UNIQUELY LABELED AMINO ACIDS FOR PROTEIN NMR

这项提案的主要目标是开发出 将阐明多肽和蛋白质的动态结构。 无论是溶液还是固态。现在人们普遍认为， 蛋白质没有静态结构，X射线数据 必须补充关于侧链的运动信息和 如果对主链原子的性质有更全面的了解 蛋白质的相互作用是要实现的。 我们建议合成各种2H、13C和15N标记的化合物 氨基酸，并将它们结合到葡萄球菌核酸酶中， 也许是其他蛋白质。然后这些分子将被研究 高场(200-500 MHz)核磁共振。在一个项目中，(15N，1-13C)-亮氨酸 将制备并掺入核酸酶中以研究该溶液 活动站点的环境。在第二个项目中，核酸酶 含有多种人工合成的C-13甲基标记氨基酸 将准备和魔角旋转技术将被使用 要获得更详细的图像-周围地区的各种 固体中标记的甲基。最后，在第三个 项目，(D6)-将使用Proline来尝试延长 我们已经开发了一种新的方法，用于在 多肽的质子光谱。 为了实施这些项目，我请求支持三个项目 五年的本科生。他们将承担初选 负责所有标记氨基酸的合成和 多肽，他们会常规地纯化和分析他们的产品 通过层析和核磁共振分析。此外，约克最近收购了一家 200 MHz宽带FT-核磁共振(借助MBRS补充 乐器奖)将使这些学生能够参与 完整地表征了他们的C-13，N-15和氚标记的 产品和收集和解释松弛时间，核 Overhaser效应，并使用复杂的脉冲交换数据 本科生一般不会掌握的技巧。

The major objective of this proposal is to develop methods that will elucidate the dynamic structure or peptides and proteins in both solution and the solid state. It is now generally recognized that proteins do not have static structures and that X-ray data must be supplemented by motional information about sidechain and backbone atoms if a more complete understanding of the nature of protein interactions is to be achieved. We propose to synthesize a variety of 2H-,13C-, and 15N-labeled amino acids and incorporate them into Staphylococcal Nuclease and, perhaps, other proteins. These molecules will then be studied by high field (200-500 MHz) NMR. In one project, (15N, 1-13C)-leucine will be prepared and incorporated in Nuclease to study the solution environment of the active site. In a second project, Nuclease containing a variety of synthetic C-13 methyl-labeled amino acids will be prepared and magic angle spinning techniques will be used to obtain a more detailed picture- of the region around the various labeled methyl groups in the solid state. Finally, in the third project, (d6)-proline will be employed in an attempt to extend a new method that we have developed for assigning cis signals in the proton spectra of peptides. To implement these projects, I am requesting support for three undergraduate students for five years. They will bear primary responsibility for the synthesis of all the labeled amino acids and peptides and they will routinely purify and analyze their products by chromatography and NMR. Also, York's recent acquisition of a 200 MHz broadband FT-NMR (with the aid of an MBRS supplemental instrumentation award) will enable these students to participate fully in characterizing their C-13, N-15, and deuterium-labeled products and in gathering and interpreting relaxation time, Nuclear Overhauser Effect, and exchange data using sophisticated pulse techniques not generally available to undergraduates.