To study the sequence determinants governing protein fold evolution, we generated hybrid sequences from two homologous proteins with 40% identity but different folds: Pfl 6 Cro, which has a mixed α + β structure, and Xfaso 1 Cro, which has an all α-helical structure. First, we first examined eight chimeric hybrids in which the more structurally conserved N-terminal half of one protein was fused to the more structurally divergent C-terminal half of the other. None of these chimeras folded, as judged by circular dichroism spectra and thermal melts, suggesting that both halves have strong intrinsic preferences for the native global fold pattern, and/or that the interfaces between the halves are not readily interchangeable. Second, we examined 10 hybrids in which blocks of the structurally divergent C-terminal region were exchanged. These hybrids showed varying levels of thermal stability and suggested that the key residues in the Xfaso 1 C terminus specifying the all-α fold were concentrated near the end of helix 4 in Xfaso 1, which aligns to the end of strand 2 in Pfl 6. Finally, we generated hybrid substitutions for each individual residue in this critical region and measured thermal stabilities. The results suggested that R47 and V48 were the strongest factors that excluded formation of the α + β fold in the C-terminal region of Xfaso 1. In support of this idea, we found that the folding stability of one of the original eight chimeras could be rescued by back-substituting these two residues. Overall, the results show not only that the key factors for Cro fold specificity and evolution are global and multifarious, but also that some all-α Cro proteins have a C-terminal subdomain sequence within a few substitutions of switching to the α + β fold.

Insertions and deletions in protein sequences, or indels, can disrupt structure and may result in changes in protein folds during evolution or in association with alternative splicing. Pfl 6 and Xfaso 1 are two proteins in the Cro family that share a common ancestor but have different folds. Sequence alignments of the two proteins show two gaps, one at the N terminus, where the sequence of Xfaso 1 is two residues shorter, and one near the center of the sequence, where the sequence of Pfl 6 is five residues shorter. To test the potential importance of indels in Cro protein evolution, we generated hybrid variants of Pfl 6 and Xfaso 1 with indels in one or both regions, chosen according to several plausible sequence alignments. All but one deletion variant completely unfolded both proteins, showing that a longer N-terminal sequence was critical for Pfl 6 folding and a longer central region sequence was critical for Xfaso 1 folding. By contrast, Xfaso 1 tolerated a longer N-terminal sequence with very little destabilization, and Pfl 6 tolerated central region insertions, albeit with substantial effects on thermal stability and some perturbation of the surrounding structure. None of the mutations appeared to convert one stable fold into the other. Based on this two-protein comparison, short insertion and deletion mutations probably played a role in evolutionary fold change in the Cro family, but were also not the only factors.