Structural stability based deep sequencing of 5 protein targets

We developed an efficient, cost-effective assay for Structural Inference By Structure-stability-based selection of deep mutation variants and high throughput sequencing (Sibs-Seq). We demonstrated that unlike naturally occurring homologous sequences evolved over billions of years, these stability-selected, artificial homologs in a single-round 24 or 36 hours of mutation selections is often sufficient for producing high-accuracy structure prediction with < 2 angstrom root-mean-squared deviation (RMSD) to the native structure. Overall design: A target protein of interest (POI) is subjected to error prone PCR (EP-PCR) to construct a mutation library (>106) that is inserted between two assisted-complementary fragments of the murine dihydrofolate reductase. A functional mDHFR will then produce tetrahydrofolate (THF) essential for Trimethoprim(TMP) resistance and allow growth of TMP-sensitive E. coli in the presence of TMP. By contrast, if the POI is in an unstable structural state, two fragments of mDHFR would be too far apart to reconstitute into a functional mDHFR. Lacking a functional mDHFR will inhibit the growth of TMP-sensitive E. coli cells when treated with TMP. Thus, when inserting a library of randomly mutated POIs, those stably folded variants will lead to TMP-resistant E. coli that can grow in the presence of TMP. The sequence counts of each mutant available from high-throughput sequencing will allow to calculate its fitness score, which provides an estimation for its stability.