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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Two-dimensional IR spectroscopy and segmental ~(13)C labeling reveals the domain structure of human γD-crystallin amyloid fibrils
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Two-dimensional IR spectroscopy and segmental ~(13)C labeling reveals the domain structure of human γD-crystallin amyloid fibrils

机译：二维红外光谱和〜（13）C分段标记揭示了人γD-晶状蛋白淀粉样蛋白原纤维的结构域

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摘要

The structural eye lens protein YD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly ~(13)C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid p-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the p-sheets. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent β-strands to each β-sheet. From these observations, we propose an initial structural model for γD-crystal-lin amyloid fibrils. Because only 1 μg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins.
机译：结构性晶状体蛋白YD-晶状体蛋白是白内障的主要成分，但聚集时其构象尚不清楚。使用表达的蛋白质连接，我们统一〜（13）C标记两个希腊关键域之一，以便它们分别在二维（2D）红外光谱中解析，以进行结构和动力学分析。在酸诱导的淀粉样蛋白原纤维形成后，二维红外光谱显示C端结构域形成淀粉样蛋白p-折叠，而N端结构域变得极为无序，但紧邻p-折叠。二维IR动力学实验表明，原纤维成核和延伸仅发生在C末端区域。这些结果是出乎意料的，因为N端结构域在单体形式中不稳定。同位素稀释实验表明，每个C末端结构域为每个β折叠贡献了两个或更少的相邻β链。从这些观察结果，我们提出了γD-晶体-林淀粉样蛋白原纤维的初始结构模型。由于2D红外光谱仅需要1μg蛋白质，因此使用这种方法可以在许多条件下研究表达不佳的蛋白质。因此，我们相信二维红外和蛋白质连接将对许多可以直接应用红外光谱的蛋白质系统（例如膜和淀粉样蛋白）的结构和动力学研究有用。

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    Sean D. Moran; Ann Marie Woys; Lauren E. Buchanan; Eli Bixby; Sean M. Decatur; Martin T. Zanni;

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    Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wl 53706,Department of Chemistry and Biochemistry, Oberlin College, 119 Woodland Street, A263, Oberlin, OH 44074;

    Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wl 53706;

    Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wl 53706;

    Department of Chemistry and Biochemistry, Oberlin College, 119 Woodland Street, A263, Oberlin, OH 44074;

    Department of Chemistry and Biochemistry, Oberlin College, 119 Woodland Street, A263, Oberlin, OH 44074;

    Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wl 53706;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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