Quantifying β-Sheet Stability by Phage Display

The small immunoglobulin G (IgG)-binding protein GB1 is a favored model system for the study of individual residue contributions to the stability of β-sheets. Nevertheless, only a few of the many possible combinations of mutations have been characterized, leaving many questions unanswered. In order to allow the simultaneous evaluation of libraries of mutants, we have adapted a phage-display method, called shotgun scanning. This method combines a binding (i.e. stability) selection with high-throughput sequence analysis. Relative folding free energies determined from GB1-phage sequence data agree well with published GB1 thermal stability studies, validating the use of phage display to conduct quantitative stability studies on GB1, and further suggesting that this method is generally applicable to mutational analysis of protein stability. Examination of residue pairing in our large collection of GB1 mutants indicates that specific side-chain–side-chain interactions are much less important to β-sheet stability than individual residue contributions. The discrepancy between this observation and published studies can be traced to anomalous stability of the alanine-substituted GB1 variants typically used as reference states in double mutant-cycle analyses. Finally, the combination of large library sizes and a quantitative stability selection should allow phage-based “computation” to be applied to protein design problems.