Peptides and proteins self-assemble into β-sheet-rich fibrils, amyloid, which extends its structure by incorporating peptide/protein molecules from solution. At the elongation edge, the peptide/protein molecule binds to the edge of the amyloid β-sheet. Such processes are transient and elusive when observing molecular details by experimental methods. We used a model protein system, peptide self-assembly mimic (PSAM), which mimics an amyloid-like structure within a globular protein by capping both edges of single-layer β sheet (SLB) with certain domains. We constructed a PSAM variant that lacks the capping domain on the C-terminal side to observe the structure of the β-sheet edge of the peptide self-assembly. This variant, which we termed PSAM-edge, proved to be soluble with a monomeric form. Urea-induced unfolding experiments revealed that PSAM-edge displayed two-state cooperative unfolding, indicating the N-terminal capping domain and extended SLB folded as one unit. The crystal structure showed that SLB was almost completely structured except for a few terminal residues. A molecular dynamics simulation results revealed that the SLB structure was retained while the C-terminal four residues fluctuated, which was consistent with the crystal structure. Our findings indicate that SLB is stable even when one side of the β-sheet edge is exposed to a solvent. This stability may prevent the dissociation of the attached peptide from the peptide self-assembly. Because of the scarcity of SLB proteins with exposed β-sheet edges in nature, successful construction of the PSAM-edge expands our understanding of protein folding and design.

中文翻译：

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使用模型蛋白对肽自组装的 β-折叠边缘进行结构分析

肽和蛋白质自组装成富含 β-折叠的原纤维，即淀粉样蛋白，通过结合溶液中的肽/蛋白质分子来扩展其结构。在延伸边缘，肽/蛋白质分子结合到淀粉样蛋白 β-折叠的边缘。当通过实验方法观察分子细节时，这样的过程是短暂的和难以捉摸的。我们使用了一种模型蛋白质系统，肽自组装模拟物 (PSAM)，它通过用某些结构域覆盖单层 β 折叠 (SLB) 的两个边缘来模拟球状蛋白质中的淀粉样蛋白结构。我们构建了一个 PSAM 变体，该变体在 C 端侧缺少加帽域，以观察肽自组装的 β-折叠边缘的结构。这种我们称为 PSAM-edge 的变体被证明可以以单体形式溶解。尿素诱导的展开实验表明，PSAM-edge 显示出两种状态协同展开，表明 N 端加帽域和扩展的 SLB 折叠为一个单元。晶体结构表明，除了少数末端残基外，SLB几乎完全结构化。分子动力学模拟结果表明，SLB结构保留，C端4个残基波动，与晶体结构一致。我们的研究结果表明，即使 β-折叠边缘的一侧暴露于溶剂中，SLB 也是稳定的。这种稳定性可以防止连接的肽从肽自组装中解离。由于自然界中很少有具有暴露的 β-折叠边缘的 SLB 蛋白，PSAM 边缘的成功构建扩展了我们对蛋白质折叠和设计的理解。