ESR STUDIES OF SPIN-LABELED MYCOBACTERIUM TUBERCULOSIS PROTEASE

自旋标记结核分枝杆菌蛋白酶的 ESR 研究

• 批准号: 7723997
• 负责人: KEITH A EARLE
• 金额: $ 0.16万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723997
• 关键词: Bacteria; Computer Retrieval of Information on Scientific Projects Database; Consultations; Cysteine; Endopeptidases; Funding; Goals; Grant; Institution; Label; Measurement; Modeling; Mutate; Mycobacterium tuberculosis; Noise; Numbers; Peptide Hydrolases; Play; Preparation; Proteins; Protocols documentation; Relative (related person); Research; Research Personnel; Resources; Role; Sampling; Signal Transduction; Source; Spin Labels; Structure; Sulfhydryl Compounds; United States National Institutes of Health; X-Ray Crystallography; improved; interest; research study

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 结核分枝杆菌 (MBT) 蛋白酶是一种 14kD 蛋白质，在防御细菌免遭宿主根除方面发挥作用。 从 X 射线晶体学中得知，该蛋白酶具有有趣的环形结构，由交替的 α 和 β 环以及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。 蛋白酶功能的一种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，我们对单标记 MBT 蛋白酶进行了自旋标记实验，其中除一个非功能性半胱氨酸外的所有非功能性半胱氨酸均已突变为不含硫醇基团的残基，因此每个蛋白酶只能有一个自旋标记。 X 波段的初步结果表明，由于信噪比较低，MBT 蛋白酶的自旋标记方案需要改进。 与 ACERT 工作人员的协商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，以解析与 α 环和 β 环不同相对方向相对应的距离分布。