Modulation of soluble guanylyl cyclase by endogenous elements and exogenous facto

单个细胞相互通信的能力是使它们能够 在多细胞生物体的背景下共存。一氧化氮(NO)是一种信号转导分子。 与相邻单元进行通信。一种特殊的异二聚体血红蛋白-可溶性鸟苷酸环化酶(SGC)- 将这种胞外NO信号转化为胞内信使3‘-5’环鸟苷一磷酸。 尽管有许多与NO相互作用抑制血红素蛋白的例子，但sGC是唯一的 被NO结合激活的血红蛋白。虽然sGC作为NO受体的功能很好 已经确定的催化激活、信号偶联和酶失活的过程还远远没有 明白了。此外，目前还不清楚这种在富氧环境中发挥作用的血红蛋白是如何发挥作用的。 选择性地以高亲和力结合NO，但不结合氧。这项提议是基于一个中央 配体特异性由两个sGC亚基形成的血红素口袋支架决定的假设 酶的整体活性受血红素构象、连接和/或氧化还原的影响 NO和其他效应分子诱导的状态。使用光谱方法的组合(UV-VIS， 荧光、EPR、共振拉曼)，我们将评估气体的结合机理和催化效果 配体(NO、CO、O2)与铁的SGC和阴离子配体不同几何构型的铁SGC。去调查 NO结合和cGMP合成之间的耦合机制我们将通过光谱监测 NO结合的过程、NO-血红素复合体的转变、蛋白质构象的变化及其相关性 这一信息与cGMP的形成动力学有关。NO结合与cGMP的扩增比 阵型将在单次或几次失误情况下的研究中确定。分析SGC的作用机制 我们将记录NO清除剂氧合血红蛋白对NO-sGC复合体的失活动力学 使用光学和EPR光谱结合测量酶活性的变化。我们还将 研究结扎和未结扎的血红素在激活/失活过程中红牛状态的变化 周期，以检验这是否是NO依赖调控的驱动机制。 智力价值：该提议的主要智力价值是阐明了新颖而独特的分子 调控该酶功能的机制，包括特定的配体选择和NO诱导 酶的激活。这项提议将提供对生物物理和生化细节的深刻见解 NO依赖的信号转导从最初的NO结合到最终的cGMP形成步骤。 更广泛的影响：此外，了解sGC配体的选择性将提供新的基础 了解气体分子与天然传感器的相互作用，并提供技术知识 在检测有毒气体存在的高灵敏度设备中设计新的纳米传感器。了解 调控sGC激活的机制可能使针对NO/cGMP的新药设计成为可能。 依赖于信号。该项目将作为培训博士后和研究生的工具。这个 培训的目标是发展学生和同事的技术和批判性思维，并加强他们的 科学兴趣：可溶性鸟酰环化酶是调节血管平滑肌松弛、血液循环的关键酶 压力、血小板聚集、血管生成等。在这项建议中，我们将确定和分析这些因素 对sGC活动周期的不同阶段至关重要。理解sGC功能的调控机制 (配体选择性、酶激活或失活)对于改进现有方案和 开发新的sGC导向疗法。

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.