title: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721-0088, USA.
authors.forename: Karen V
authors.initials: KV
authors.lastname: Eaton
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721-0088, USA.
authors.forename: William J
authors.initials: WJ
authors.lastname: Anderson
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721-0088, USA.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Dubrava
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721-0088, USA.
authors.forename: Vlad K
authors.initials: VK
authors.lastname: Kumirov
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721-0088, USA.
authors.forename: Emily M
authors.initials: EM
authors.lastname: Dykstra
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721-0088, USA cordes@email.arizona.edu.
authors.forename: Matthew H J
authors.initials: MH
authors.lastname: Cordes
mesh_terms: D019020:Directed Molecular Evolution; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins
keywords: chimera; folding specificity; hybrid sequence; structural evolution