Hydrophobic Surface Burial Is the Major Stability Determinant of a Flat, Single-layer β-Sheet

Formation of a flat β-sheet is a fundamental event in β-sheet-mediated protein self-assembly. To investigate the contributions of various factors to the stability of flat β-sheets, we performed extensive alanine-scanning mutagenesis experiments on the single-layer β-sheet segment of Borrelia outer surface protein A (OspA). This β-sheet segment consists of β-strands with highly regular geometries that can serve as a building block for self-assembly. Our Ala-scanning approach is distinct from the conventional host-guest method, in that it introduces only conservative, truncation mutations that should minimize structural perturbation. Our results showed very weak correlation with experimental β-sheet propensity scales, statistical β-sheet propensity scales, or cross-strand pairwise correlations. In contrast, our data showed strong positive correlation with the change in buried non-polar surface area. Polar interactions including prominent Glu-Lys cross-strand pairs contribute marginally to the β-sheet stability. These results were corroborated by results from additional non-Ala mutations. Taken together, these results demonstrate the dominant contribution of non-polar surface burial to flat β-sheet stability even at solvent-exposed positions. The OspA single-layer β-sheet achieves efficient hydrophobic surface burial without forming a hydrophobic core by a strategic placement of a variety of side-chains. These findings further suggest the importance of hydrophobic interactions within a β-sheet layer in peptide self-assembly.