LETTERS Lack of Evolutionary Conservation at Positions Important for Thermal Stability in the Yeast ODCase Protein

Mutations destabilizing the spatial structure of proteins can persist in populations if they are fixed by drift or compensated by other mutations. The prevalence and dynamics of these processes remain largely unrecognized. A suitable target to screen for both deleterious and compensatory mutations is the URA3 gene in yeast. We identified 13 positions in which a single missense substitution causes substantially strong thermal sensitivity. We then applied mild mutagenesis resulting in roughly one base substitution per gene and found that only reversions to an original amino acid can compensate for the thermal instability. However, the 13 positions are not visibly conserved across 53 species of Ascomycota, despite that the gene product is an enzyme of stable function and high efficiency. This shows how much fitness penalties for amino acid substitutions are background dependent, underscoring the role of complex intragenic interactions in the evolution of proteins.