Aromatic cluster mutations produce focal modulations of β-sheet structure

Protein Sci. 2015 May;24(5):841-9. doi: 10.1002/pro.2657. Epub 2015 Mar 25.


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.

Keywords: beta-sheet formation; conformational stability; protein design; secondary structure.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acids, Aromatic / chemistry
  • Amino Acids, Aromatic / genetics
  • Antigens, Surface / chemistry*
  • Antigens, Surface / genetics
  • Bacterial Outer Membrane Proteins / chemistry*
  • Bacterial Outer Membrane Proteins / genetics
  • Bacterial Vaccines / chemistry*
  • Bacterial Vaccines / genetics
  • Borrelia / chemistry*
  • Borrelia Infections / genetics
  • Borrelia Infections / microbiology*
  • Crystallography, X-Ray
  • Humans
  • Lipoproteins / chemistry*
  • Lipoproteins / genetics
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Structure, Secondary*


  • Amino Acids, Aromatic
  • Antigens, Surface
  • Bacterial Outer Membrane Proteins
  • Bacterial Vaccines
  • Lipoproteins
  • OspA protein

Associated data

  • PDB/2OY5
  • PDB/2PI3
  • PDB/3AUM