ATOMIC STRUCTURES OF SINGLE-LAYER BETA-SHEET IN OSPA USING SURFACE MUTATIONS

使用表面突变的 OSPA 中单层 β-片层的原子结构

• 批准号: 7725993
• 负责人: KOKI MAKABE
• 金额: $ 0.39万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7725993
• 关键词: Borrelia burgdorferi; Computer Retrieval of Information on Scientific Projects Database; Condition; Funding; Grant; Housing; Institutes; Institution; KLK3 gene; Methods; Molecular Structure; Mutation; OspA protein; Peptides; Range; Research; Research Personnel; Resources; Screening procedure; Solvents; Source; Structure; Surface; United States National Institutes of Health; Variant; Vertebral column; base; beta pleated sheet; design; high throughput screening; improved; interest; synchrotron radiation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Outer surface protein A (OspA) from Borrelia burgdorferi contains a unique, single-layer beta-sheet that connects two globular domains. Although this beta-sheet segment is exposed to the solvent and thus it does not contain a hydrophobic core, it is highly stable. Therefore, OspA provides attractable platform for investigating the structure and energetics of nonglobular beta-sheets. The beta-sheet segment comprises two homologous beta-hairpins. We have previously demonstrated that the single-layer beta-sheet can be stably extended, in the context of OspA, by inserting copies of the beta-hairpin unit, reminiscent of the propagation of the cross-beta structure in peptide fibrils (cf. attached pdf file). Detailed molecular structures of the extended OspA would be very important for understanding the effects of the sheet extension and for further improving the design of single-layer beta-sheets. Our previous NMR characterization focused on the backbone structure of OspA+1bh, and it has been difficult to precisely define the structure of the single-layer beta-sheet region using NMR methods, because this region inherently has very few long-range interactions that are critical in NMR-based structure determination. Also NMR assignments of OspA+2bh has been technically challenging due to the presence of two 23-residue segments with an identical sequence. Thus the determination of precise crystal structures using APS synchrotron radiation is of a great interest. We have successfully crystallized OspA variants, each containing several copies of the hairpin inserts by optimizing initial conditions found in high-throughput screening at the Hauptman-Woodward institute and in-house screening.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 来自伯氏疏螺旋体的外表面蛋白A（OspA）包含连接两个球状结构域的独特的单层β-折叠。 尽管该β-折叠片段暴露于溶剂中，因此不含疏水核，但其高度稳定。 因此，OspA为研究非小叶β折叠的结构和能量学提供了一个有吸引力的平台。β-折叠区段包含两个同源的β-发夹。 我们先前已经证明，在OspA的背景下，通过插入β-发夹单元的拷贝，单层β-折叠可以稳定地延伸，这让人想起肽原纤维中交叉β结构的传播（参见图10）。附pdf档案）。 详细的分子结构的扩展OspA将是非常重要的理解的影响，片扩展和进一步改善设计的单层β-片。 我们之前的NMR表征集中在OspA+1bh的骨架结构上，并且很难使用NMR方法精确定义单层β-折叠区域的结构，因为该区域固有地具有非常少的长程相互作用，这在基于NMR的结构测定中是至关重要的。 此外，OspA+2bh的NMR分配在技术上具有挑战性，因为存在两个具有相同序列的23个残基片段。因此，使用APS同步辐射测定精确的晶体结构是一个很大的兴趣。我们已经成功地使OspA变体结晶，每个变体包含发夹插入物的几个拷贝，通过优化在Hauptman-Woodward研究所和内部筛选的高通量筛选中发现的初始条件。