Residues participating in the protein folding nucleus do not exhibit preferential evolutionary conservation

To what extent does natural selection act to optimize the details of protein folding kinetics? In an effort to address this question, the relationship between an amino acid's evolutionary conservation and its role in protein folding kinetics has been investigated intensively. Despite this effort, no consensus has been reached regarding the degree to which residues involved in native-like transition state structure (the folding nucleus) are conserved. Here we report the results of an exhaustive, systematic study of sequence conservation among residues known to participate in the experimentally (φ-value) defined folding nuclei of all of the appropriately characterized proteins reported to date. We observe no significant evidence that these residues exhibit any anomalous sequence conservation. We do observe, however, a significant bias in the existing kinetic data: the mean sequence conservation of the residues that have been the subject of kinetic characterization is greater than the mean sequence conservation of all residues in 13 of 14 proteins studied. This systematic experimental bias gives rise to the previous observation that the median conservation of residues reported to participate in the folding nucleus is greater than the median conservation of all of the residues in a protein. When this bias is corrected (by comparing, for example, the conservation of residues known to participate in the folding nucleus with that of other, kinetically characterized residues) the previously reported preferential conservation is effectively eliminated. In contrast to well-established theoretical expectations, both poorly and highly conserved residues are apparently equally likely to participate in the protein-folding nucleus.