Studying protein fold evolution with hybrids of differently folded homologs

Abstract

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.

The Role of Evolutionary Selection in the Dynamics of Protein Structure Evolution

“…Among Cro bacteriophage transcription factors, a pair of homologous proteins with 40% sequence identity (indicating that they share a recent common ancestor) but different structures was found. Subsequent studies showed that some Cro proteins might be just a few mutations away from changing fold (18,21). Similarly, the structure of a protein naturally adopting a 3a fold was converted into a 4bþa fold via a series of carefully engineered point mutations.…”

Evidence for the recent origin of a bacterial protein-coding, overlapping orphan gene by evolutionary overprinting

“…This is corroborated by findings that long non-coding RNA may serve as a novelty pool and that ribosomes indeed translate novel ORFs [ 6 , 7 ]. It is hypothesized that this mechanism might produce novel domains or folds, which are added to existing genes or assembled to new genes [ 8 , 9 ].…”

The role of negative selection in protein evolution revealed through the energetics of the native state ensemble

“…One particularly interesting aspect of this problem is revealed by the repeated observation of “chameleon sequences”, which can adopt different folds, and the emerging discovery of “metamorphic proteins”, which change fold as part of their function. Such extremes of conformational specificity, which have already been shown to be amenable to protein engineering, may prove to be an evolutionarily important mechanism for both fold change and functional versatility (as a prominent sub‐class of “moonlighting proteins”). However, current bioinformatics tools and molecular dynamics simulations, using sequence or structure information, fail to reliably identify chameleon or metamorphic proteins .…”