Why do certain proteins evolve much slower than others? We compared not only rates per protein, but also rates per position within individual proteins. For ∼90% of proteins, the distribution of positional rates exhibits three peaks: a peak of slow evolving residues, with average log 2 [normalized rate], log 2 μ, of ca. −2, corresponding primarily to core residues; a peak of fast evolving residues (log 2 μ ∼ 0.5) largely corresponding to surface residues; and a very fast peak (log 2 μ ∼ 2) associated with disordered segments. However, a unique fraction of proteins that evolve very slowly exhibit not only a negligible fast peak, but also a peak with a log 2 μ ∼ −4, rather than the standard core peak of −2. Thus, a "freeze" of a protein's surface seems to stop core evolution as well. We also observed a much higher fraction of substitutions in potentially interacting residues than expected by chance, including substitutions in pairs of contacting surface-core residues. Overall, the data suggest that accumulation of surface substitutions enables the acceptance of substitutions in core positions. The underlying reason for slow evolution might therefore be a highly constrained surface due to protein-protein interactions or the need to prevent misfolding or aggregation. If the surface is inaccessible to substitutions, so becomes the core, thus resulting in very slow overall rates. core mutation | correlated mutation | protein mutations | surface mutation P rotein evolutionary rates-that is, the rate by which protein sequences change over time-differ by factors of 100 to 1,000, even within the same organism (1-9). Early theories surmised that rates are determined by the proportion of protein sites that are involved in function, and are therefore under strong purifying selection (10). However, although many plausible functional and biophysical constraints have been examined in relation to evolutionary rates, only weak correlations were found. Functional constraints manifested in gene essentiality show no correlation with evolutionary rates (7, 11). However, proteins with a larger fraction of interactive surface area tend to be more conserved (4, 12), supporting the notion that binding interfaces evolve slower than nonbinding surfaces (3). Unexpectedly, expression levels seem to be the strongest predictor of evolutionary rates (13,14). Highly expressed proteins tend to evolve slowly, possibly because of their higher sensitivity to mistranslation and misfolding (2). Nonetheless, the divergence rates of protein sequences and of their expression levels are not correlated (6). Overall, the variety of functional and biophysical factors that constrain protein evolution complicates the understanding of evolutionary rates (1).Evolutionary rates were thus far analyzed only as overall rates per protein (2-8). However, within a given protein, each position is under a different type and magnitude of selection pressure (10). A buried, core position would be under strong selection to maintain the protein's configurational stability; a s...