Nitric Oxide Signaling with Heme Proteins

这项建议是对HL62969授权的竞争性续展。我们的研究集中在一氧化氮(NO)及其与血红素蛋白的相互作用，使用了各种蛋白质例子和许多主要是生物物理的方法。NO调节所有哺乳动物组织的活动，并在多种领域发挥核心作用，包括血管调节、凝血、记忆形成、氧应激、细胞凋亡和入侵物种的巨噬细胞杀伤。NO由一氧化氮合酶产生，并由可溶性鸟苷环化酶(SGC)检测，两者都通过血红素与NO相互作用。其他可能受一氧化氮影响的分子包括细胞色素氧化酶、血红蛋白和含有游离硫醇的基因调节蛋白。我们进入这一领域是通过一类昆虫的Herne蛋白，称为Nitphorins，它从昆虫的唾液中运输NO 对潜在受害者的组织，导致血管扩张，减少血液凝固，并为昆虫提供令人满意的血液食物。我们已经表达并鉴定了五种亚硝酚，其中四种来自Rhodnius prolixus，它是一种接吻的臭虫，它运输恰加斯病的锥虫，一种来自Cimex lectularius，一种是臭虫。我们已经测定了四个硝基化合物的晶体结构，分辨率高达0.85A，并正在进行动力学、光谱、热力学、诱变、理论和计算研究，以将结构与NO运输中的功能联系起来。因此，研究揭示了血红素分子是高度 扭曲(褶皱)，这可能有助于稳定硝基酚功能所需的铁氧化状态。 他们还揭示了NO诱导的亚硝酚类分子的大量构象变化，这显然是为了使NO脱溶并保护NO免受水和氧气的反应。我们还将我们的研究扩展到其他NO感觉蛋白，包括sGC。在下一个资金阶段，我们建议进行以下实验：(1)了解血红素扭曲在亚硝基复合体形成和信号传递中的作用；(2)了解蛋白质构象变化在亚硝基结合和释放NO中的作用；(3)揭示Cimex nitphorin转运NO的机制，这似乎涉及可逆的S-亚硝化反应；以及(4)生产和鉴定重组sGC，这是一种对NO信号转导至关重要的复杂蛋白质。 这些研究将为NO信号的复杂化学提供前所未有的洞察力。

This proposal is the competitive renewal of grant HL62969. Our studies center on nitric oxide (NO) and its interactions with heme proteins, using a variety of protein examples and numerous, largely biophysical, approaches. NO regulates activities in all mammalian tissues and plays a central role in diverse areas, including vasodUation, blood coagulation, memory formation, oxygen stress, apoptosis, and macrophage killing of invading species. NO is produced by NO synthase and detected by soluble guanylate cyclase (sGC), both of which interact with NO through heme. Other molecules possibly influenced by NO include cytochrome oxidase, hemoglobin, and gene regulatory proteins with free thiols. We have entered this field through a family of insect herne proteins, called nitrophorins, that transport NO from the saliva of the insect to the tissues of potential victims, leading to vasodilation, reduced blood coagulation and a satisfying blood meal for the insect. We have expressed and characterized five nitrophorins, four from Rhodnius prolixus, the kissing bug, which transports the trypanosome for Chagas' disease, and one from Cimex lectularius, the bed bug. We have determined crystal structures of four nitrophodns, to as high as 0.85 A resolution, and have kinetic, spectroscopic, thermodynamic, mutagenic, theoretical and computational studies underway to link structure to function in NO transport. Thus studies have revealed heme molecules that are highly distorted (ruffled), which may serve to stabilize the ferric oxidation state required for nitrophorin function. They have also revealed a substantial NO-induced conformational change in the nitrophorins that apparently serves to desolvate and protect NO from reaction with water and oxygen. We are also extending our studies to other NO sensing proteins, including sGC. In the next funding period, we propose experiments to (1) understand the role of heme distortion in nitrosyl complex formation and signaling; (2) understand the role of protein conformational change in NO binding and release by the Rhodnius nitrophorins; (3) uncover the mechanism for NO transport by Cimex nitrophorin, which appears to involve reversible S-nitrosylation; and (4) produce and characterize recombinant sGC, a complicated protein of central importance to NO signaling. These studies will provide unprecedented insight into the complicated chemistry of NO signaling.