title: Aromatic cluster mutations produce focal modulations of β-sheet structure.
abstract: Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model β-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain "clusters" at highly solvent-exposed positions in the flat, single-layer β-sheet of Borrelia outer surface protein A (OspA). This unusual β-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the β-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of β-sheets. Surprisingly, X-ray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA β-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating β-sheet conformation in protein design.
authors: Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, Illinois, 60637.
authors: Matthew
authors: M
authors: Biancalana
authors: Koki
authors: K
authors: Makabe
authors: Shude
authors: S
authors: Yan
authors: Shohei
authors: S
authors: Koide
mesh_terms: D024322:Amino Acids, Aromatic / Q000737:chemistry / Q000235:genetics; D000954:Antigens, Surface / Q000737:chemistry* / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D001428:Bacterial Vaccines / Q000737:chemistry* / Q000235:genetics; D001898:Borrelia / Q000737:chemistry*; D001899:Borrelia Infections / Q000235:genetics / Q000382:microbiology*; D018360:Crystallography, X-Ray; D006801:Humans; D008074:Lipoproteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017433:Protein Structure, Secondary
chemical_list: D024322:Amino Acids, Aromatic; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D008074:Lipoproteins; C072045:OspA protein
keywords: beta-sheet formation; conformational stability; protein design; secondary structure
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