Heme Protein Sensors in Prokaryotes

描述（由申请人提供）：对关键信号蛋白如何区分一氧化氮（NO）和氧气的分子水平理解是本提案的中心焦点。一氧化氮是一种关键的信号分子，在血管扩张、阴茎勃起和其他平滑肌相关反应中起着核心作用。一氧化氮也是中枢神经系统的一种信号剂，被免疫系统用来杀死感染性生物和肿瘤细胞。一氧化氮是有毒的，化学反应性强，主要问题仍然是如何在人类和其他动物身上特异性地使用这种分子。最近的观察清楚地表明，细菌含有一个与鸟苷酸环化酶密切相关的蛋白质家族，鸟苷酸环化酶是人类的NO受体（传感器）。然而，虽然在某些情况下，我们的假设是这些原核蛋白参与NO传感，但在其他情况下，它们似乎是O2传感器。这些蛋白质如何能够区分一氧化氮和氧气是本提案的总体目标，也是了解功能的总体目标。获得的结果不仅可能解释细菌如何感知NO和氧，而且还将阐明这些分子如何被感知并在人类和其他动物中使用。此外，这项工作将为为什么一些细菌病原体不被NO杀死提供新的数据。实验上，本项目将涉及分子生物学和蛋白质表征的一般工具以及先进的光谱技术，如共振拉曼光谱。此外，还将利用基因转染大肠杆菌进行蛋白质结晶结构测定和功能研究。允许这种困难的区分的结构决定因素并不明显，并且仍然是生物识别和信号特异性的核心。在过去的十年中出现的结合NO的蛋白质特征现在已经扩展到包括氧。CO信号是一个活跃的研究领域，缺乏特定的受体。这个家族中的蛋白质很可能是这个关键的缺失成分的可能性。此外，当病原体对来自免疫系统的NO挑战作出反应时，它们可以使用类似于假设包含在许多兼性好氧细菌中的表观NO信号系统的受体系统。

DESCRIPTION (provided by applicant): A molecular level understanding of how key signaling proteins distinguish between nitric oxide (NO) and oxygen is the central focus of this proposal. NO is a key signaling molecule that plays a central role in blood vessel dilation, penile erections and other smooth muscle related responses. NO also is a signaling agent in the central nervous system and is used by the immune system to kill infectious organisms and tumor cells. NO is toxic and chemically reactive and major questions remain concerning how this molecule is used with specificty in humans and other animals. Recent observations clearly show that bacteria contain a family of proteins that are closely related to the enzyme guanylate cyclase, the NO receptor (sensor) in humans. However, while in some cases our hypothesis is that these prokaryotic proteins are involved in NO sensing, in others is appears that they are O2 sensors. How are these proteins able to distinguish NO from oxygen is the broad overall goal of this proposal as well as understanding function. The results obtained are likely to explain not only how NO and oxygen are sensed in bacteria but will also shed light on how these molecules are sensed and used in humans and other animals. Further, the work will provide novel data on why some bacterial pathogens are not killed by NO. Experimentally this project will involve general tools of molecular biology and protein characterization plus advanced spectroscopic techniques such as resonance Raman spectroscopy. In addition, protein crystallization structure determination and functional studies by gene transfection into E. coli will be used. The structural determinants that allow for this difficult discrimination are not obvious and remain at the heart of biological recognition and specificity of signaling. What appeared for the last 10 years to be a characterization of proteins that bind NO has now been expanded to include oxygen. CO signaling is an active area of research lacking among other things a specific receptor. Proteins in this family are likely possibilities for this key, missing component. Furthermore, when pathogens respond to a NO challenge from the immune system, they could use a receptor system similar to the apparent NO signaling system hypothesized to be contained in a number of facultative aerobic bacteria.