Yan Shude, Gawlak Grzegorz, Makabe Koki, Tereshko Valentina, Koide Akiko, Koide Shohei
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
J Mol Biol. 2007 Apr 20;368(1):230-43. doi: 10.1016/j.jmb.2007.02.003. Epub 2007 Feb 7.
Formation of a flat beta-sheet is a fundamental event in beta-sheet-mediated protein self-assembly. To investigate the contributions of various factors to the stability of flat beta-sheets, we performed extensive alanine-scanning mutagenesis experiments on the single-layer beta-sheet segment of Borrelia outer surface protein A (OspA). This beta-sheet segment consists of beta-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 beta-sheet propensity scales, statistical beta-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 beta-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 beta-sheet stability even at solvent-exposed positions. The OspA single-layer beta-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 beta-sheet layer in peptide self-assembly.
扁平β-折叠的形成是β-折叠介导的蛋白质自组装中的一个基本事件。为了研究各种因素对扁平β-折叠稳定性的贡献，我们对伯氏疏螺旋体表面蛋白A（OspA）的单层β-折叠片段进行了广泛的丙氨酸扫描诱变实验。这个β-折叠片段由具有高度规则几何形状的β-链组成，可作为自组装的构建模块。我们的丙氨酸扫描方法与传统的主客体方法不同，因为它只引入保守的截断突变，这种突变应能使结构扰动最小化。我们的结果与实验性β-折叠倾向量表、统计性β-折叠倾向量表或跨链成对相关性显示出非常弱的相关性。相比之下，我们的数据与埋藏的非极性表面积的变化显示出强烈的正相关。包括突出的Glu-Lys跨链对在内的极性相互作用对β-折叠稳定性的贡献微乎其微。这些结果得到了其他非丙氨酸突变结果的证实。综上所述，这些结果表明即使在溶剂暴露位置，非极性表面埋藏对扁平β-折叠稳定性也有主要贡献。OspA单层β-折叠通过多种侧链的策略性排列，在不形成疏水核心的情况下实现了有效的疏水表面埋藏。这些发现进一步表明了β-折叠层内疏水相互作用在肽自组装中的重要性。
