Heme Protein Sensors in Prokaryotes

原核生物中的血红素蛋白传感器

• 批准号: 7125140
• 负责人: MICHAEL A. MARLETTA
• 金额: $ 33.42万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-22 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7125140
• 关键词: Escherichia coli; Raman spectrometry; bacterial proteins; biological signal transduction; crystallization; enzyme activity; gene expression; hemoprotein; histidine; ligands; molecular cloning; nitric oxide; oxygen; point mutation; prokaryote; protein binding; protein kinase; protein protein interaction; protein purification; protein structure function; transfection

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.

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核心焦点。 NO 是一种关键信号分子，在血管扩张、阴茎勃起和其他平滑肌相关反应中发挥核心作用。 NO 也是中枢神经系统中的信号剂，被免疫系统用来杀死传染性生物体和肿瘤细胞。 NO 有毒且具有化学反应性，主要问题仍然是如何在人类和其他动物中特异性地使用该分子。最近的观察清楚地表明，细菌含有一个与鸟苷酸环化酶（人类的 NO 受体（传感器））密切相关的蛋白质家族。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。这些蛋白质如何区分一氧化氮和氧气是该提案的总体目标以及理解功能。获得的结果不仅可能解释细菌如何感知一氧化氮和氧气，还将揭示人类和其他动物如何感知和使用这些分子。此外，这项工作还将提供关于为什么某些细菌病原体不能被一氧化氮杀死的新数据。在实验上，该项目将涉及分子生物学和蛋白质表征的通用工具以及先进的光谱技术，例如共振拉曼光谱。此外，还将使用通过基因转染至大肠杆菌进行的蛋白质结晶结构测定和功能研究。允许这种困难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传导特异性的核心。过去十年中出现的与一氧化氮结合的蛋白质的特征现在已经扩展到包括氧。 CO信号传导是一个活跃的研究领域，但除其他外还缺乏特定的受体。这个家族中的蛋白质很可能是这个关键的、缺失的成分的可能性。此外，当病原体对来自免疫系统的一氧化氮攻击做出反应时，它们可以使用类似于表面一氧化氮信号系统的受体系统，该系统被假设包含在许多兼性需氧细菌中。