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

McDermottMCB 9983581本项目的目的是开发固态NMR方法，以泛素、BPTI和margatoxin作为模型系统，对13 C、15 N标记和选择性2H标记的蛋白质进行均匀表征。 我们的目标是建立可靠的分配方法来研究未表征的膜系统。高分辨率，魔角旋转技术将在分配协议中强调，以允许通过各向同性碳位移确认侧链身份，类似于溶液NMR。 最近已经证明，高分辨率多维固态NMR方法可用于关联水合微晶U-13 C和15 N BPTI的主链和侧链13 C和15 N化学位移。 分配的方法将优化和开发的长范围的距离约束和动态约束，是一致的统一标记。 这项研究的目标是进一步发展固态核磁共振方法来研究大型和复杂的生物分子。目前所收集的特征分子并不完整，其特殊的盲点是位于细胞周边的蛋白质，在其含有油脂的膜中;它们在这里监督细胞之间的通信和交通。膜蛋白占细胞蛋白质的1/3，但只有少数被鉴定。

McDermottMCB 9983581The objective of this project is to develop solid state NMR methods to characterize uniformly 13C, 15N labeled and selectively 2H labeled proteins using ubiquitin, BPTI, and margatoxin as model systems. The goal is to establish reliable assignment methods to study uncharacterized membrane systems. High resolution, magic angle spinning techniques will be emphasized in the assignment protocol in order to allow confirmation of sidechain identity through isotropic carbon shifts, in analogy to solution NMR. It has been recently demonstrated that high-resolution multidimensional solid state NMR methods can be used to correlate backbone and sidechain 13C and 15N chemical shifts of hydrated micro-crystalline U-13C and 15N BPTI. Methods for assignment will be optimized and developed for long range distance constraints and for dynamical constraints that are consistent with uniform labeling. The goal of this research is to further develop solid state nuclear magnetic resonance methods to study large and complex biological molecules. The present collection of characterized molecules is incomplete, and its particular blind spot is the fascinating proteins that rest at the perimeter of the cell, in its grease-containing membrane; here they supervise the communication and traffic between cells. Membrane proteins constitute 1/3 of the proteins of a cell, and yet only a handful of them are characterized.