title: Sequence replacements in the central beta-turn of plastocyanin.
abstract: The role of beta-turns in dictating the structure of a beta-barrel protein is assessed by probing the tolerance of the central beta-turn of poplar plastocyanin to substitution by arbitrary sequences. Native plastocyanin binds copper and is colored bright blue. However, when the wild-type Pro47-Ser48-Gly49-Val50 turn sequence is replaced by arbitrary tetrapeptides, the vast majority (92/98 = 94%) of mutant proteins cannot fold into the native blue structure. Characterization of the colorless mutant proteins demonstrates that the majority of substitutions in this type II beta-turn disrupt the native structure severely. Gross structural changes are indicated by major differences in the CD spectra of the mutants relative to the wild-type protein, and by the much larger apparent size of mutant proteins in gel filtration experiments. These mutant proteins do not bind copper. Furthermore, Cys84 forms a disulfide bond readily in the colorless mutant proteins, indicating that it has moved away from the buried position it occupies in the native copper binding site and has become exposed. These results indicate that the central beta-turn in plastocyanin is not merely a default structure arising in response to the surrounding context; rather, sequence information in this turn plays an active role in dictating the location of a chain reversal in the beta-barrel structure. These findings are discussed in terms of their implications for the folding of natural proteins, as well as the design of de novo proteins.
authors.affiliation: Department of Chemistry, Princeton University, New Jersey 08544-1009, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Ybe
authors.forename: M H
authors.initials: MH
authors.lastname: Hecht
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003124:Colorimetry; D003300:Copper / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005813:Genes, Synthetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009010:Monte Carlo Method; D016297:Mutagenesis, Site-Directed; D010970:Plastocyanin / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D011993:Recombinant Fusion Proteins; D003300:Copper; D010970:Plastocyanin