment. Our protocol combines protein randomization, Yeast Surface Display technology, deep sequencing, and a few experimental ΔΔG(bind) data points on purified proteins to generate ΔΔG(bind) values for the remaining numerous mutants of the same protein complex. Using this methodology, we comprehensively map the single-mutant binding landscape of one of the highest-affinity interaction between BPTI and Bovine Trypsin (BT). We show that ΔΔG(bind) for this interaction could be quantified with high accuracy over the range of 12 kcal mol(−1) displayed by various BPTI single mutants.