Genetic Drift and Structural Constraints: The Determinants of Compensatory Mutations in Mammalian Proteins

Proteins must maintain function integrity in the face of relentless mutational challenges. One key mechanism is compensatory mutation, a form of intra-protein coevolution where a subsequent mutation at a different site restores fitness lost to a prior deleterious mutation. While the biophysical basis of this process is wellstudied, the population genetic forces that dictate its prevalence across the tree of life remain poorly understood. This thesis confronts this gap by testing the central hypothesis that effective population size () is a primary determinant of the rate of compensatory mutation, a process with conflicting theoretical dependencies on population size. To test this hypothesis, we developed a comprehensive analytical framework integrating genomic, phylogenetic, and structural data from 78 mammalian species. We first validated the branch-specific ratio of non-synonymous to synonymous substitutions (/) as a robust proxy for . Compensatory events for three key biochemical properties (polarity, volume, and charge) were then identified using CoMap. A novel branch-specific parametric bootstrap test was developed to map these events to specific lineages, allowing for a direct correlation with our proxy. Finally, we integrated these findings with high-resolution experimental and predicted protein structures to provide a mechanistic, biophysical context. Our results establish a strong, positive, and highly significant correlation between / and the accumulation of compensatory mutations. This finding—supported by control analyses using direct estimates and body mass data—indicates that lineages with smaller effective population sizes accumulate significantly more compensatory mutations. Structural analysis confirmed that this effect is a primary predictor of compensation and further revealed that the compact structural core of proteins is a hotspot for such events. This study provides the first large-scale, genomic evidence that genetic drift is a key driver of compensatory evolution. Our findings strongly suggest that the initial fixation of a slightly deleterious mutation, a process facilitated by drift, is the rate-limiting step. This work offers a plausible molecular mechanism for the "valley crossing" component of Sewall Wright’s Shifting Balance Theory and provides a fundamental population-genetic basis for understanding antimicrobial resistance and protein structure prediction.