Modulation of soluble guanylyl cyclase by endogenous elements and exogenous facto

描述（由申请人提供）：单个细胞相互通信的能力是允许它们在多细胞生物体环境中共存的基本特性。一氧化氮（NO）是一种用于与相邻细胞进行通讯的信号分子。一种特殊的异源二聚血红素蛋白-可溶性鸟苷酸环化酶（sGC）-将这种细胞外NO信号转化为细胞内信使3 '-5'环鸟苷一磷酸。虽然有许多血红素蛋白通过与NO相互作用而被抑制的例子，但sGC是唯一通过NO结合而被激活的血红素蛋白。虽然sGC作为NO受体的功能已经被很好地确定，但是催化活化、信号耦合和酶失活的过程还远未被理解。此外，尚不清楚这种在富氧环境中起作用的血红素蛋白如何以高亲和力选择性地结合NO，但不结合氧。该建议是基于一个中心的假设，即配体的特异性是由两个sGC亚基形成的血红素口袋支架和酶的整体活性的影响血红素构象和连接和/或氧化还原状态的变化诱导的NO和其他效应分子。使用光谱方法（UV-Vis、荧光、EPR、共振拉曼）的组合，我们将评估气态配体（NO、CO、O2）与亚铁sGC和具有不同几何形状的阴离子配体与铁sGC的结合机制和催化效果。为了研究NO结合与cGMP合成之间的偶联机制，我们将通过光谱监测NO结合、NO-血红素复合物的转变、蛋白质构象变化的过程，并将这些信息与cGMP形成的动力学相关联。NO结合和cGMP形成之间的放大率将在单次或少量周转下进行的研究中确定。为了分析sGC失活的机制，我们将使用光学和EPR光谱结合酶活性变化的测量记录NO清除剂氧合血红蛋白对NO-sGC复合物的失活动力学。我们还将研究在激活/失活循环过程中连接和未连接的血红素的red-ox状态的变化，以测试这是否是NO依赖性调节的驱动机制。智力优势：该建议的主要智力价值是阐明了这种酶的功能，包括特定的配体选择和NO诱导的酶活化的新颖和独特的分子机制。这一建议将提供深刻的洞察到NO依赖的信号转导的生物物理和生物化学的细节，从第一个NO结合到最终的cGMP形成步骤。 更广泛的影响：此外，对sGC配体选择性的理解将提供有关气体分子与天然传感器相互作用的新的基础知识，并提供技术知识来设计用于检测有毒气体存在的高灵敏度设备中的新纳米传感器。对支配sGC活化的机制的理解可以使得能够设计靶向NO/cGMP依赖性信号传导的新药。该项目将作为培养博士后和研究生的工具。培训的目标是培养学生和研究员的技术和批判性思维，并加强他们对科学的兴趣。可溶性鸟苷酸环化酶是调节血管平滑肌松弛、血压、血小板聚集、血管生成等的关键酶。了解调控sGC功能的机制（配体选择性、酶激活或失活）对于改善现有方案和开发新的sGC导向疗法至关重要。

DESCRIPTION (provided by applicant): The ability of individual cells to communicate with each other is a fundamental property that allows them to coexist in a context of multicellular organisms. Nitric oxide (NO) is one of the signaling molecules used to communicate with adjacent cells. A special heterodimeric hemeprotein - soluble guanylyl cyclase (sGC) - converts this extracellular NO signal into intracellular messenger 3'-5' cyclic guanosine monophosphate. Although there are many examples of hemeproteins inhibited by interaction with NO, sGC is the only hemeproteins which is activated by NO binding. Although the function of sGC as NO receptor is well established, the processes of catalytic activation, signal coupling and enzyme deactivation are far from understood. Moreover, it is not understood how this hemeprotein which function in oxygen-rich environment selectively binds NO with high affinity, but does not bind oxygen. The proposal is based on a central hypothesis that the ligand specificity is determined by the heme pocket scaffold shaped by both sGC subunits and overall activity of the enzyme is affected by the changes of heme conformation and ligation and/or redox state induced by NO and other effector molecules. Using a combination of spectroscopic methods (UV-Vis, fluorescence, EPR, resonance Raman) we will assess the binding mechanism and catalytic effect of gaseous ligands (NO, CO, O2) to ferrous sGC and anionic ligands with different geometry to ferric sGC. To investigate the coupling mechanism between the binding of NO and cGMP synthesis we will spectroscopically monitor the processes of NO binding, transition of NO-heme complexes, protein conformational changes and will correlate this information with the dynamics of cGMP formation. The amplification ratio between NO binding and cGMP formation will be determined in studies done under single or few turnovers. To analyze the mechanism of sGC deactivation we will record the deactivation kinetics of NO-sGC complex by NO scavenger oxyhemoglobin using optical and EPR spectroscopy coupled with measurements of changes in enzyme activity. We will also investigate the changes in the red-ox state of the ligated and unligated heme during activation/deactivation cycle to test whether this is the driving mechanism of NO-dependent regulation. Intellectual Merit: The main intellectual merit of the proposal is elucidation of the novel and unique molecular mechanisms that govern the function of this enzyme, including specific ligand selection and NO induced enzyme activation. This proposal will provide incisive insight into the biophysical and biochemical specifics of NO-dependent signal transduction from the first NO binding to the final cGMP formation steps. Broader Impacts: In addition, understanding of the sGC ligand selectivity will provide new fundamental knowledge about the interaction of gaseous molecules with natural sensor and provide technical knowledge to design new nanosensors in high-sensitivity devices detecting the presence of toxic gases. Understanding of the mechanisms that govern activation of sGC may enable design for new drugs targeting the NO/cGMP- dependent signaling. The project will serve as a vehicle for training postdoctoral and graduate students. The goal of the training is to develop the student's and fellow's technical and critical thinking and reinforce their interest in science. Soluble guanylyl cyclase is a key enzyme in regulation of vascular smooth muscle relaxation, blood pressure, platelet aggregation, angiogenesis etc. In this proposal we will determine and analyze the factors crucial for different phases of sGC activity cycle. Understanding the mechanisms governing sGC function (ligand selectivity, enzyme activation or deactivation) is essential for improving existing regimens and developing new sGC-directed therapies.