PROTEIN-NO INTERACTIONS PROBED BY CONVENTIONAL AND TIME-RESOLVED X-RAY CRYSTA

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。 自80年代末以来，一氧化氮被认为具有许多重要的生理作用。例如，NO具有重要的信号功能，是一种有效的抗菌剂，对多种细菌、病毒、真菌甚至寄生虫都有活性。一氧化氮？S的生理功能几乎总是涉及与蛋白质的特定相互作用；在确定这种蛋白质-NO相互作用的机制方面已经取得了很大进展，但仍有许多问题。在这里，我们建议开发广泛适用的方法，在蛋白质晶体中光化学产生游离NO，以便使用时间分辨蛋白质X射线结晶学实时监测NO与蛋白质的后续相互作用。这些方法将是研究NO信号和抗微生物现象的详细机制的宝贵工具。该项目将分为两个阶段。在第一阶段中，将在谢尔曼丙酸杆菌的牛肝过氧化氢酶和超氧化物歧化酶(SOD)晶体上测试各种产生NO的方法。这两种蛋白质都是容易获得的蛋白质，其晶体结构已知，并且与NO有强烈的相互作用。在该项目的第二阶段，将使用最好的NO光生系统来研究NO与来自结核分枝杆菌的含氧hBN的相互作用。人血清白蛋白是一种蛋白质，被认为可以保护结核分枝杆菌不受人体的伤害？S认为，这种蛋白质可以让结核病潜伏数年。这一阶段的目的将是表明我们的方法可以应用于医学和公共卫生中相当重要的系统。

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. Since the late 80s nitric oxide has been recognized to have many important physiological roles. For example, NO has important signaling functions, and is a potent antimicrobial agent active against a wide range of bacteria, viruses, fungi and even parasites. Nitric oxide?s physiological functions almost invariably involve specific interactions with proteins; much progress has been made in identifying the mechanisms of such protein-NO interactions, but many questions remain. Herein we propose to develop broadly applicable methodologies for photochemically generating free NO within a protein crystal, so that subsequent interactions of NO with the protein can be monitored in real time using time-resolved protein X-ray crystallography. Such methodologies will be invaluable tools for studying the detailed mechanisms of NO signaling and antimicrobial phenomena. The project will be divided into 2 phases. In phase 1 various methods for NO photogeneration will be tested on crystals of bovine liver catalase and superoxide dismutase (SOD) from Propionibacterium shermanii. Both are readily available proteins whose crystal structures are known, and that interact strongly with NO. In the second phase of the project the best NO photogenerating systems will be used to investigate NO interaction with oxygenated HbN from M. tuberculosis. HbN is a protein that is thought to protect M. tuberculosis from the body?s defenses, and allow TB to persist for years in a latent state. The aim of this phase will be to show that our method can be applied to a system of considerable importance in medicine and public health.