The pSUFER section of the CSFp strategy was used to diagnose and improve suboptimal regions in the design protein which detects energetically suboptimal sites in the design by means of the Rosetta ato

The pSUFER segment of the CSFp strategy was employed to diagnose and enhance suboptimal regions in the design protein, which identifies energetically suboptimal sites in the design through the use of the Rosetta atomic computational mutation scan method. Several frustrated residues were detected in the overall structure of D-Lac (Figure 4A), while the frustrated residues in close proximity to the binding pocket were further scrutinized in subsequent evolution (Figure 4B). Residues E80, N146, N252, A271, F308, and D311 were identified as 'suboptimal', with E80, N252 and D311 being excluded from evolution as calcium ligand residues. Moreover, F308 was converted to glycine in the previous evolution, while N146T and N146Q resulted in a loss of activity (Figure 3G). However, A271 was deemed an important evolvable residue due to its suboptimal markers in pSUFER over 14, and the fact that both A271D and A271N exhibited synergistic mutagenesis with F308G in the sequence-dependent evolution analysis of FuncLib (Figure 4C). As such, A271 was mutated to E, L, and Q, respectively, as these residues are present in the sequence space of pSUFER and possess distinct side chain properties. This resulted in an 18-fold increase in activity for the double mutation A271E/F308G (Figure 4E). Finally, the optimal double mutation A271E/F308G served as a template for combined mutation with N96S, and the mutant N96S/A271E/F308G demonstrated a 35-fold increase in activity (Figure 4E)