Mapping The Domain Architecture And Conformations of Soluble Guanylate Cyclase

描述（由申请人提供）：一氧化氮（NO）信号通路介导多种生理功能，包括血管舒张、神经传递、心肌功能和血小板聚集。可溶性鸟苷酸环化酶（sGC）是NO的主要受体。NO-sGC信号通路的功能障碍可导致心脏病、勃起功能障碍、中风和高血压。了解NO诱导的sGC活化的分子细节对于开发这些疾病状态的治疗至关重要。哺乳动物sGC是一种多结构域异二聚体血红素蛋白。由于全长sGC已被证明难以进行高分辨率结构分析（例如，X射线晶体学），域架构和NO诱导的构象变化知之甚少。本文提出的研究的具体目标集中在通过平行的互补蛋白质作图方法阐明sGC的结构域组织和构象变化。氢-氘交换质谱（HDX-MS）是通过测量酰胺质子与氘代溶剂交换速率的变化来绘制蛋白质表面的溶剂暴露的强有力策略。HDX-MS将用于绘制sGC结构域截短的相互作用表面。将进行丙氨酸扫描以确定对sGC结构域间相互作用至关重要的单个残基。为了表征sGC结构域的相对取向，将采用羟基自由基足迹法。在结构域间相互作用附近拴系的试剂处产生的羟基自由基可以在近端残基处切割多肽主链。这些切割模式报告了sGC结构域表面之间的接近性。HDX-MS、丙氨酸扫描和羟基自由基足迹法的结果将被整合以开发全长sGC的结构域结构的模型。然后可以在全长sGC中评估由NO刺激诱导的sGC结构域结构的变化。HDX-MS将用于绘制NO诱导的sGC表面可及性变化，以开发控制sGC环化酶活性的构象变化模型。 公共卫生相关性：通过可溶性鸟苷酸环化酶（sGC）的一氧化氮（NO）信号传导介导对循环和神经功能至关重要的多种生理过程。NO/sGC信号传导的中断与心脏病、中风、勃起功能障碍和神经变性有关。拟议的研究旨在阐明NO诱导的sGC激活的分子细节，这是开发NO/sGC功能障碍相关疾病治疗的先决条件。

DESCRIPTION (provided by applicant): Nitric oxide (NO) signaling pathways mediate diverse physiological functions, including vasodilation, neurotransmission, myocardial function, and platelet aggregation. Soluble guanylate cyclase (sGC) is the primary receptor of NO. Dysfunctions in the NO-sGC signaling pathway can lead to heart disease, erectile dysfunction, stroke, and hypertension. Understanding the molecular details of NO-induced sGC activation is crucial for developing treatments for these disease states. Mammalian sGC is a multi-domain, heterodimeric hemoprotein. Because full-length sGC has proven intractable to high resolution structure analysis (e.g., X-ray crystallography), the domain architecture and NO-induced conformational changes are poorly understood. The specific aims of the research proposed herein are focused on illuminating the domain organization and conformational changes of sGC through parallel, complementary protein mapping approaches. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful strategy for mapping the solvent exposure of protein surfaces by measuring changes in the rates of amide proton exchange with a deuterated solvent. HDX-MS will be used to map the interaction surfaces of sGC domain truncations. Alanine scanning will be performed to define individual residues that are crucial to sGC inter-domain interactions. To characterize the relative orientations of the sGC domains, hydroxyl radical footprinting will be employed. Hydroxyl radicals generated at a reagent tethered near an inter-domain interaction can cleave polypeptide backbones at proximal residues. These cleavage patterns report on the proximities between surfaces of sGC domains. The results of the HDX-MS, alanine scanning, and hydroxyl radical footprinting will be integrated to develop a model of the domain architecture of full-length sGC. Changes in the sGC domain architecture induced by NO stimulus can then be assessed in full-length sGC. HDX-MS will be used to map NO-induced changes in sGC surface accessibility to develop a model of the conformational changes that control sGC cyclase activity. PUBLIC HEALTH RELEVANCE: Nitric oxide (NO) signaling via soluble guanylate cyclase (sGC) mediates diverse physiological processes crucial to circulatory and neurological function. Disruptions in NO/sGC signaling have been linked to heart disease, stroke, erectile dysfunction, and neurodegeneration. The proposed research aims to illuminate the molecular details of NO-induced sGC activation, a prerequisite for developing treatments for diseases related to NO/sGC dysfunction.