ESR STUDIES OF SPIN-LABELED MYCOBACTERIUM TUBERCULOSIS PROTEASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 结核分枝杆菌蛋白(MBT)是一种14kD的蛋白质，在抵抗宿主根除细菌的过程中发挥着重要作用。从X射线结晶学中已知该酶具有有趣的环状结构，由交替的α环和β环以及一些非功能性半胱氨酸组成，这些半胱氨酸是自旋标记的候选物质。蛋白水解酶功能的一种模型是环相对于另一个环旋转，导致进入环状区的蛋白质分解。作为验证这一假说的第一步，已经对单标记的MBT蛋白酶进行了自旋标记实验，其中除了一个之外，所有不起作用的半胱氨酸都被突变为没有硫醇基团的残基，使得每个蛋白酶只有一个自旋标记是可能的。在X波段的初步结果表明，由于信噪比较低，用于MBT蛋白酶的自旋标记的方法需要改进。与ACERT工作人员的协商导致改进了样品制备方案。最终目标是对双标记MBT蛋白酶进行距离测量，以解决与α环和β环不同相对取向对应的距离分布。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mycobacterium Tuberculosis (MBT) Protease is a 14kD protein that plays a role in the defense of the bacterium against eradication by the host. The protease is known from X-ray crystallography to have an interesting annular structure, composed of alternating alpha and beta rings with a number of non-functional cysteines that are candidates for spin labeling. One model of protease function is that the rings rotate with respect to one another leading to the decomposition of proteins that enter the annular region. As a first step to check this hypothesis, spin-labeling experiments have been performed on singly-labeled MBT protease, where all but one of the non-functional cysteines have been mutated to a residue without a thiol group, such that only one spin label per protease is possible. Initial results at X-band indicate that the protocol for spin-labeling the MBT protease needs to be improved, as the signal to noise ratio was low. Consultations with ACERT staff have led to improved sample preparation protocols. The eventual goal is to perform distance measurements on bi-labeled MBT protease, to resolve the distribution of distances corresponding to different relative orientations of the alpha and beta rings.