ESR STUDIES OF SPIN-LABELED MYCOBACTERIUM TUBERCULOSIS PROTEASE

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 结核分枝杆菌（MBT）蛋白酶是一种14 kD的蛋白质，在防御细菌免受宿主根除中发挥作用。 从X射线晶体学中已知该蛋白酶具有有趣的环状结构，由交替的α和β环与许多非功能性半胱氨酸组成，这些半胱氨酸是自旋标记的候选者。 蛋白酶功能的一个模型是环相对于彼此旋转，导致进入环形区域的蛋白质分解。 作为检验这一假设的第一步，已经在单标记的MBT蛋白酶上进行了自旋标记实验，其中除了一个之外的所有非功能性半胱氨酸已经突变为没有巯基的残基，使得每个蛋白酶仅一个自旋标记是可能的。 在X波段的初步结果表明，用于自旋标记的MBT蛋白酶的协议需要改进，因为信噪比低。 与ACERT工作人员的协商改进了样品制备方案。 最终的目标是对双标记的MBT蛋白酶进行距离测量，以解析对应于α和β环的不同相对取向的距离分布。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.