In the Cro protein family, an evolutionary change in secondary structure has converted an α-helical fold to a mixture of α-helix and β-sheet. P22 Cro and λ Cro represent the ancestral all-α and descendant α+β folds, respectively. The major structural differences between these proteins are at the C-terminal end of the domain (residues 34-56), where two α-helices in P22 Cro align with two β-strands in λ Cro. We sought to assess the possibility that smooth evolutionary transitions could have converted the all-α structure to the α+β structure through sequences that could adopt both folds. First, we used scanning mutagenesis to identify and compare patterns of key stabilizing residues in the C-terminal regions of both P22 Cro and λ Cro. These patterns exhibited little similarity to each other, with structurally important residues in the two proteins most often occurring at different sequence positions. Second, "hybrid scanning" studies, involving replacement of each wild-type residue in P22 Cro with the aligned wild-type residue in λ Cro and vice versa, revealed five or six residues in each protein that strongly destabilized the other. These results suggest that key stability determinants for each Cro fold are quite different and that the P22 Cro sequence strongly favors the all-α structure while the λ Cro sequence strongly favors the α+β structure. Nonetheless, we were able to design a "structurally ambivalent" sequence fragment (SASF1), which corresponded to residues 39-56 and simultaneously incorporated most key stabilizing residues for both P22 Cro and λ Cro. NMR experiments showed SASF1 to stably fold as a β-hairpin when incorporated into the λ Cro sequence but as a pair of α-helices when incorporated into P22 Cro.
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