High-throughput analysis of the protein sequence-stability landscape using a quantitative yeast surface two-hybrid system and fragment reconstitution

Stability evaluation of many mutants can lead to a better understanding of the sequence determinants of a structural motif and of factors governing protein stability and protein evolution. The traditional biophysical analysis of protein stability is low throughput, limiting our ability to widely explore sequence space in a quantitative manner. In this study, we have developed a high-throughput library screening method for quantifying stability changes, which is based on protein fragment reconstitution and yeast surface display. Our method exploits the thermodynamic linkage between protein stability and fragment reconstitution and the ability of the yeast surface display technique to quantitatively evaluate protein-protein interactions. The method was applied to a fibronectin type III (FN3) domain. Characterization of fragment reconstitution was facilitated by the co-expression of two FN3 fragments, thus establishing a yeast surface two-hybrid method. Importantly, our method does not rely on competition between clones and thus eliminates a common limitation of high-throughput selection methods in which the most stable variants are recovered predominantly. Thus, it allows for the isolation of sequences that exhibit a desired level of stability. We identified more than 100 unique sequences for a β-bulge motif, which was significantly more informative than natural sequences of the FN3 family in revealing the sequence determinants for the β-bulge. Our method provides a powerful means for the rapid assessment of the stability of many variants, for the systematic assessment of the contribution of different factors to protein stability, and for enhancement of the protein stability.