Modulation of soluble guanylyl cyclase by endogenous elements and exogenous facto

描述（由申请人提供）：单个细胞相互沟通的能力是使它们能够在多细胞生物环境中共存的基本特性。一氧化氮（NO）是一种用于与邻近细胞沟通的信号分子。一种特殊的异二聚体血红蛋白-可溶性鸟苷环化酶(sGC) -将细胞外NO信号转化为细胞内信使3‘-5’环鸟苷单磷酸。虽然有许多血红蛋白被NO相互作用抑制的例子，但sGC是唯一被NO结合激活的血红蛋白。虽然sGC作为NO受体的功能已经确定，但其催化活化、信号偶联和酶失活的过程尚不清楚。此外，这种在富氧环境中起作用的血红蛋白是如何高亲和力地选择性结合NO而不结合氧的，目前还不清楚。该提议基于一个中心假设，即配体特异性是由sGC亚基形成的血红素口袋支架决定的，酶的总体活性受到NO和其他效应分子诱导的血红素构象和连接的变化和/或氧化还原状态的影响。利用紫外可见光谱、荧光光谱、EPR光谱、共振拉曼光谱等多种光谱方法，研究了气态配体（NO、CO、O2）对亚铁sGC和不同几何形状阴离子配体对铁sGC的结合机理和催化作用。为了研究NO结合与cGMP合成之间的耦合机制，我们将光谱监测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形成的动力学联系起来。NO结合和cGMP形成之间的扩增比率将在单次或少量轮换下进行的研究中确定。为了分析sGC失活的机理，我们将利用光学和EPR光谱结合酶活性变化的测量记录NO-sGC复合物在NO清除剂氧化血红蛋白作用下的失活动力学。我们还将研究在激活/失活周期中结扎血红素和未结扎血红素的红牛状态的变化，以测试这是否是no依赖性调节的驱动机制。智力优势：该提案的主要智力优势是阐明了控制该酶功能的新颖和独特的分子机制，包括特异性配体选择和NO诱导的酶激活。这一建议将为从第一个NO结合到最后的cGMP形成步骤的NO依赖信号转导的生物物理和生化特性提供深刻的见解。

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.