STRUCTURAL/FUNCTIONAL MODULARITY IN NITRIC OXIDE SYNTHAS

揭开一氧化氮合酶(NOSS)的结构秘密 成为一个重要的目标，目的是了解它们如何 由特定底物或 抑制剂。大鼠小脑一氧化氮合酶的克隆、表达形式是 这项提案的主题。L-瓜氨酸的形成与NO.从 氨基酸L-精氨酸需要在体内从NADPH中减少当量 需要两个单氧化步骤的总反应，由一个 酵素。神经元一氧化氮合酶的分子质量约为160 kDa，是 在脑组织中的各种细胞类型中表达，以及在其他 组织，如胃肠道系统。实验假说 这种高度复杂的蛋白质含有FAD、FMN和Fe- 原卟啉IX作为修复基，四氢生物蝶呤和 单个多肽链中的钙调蛋白结合序列包括 在基因融合过程中结合在一起的结构域 独立的折叠属性。 为了检验这一假设，计划了以下实验目标： 1.证明了独立折叠整环的存在性 小脑一氧化氮合酶的几种类型的实验 实施：a.显微切割分子克隆方法，其中 神经元型一氧化氮合酶(NNOS)的推测独立结构域在E. 将继续追求，以获得足够的量 通过生物物理技术对每一种材料进行表征。 提供了初步和已公布的数据，以显示 这一方法的成功。B.成功表达了N-和C- Termini，代表nNOS的血红素和黄素结合域， 和二氢叶酸还原酶(DHFR)基序。 正在进行的实验中，PI将尝试表达其他 子域。例如，试图表达nNOS的N末端， 它不存在于可诱导的巨噬细胞类型的亚型 构成内皮细胞的亚型，将被制造出来。此外，一名 由建模技术(AS)确定的推定的“抑制性多肽” 是DHFR基序)作为Ca+2/钙调蛋白诱导的一个独特特征 异构体，将通过分子克隆的显微解剖进行检测 和表情。这些领域的特征将利用各种 光谱技术，包括荧光和光学 吸收、圆二色(CD)和电子顺磁(EPR)和 核磁共振(核磁共振)光谱。 2.确定各个域如何在三维中折叠 通过对那些承载着重要意义的领域的结构进行建模来实现空间 与有结构信息的酶序列同源 来自X射线结晶学或2D核磁共振。初步数据提交给 显示各种二氢叶酸还原酶和几个 芳香氨基酸羟基酶与神经元型一氧化氮合酶的序列。 这些模型化结构的比较将用于确定目标 用于定点突变和缺失突变。野生型和 突变结构域还将通过荧光、吸光度、EPR、 以及核磁共振光谱学(如果适用)。核磁共振技术可以产生3D 较小域名的结构信息。 3.在各种情况下，尝试重建 表格上的编号。和L-从分离纯化的结构域中提取的瓜氨酸 碎片。活性与结构的关系 独立表达的模块的活动和结构 完整的全酶将被测定。

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.