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

在这个项目中，PI将开发新的固态NMR方法来研究蛋白质结构。 在上一个资助期内，主要研究者已大致完成了微晶人泛素的位点特异性NMR共振归属，为通过固态NMR进行从头结构测定的第一阶段提供了原理证明。 在本项目中，PI将确认现有分配，并定位移动的部分的信号。随着最近高磁场强度的可用性，她的团队打算尝试更具挑战性和生物学意义的目标;另外两种蛋白质将被分配为固相沉淀物：钙调素和磷酸丙糖异构酶。最后，PI将利用几个实验数据来源，基于固态NMR数据确定泛素的3D结构。扭转约束可从化学位移本身，这将使用统计数据库进行分析，从解决方案NMR以及从计算的位移。氢键相关性将通过N-H测量. C相关性。三级接触将使用自旋扩散以及选择性重新耦合实验来测量。虽然结构基因组学进展迅速，但许多重要的不溶性蛋白质并不容易表征; PI的实验室将在通过这项工作建立方法时尝试表征一些不溶性靶点。PI参与了与几个学术和工业实验室的合作，这将促进通过本研究获得的信息的转移。然而，核心工作是培养优秀的研究生。高中和本科生将在PI的实验室接受结构生物学和磁共振的培训。

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.