The repertoire of proteins and nucleic acids in the living world is determined by evolution; their properties are determined by the laws of physics and chemistry. Explanations of these two kinds of causality — the purviews of evolutionary biology and biochemistry, respectively — are typically pursued in isolation, but many fundamental questions fall squarely at the interface of fields. Here we articulate the paradigm of evolutionary biochemistry, which aims to dissect the physical mechanisms and evolutionary processes by which biological molecules diversified and to reveal how their physical architecture facilitates and constrains their evolution. We show how an integration of evolution with biochemistry moves us towards a more complete understanding of why biological molecules have the properties that they do.

Phylogenetic reconstruction of the structure and function of the ancestor of the nuclear receptor protein family reveals how functional diversity evolves by subtle tinkering with an ancestral template.

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Many functionally essential ionizable groups are buried in the hydrophobic interior of proteins. A systematic study of Lys, Asp, and Glu residues at 25 internal positions in staphylococcal nuclease showed that their pK a values can be highly anomalous, some shifted by as many as 5.7 pH units relative to normal pK a values in water. Here we show that, in contrast, Arg residues at the same internal positions exhibit no detectable shifts in pK a ; they are all charged at pH ≤ 10. Twenty-three of these 25 variants with Arg are folded at both pH 7 and 10. The mean decrease in thermodynamic stability from substitution with Arg was 6.2 kcal∕mol at this pH, comparable to that for substitution with Lys, Asp, or Glu at pH 7. The physical basis behind the remarkable ability of Arg residues to remain protonated in environments otherwise incompatible with charges is suggested by crystal structures of three variants showing how the guanidinium moiety of the Arg side chain is effectively neutralized through multiple hydrogen bonds to protein polar atoms and to site-bound water molecules. The length of the Arg side chain, and slight deformations of the protein, facilitate placement of the guanidinium moieties near polar groups or bulk water. This unique capacity of Arg side chains to retain their charge in dehydrated environments likely contributes toward the important functional roles of internal Arg residues in situations where a charge is needed in the interior of a protein, in a lipid bilayer, or in similarly hydrophobic environments.

SummaryProtein families with functionally diverse members can illuminate the structural determinants of protein function and the process by which protein structure and function evolve. To identify the key amino acid changes that differentiate one family member from another, most studies have taken a "horizontal" approach, swapping candidate residues between present-day family members. This approach has often been stymied, however, by the fact that shifts in function often require multiple interacting mutations; chimeric proteins are often non-functional, either because one lineage has amassed mutations that are incompatible with key residues that conferred a new function on other lineages, or because it lacks mutations required to support those key residues. These difficulties can be overcome by using a vertical strategy, which reconstructs ancestral genes and uses them as the appropriate background in which to study the effects of historical mutations on functional diversification. In this review, we discuss the advantages of the vertical strategy and highlight several exemplary studies that have used ancestral gene reconstruction to reveal the molecular underpinnings of protein structure, function, and evolution.