Assignment and Structural Characterization of Uniformly Labeled Proteins by Solid State NMR

在这个项目中，PI将开发新的固体核磁共振方法来研究蛋白质结构。在之前的授权期内，PI已基本完成了微晶人泛素的位置特异性核磁共振指认，为固体核磁共振确定从头结构的第一阶段提供了原理证明。在本项目中，PI将确认现有的分配，并为移动部分定位信号。随着最近高磁场强度的出现，她的团队打算尝试更具挑战性和生物学意义的靶点；另外两种蛋白质将被指定为固相沉淀物：钙调蛋白和磷酸丙糖异构酶。最后，PI将根据固体核磁共振数据，利用几个实验数据来源来确定泛素的三维结构。扭转约束可以从化学位移本身获得，这将使用来自溶液核磁共振的统计数据库以及从计算的位移进行分析。氢键关联将通过N-H…C关联来测量。三级接触将通过自旋扩散和选择性重新耦合实验进行测量。结构将使用商业软件计算。虽然结构基因组学进展迅速，但许多重要的不可溶蛋白质并不容易表征；当通过这项工作建立方法时，PI的实验室将尝试表征一些不可溶靶标。PI参与了与几个学术和工业实验室的合作，以促进通过这项研究获得的信息的转移。然而，核心努力是培养优秀的研究生。高中生和本科生将接受培训，并在PI的实验室中接触结构生物学和磁共振。

In this project the PI will develop new solid state NMR methods to study protein structure. In the previous grant period, the PI has largely completed the site-specific NMR resonance assignments for microcrystalline human Ubiquitin, providing proof of principle for the first stage in denovo structure determination by solid state NMR. In this project, the PI will confirm the existing assignments, and locate signals for the mobile portions. With the recent availability of high magnetic field strengths, her group intends to attempt more challenging and biologically interesting targets; two additional proteins would be assigned as solid phase precipitates: Calmodulin and Triosephosphate Isomerase. Finally, the PI will determine the 3D structure of Ubiquitin based on solid-state NMR data, utilizing several sources of experimental data. Torsional constraints are available from the chemical shifts themselves, which will be analyzed using statistical databases from solution NMR as well as from computed shifts. Hydrogen bond correlations will be measured via N-H...C correlations. Tertiary contacts will be measured using spin diffusion as well as selective recoupling experiments. Structures would be calculated using commercially available software.While structural genomics proceeds apace, many important non-soluble proteins are not readily characterized; the PI's laboratory will attempt characterization of some of the non-soluble targets when the methods are established through this work. The PI is involved with collaborations with several academic and industrial labs that will facilitate transfer of the information obtained through this research. The core effort, however, is the training of outstanding graduate students. High school and undergraduate students will be trained and exposed to structural biology and magnetic resonance in the PI's laboratory.