Quinary structure modulates protein sequence stability in vivo

Relying on evolutionary history within 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 weakly conserved surface residues. Surface mutations can have large effects on protein quinary structure, interactions that organize protein interactions inside cells, which are largely dependent on surface charges. Here we compare the sequence, stability, and kinetics of consensus PGK and four extant PGK sequences. We find that all five sequences are stabilized in mammalian cells and in E. coli compared to in vitro. Of the five sequences, the consensus sequence was most stable and showed a larger thermodynamic stability in cells compared to the naturally occurring homologs. Perhaps unsurprisingly, the thermodynamic stabilities of the four extant sequences are near the temperatures of their native environments. Of the extant sequences, the sequence derived from E. coli had the smallest change in stability across environments, likely because it evolved to function in the highly charged bacteria cytosol. At the melting temperature, the folding kinetics of the five sequences are identical and the folding kinetics of the extant sequences are unchanged in cells compared to in vitro. These results demonstrate the significance of quinary interactions in consensus sequences, highlighting the importance of incorporating quinary structure into models for consensus sequence design.