Relying on evolutionary history recorded in thousands of homologous sequences to map important residues, consensus sequence design has emerged as an effective approach for designing biologically functional proteins with high stability. Intriguingly, sequence differences between consensus and extant homologs are predominantly located at surface residues. Surface mutations can have large effects on protein quinary structure, which organizes protein interactions inside cells. Here we compare the stability and folding kinetics of consensus phosphoglycerate kinase (PGK) and four extant PGK sequences in vitro and in cells. All PGK sequences are stabilized in mammalian cells and in <i>E. coli </i>compared to in vitro. The consensus sequence was most stable against heat denaturation in vitro and in cells compared to the naturally occurring homologs. Of the extant sequences, the sequence derived from <i>E. coli </i>had the smallest variation in stability across environments, likely because it evolved to function in the highly crowded bacteria cytosol. The sequence derived from yeast had the largest range of stability across environments, likely because of reduced evolutionary pressure to avoid non-specific interactions in the less crowded cytoplasm. Within this series, protein stability is strongly correlated with more negative net surface charge and repulsive cytoplasmic interactions, whereas environmental sensitivity is correlated with high positive surface charge and sticking (attractive) surface interactions in the cytosol. Therefore, while consensus sequence design identifies residues favorable for protein stability, it also introduces an evolutionary disadvantage by reducing the protein’s resistance to environmental perturbations. These results demonstrate the significance of quinary interactions in consensus sequences, highlighting the importance of incorporating quinary structure into models for consensus sequence design.

Interplay of surface electrostatics and cellular environment in the evolution of protein thermal stability

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ACS Fall 2022

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