Nitric Oxide Signaling with Heme Proteins

本提案是资助HL 62969的竞争性续期。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，使用各种蛋白质的例子和许多，主要是生物物理，方法。NO调节所有哺乳动物组织中的活动，并在不同的领域中发挥核心作用，包括血管调节、血液凝固、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NO合成酶产生，并由可溶性鸟苷酸环化酶（sGC）检测，两者都通过血红素与NO相互作用。可能受NO影响的其他分子包括细胞色素氧化酶、血红蛋白和具有游离巯基的基因调节蛋白。我们已经进入这一领域，通过一个家庭的昆虫赫恩蛋白，称为nitrophorins，运输NO从唾液的昆虫 这些昆虫的血液会进入潜在受害者的组织，导致血管扩张，血液凝固减少，并为昆虫提供令人满意的血液。我们已经表达并表征了五种硝化蛋白，四种来自Rhodnius prolixus，吻虫，其运输锥虫用于恰加斯病，一种来自臭虫Cimex lectularius，臭虫。我们已经确定了四个nitrophodns的晶体结构，高达0.85 A的分辨率，并有动力学，光谱学，热力学，诱变，理论和计算研究正在进行中的连接结构，功能在NO运输。因此，研究揭示了血红素分子， 扭曲的（皱褶的），这可能有助于稳定硝化蛋白功能所需的铁氧化态。 他们还揭示了大量的NO诱导的构象变化的nitroporins，显然是为了去溶剂化和保护NO与水和氧气反应。我们还将研究扩展到其他NO传感蛋白，包括sGC。在下一个资助期内，我们计划进行以下实验：（1）了解血红素畸变在亚硝酰复合物形成和信号传导中的作用;（2）了解蛋白质构象变化在Rhodnius nitrophorins结合和释放NO中的作用;（3）揭示Cimex nitrophorin转运NO的机制，这似乎涉及可逆的S-亚硝酰化;（4）研究Cimex nitrophorin的作用机制。和（4）产生和表征重组sGC，一种对NO信号传导至关重要的复杂蛋白。 这些研究将为NO信号的复杂化学提供前所未有的见解。

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.