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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 伯氏疏螺旋体外表面蛋白A(OspA)含有一个连接两个球状结构域的独特的单层β-折叠。尽管这种β-折叠片段暴露在溶剂中，因此它不包含疏水核心，但它是高度稳定的。因此，OspA为研究非小叶β-折叠的结构和能量学提供了吸引人的平台。β-折叠片段由两个同源的β-发夹组成。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，可以稳定地延伸单层β-折叠，这让人想起交叉β结构在多肽纤维中的传播(参见。附加的pdf文件)。 扩展的OspA的详细分子结构对于理解片材延伸的影响和进一步改进单层β片材的设计非常重要。我们以前的核磁共振表征主要集中在OspA 1BH的主干结构上，所以很难用核磁共振方法精确定义单层β-折叠区域的结构，因为这个区域本身就很少有在基于核磁共振的结构确定中至关重要的长程相互作用。此外，由于存在两个具有相同序列的23个残基片段，OspA 2BH的核磁共振指定在技术上具有挑战性。因此，利用APS同步辐射来确定精确的晶体结构是非常有意义的。我们通过优化Hauptman-Woodward研究所高通量筛选和内部筛选中的初始条件，成功地结晶了OspA变体，每个变体包含几个发夹插入件的副本。

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.