UNIQUELY LABELED AMINO ACIDS FOR PROTEIN NMR

该提案的主要目标是制定方法， 将阐明动态结构或肽和蛋白质， 溶液和固态。 现在人们普遍认为， 蛋白质没有固定的结构， 必须由关于侧链的运动信息补充， 骨架原子，如果一个更完整的理解的性质， 蛋白质的相互作用。 我们建议合成各种2 H-，13 C-和15 N-标记的 氨基酸并将其掺入葡萄球菌核酸酶中， 也许是其他蛋白质。 然后这些分子将被研究， 高场（200-500 MHz）NMR。 在一个项目中，（15 N，1- 13 C）-亮氨酸 将制备并掺入核酸酶中以研究溶液 活动现场的环境。 在第二个项目中，核酸酶 含有多种合成的C-13甲基标记的氨基酸 将准备和魔角旋转技术将使用 以获得更详细的图片-周围的各种区域 固态的标记甲基。 最后，在第三 项目，（d 6）-脯氨酸将被用于试图延长 新的方法，我们已经开发了分配顺式信号在 肽的质子谱。 为了实施这些项目，我请求支持三个项目， 本科生五年。 他们将承担主要 负责合成所有标记的氨基酸， 他们将定期纯化和分析他们的产品 通过色谱和核磁共振。 此外，约克最近收购了一家 200 MHz宽带FT-NMR（借助MBRS补充 仪器奖）将使这些学生参与 完全表征其C-13，N-15和氘标记的 产品和收集和解释弛豫时间，核 Overhauser效应，并使用复杂的脉冲交换数据 本科生一般不具备的技术。

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.