STRUCTURAL/FUNCTIONAL MODULARITY IN NITRIC OXIDE SYNTHAS

揭开一氧化氮合酶（NOS）的结构秘密， 成为一个重要的目标，以了解他们如何能够 受特定底物的差异调节和/或抑制， 抑制剂的大鼠小脑NOS，在其克隆，表达形式，是 这个提议的主题。瓜氨酸和NO.的形成 氨基酸，L-精氨酸，需要还原当量从NADPH中， 整个反应需要两个单加氧步骤， 酵素神经元NOS具有约160 kDa的分子量， 在脑组织中的多种细胞类型中表达，以及在其他组织中表达。 组织，如胃肠道系统。实验假设 这种高度复杂的蛋白质，含有FAD，FMN和Fe- 原卟啉IX，作为辅基，和四氢生物蝶呤， 在单个多肽链中的钙调蛋白结合序列，包含 在基因融合过程中结合并表现出 独立的折叠性能。 为了检验这一假设，计划了以下实验目标： 1.为了证明存在独立折叠的结构域， 小脑一氧化氮合酶，几种类型的实验将 执行：a.显微切割分子克隆方法，其中， 在大肠杆菌中表达了一氧化氮合酶（nNOS）非依赖性结构域。 大肠杆菌，将继续进行，以获得足够的数量， 通过生物物理技术来表征它们中的每一个。 初步和公布的数据显示， 这种做法的成功。B.在成功表达了N-和C- 末端，其代表nNOS的血红素和黄素结合结构域， 和E.杆菌 正在进行的实验，PI将尝试表达其他 子域。 例如，尝试表达nNOS的N末端， 它不存在于诱导型巨噬细胞型同种型中， 将制备组成型内皮同种型。此外，A的作用 推定的“抑制性多肽”，通过建模技术鉴定（如 是DHFR基序）作为Ca+2/钙调素诱导的 将通过分子克隆的显微切割检查亚型 和表达。这些结构域的表征将利用各种 光谱技术，包括荧光和光学 吸收、圆二色性（CD）和电子顺磁（EPR）， 核磁共振（NMR）光谱。 2.为了确定各种结构域如何在三维空间中折叠， 通过对那些具有重要意义的域的结构建模， 与存在结构信息的酶序列同源 X射线晶体学或二维核磁共振。初步数据已提交给 显示了各种二氢叶酸还原酶和几种 芳香族氨基酸羟化酶与神经元NOS中的序列。 对建模结构的这些比较将用于确定目标 用于定点诱变和缺失突变。野生型和 突变的结构域也将通过荧光，吸光度，EPR， 和NMR光谱学（如适用）。NMR技术可以产生3D 更小域的结构信息。 3. 尝试在各种条件下， 从分离纯化的结构域中形成NO.和L-瓜氨酸 片段本文还讨论了活性与结构的关系， 独立表达的模块的活动和结构， 将测定完整的全酶。

Unraveling the structural secrets of nitric oxide synthases (NOSs) has become an important goal for the purpose of understanding how they can be differentially regulated and/or inhibited by specific substrates or inhibitors. Rat cerebellar NOS, in its cloned, expressed forms, is the subject of this proposal. The formation of L-citrulline and NO. from the amino acid, L-arginine, requires reducing equivalents from NADPH in an overall reaction requiring two monooxygenation steps catalyzed by a single enzyme. Neuronal NOS has a molecular mass of approximately 160 kDa and is expressed in a variety of cell types in brain tissue, as well as in other tissues, such as the gastrointestinal system. The experimental hypothesis is that this highly complex protein, which contains FAD, FMN, and Fe- protoporphyrin IX, as prosthetic groups, and tetrahydrobiopterin and calmodulin binding sequences in a single polypeptide chain, is comprised of domains which have combined in a gene fusion process and exhibit independent folding properties. To examine this hypothesis, the following experimental aims are planned: 1. To demonstrate the existence of independently folding domains of cerebellar nitric oxide synthase, several types of experiments will be performed: a. Microdissection molecular cloning methods, in which the putatively independent domains of neuronal NOS (nNOS) are expressed in E. coli, will continue to be pursued in order to obtain sufficient quantities of material to characterize each of them by biophysical techniques. Preliminary and published data are presented to show the potential for success of this approach. b. Having successfully expressed the N- and C- termini, which represent the heme- and flavin-binding domains of nNOS, respectively, and the dihydrofolate reductase (DHFR) motif in E. coli in ongoing experiments, the PI will attempt the expression of other subdomains. For example, attempts to express the N-terminus of nNOS, which is not present in either the inducible macrophage-type isoform of the constitutive endothelial isoform, will be made. Also, the role of a putative "inhibitory polypeptide", identified by modeling techniques (as was the DHFR motif) as a unique feature of the Ca+2/calmodulin-inducible isoforms, will be examined through microdissection by molecular cloning and expression. Characterization of these domains will utilize a variety of spectroscopic techniques, including fluorescence and optical absorption, circular dichroism (CD), and electron paramagnetic (EPR) and nuclear magnetic resonance (NMR) spectroscopy. 2. To determine how the various domains are folded in three-dimensional space by modeling the structures of those domains which bear significant homology to sequences of enzymes for which there is structural information from X-ray crystallography or 2D NMR. Preliminary data are presented to show structural homologies of various dihydrofolate reductases and several aromatic amino acid hydroxylases with a sequence in the neuronal NOS. These comparisons of modeled structures will be used to determine targets for site-directed mutagenesis and deletion mutations. The wild type and mutated domains will also be compared by fluorescence, absorbance, EPR, and NMR spectroscopy when applicable. NMR techniques can yield 3D structural information on the smaller domains. 3. To attempt, under a variety of conditions, the reconstitution of the form ad on of NO. and L-citrulline from the isolated purified domain fragments. The relationship of the activity and structure of the independently expressed modules to the activity and structure of the intact holoenzyme will be determined.