Aromatic cluster mutations produce focal modulations of β‐sheet structure

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.