RESIDUE & DOMAIN SPECIFIC IR PROBES OF PROTEIN STRUCTURE

描述：本建议使用红外光谱分析中的特定探针 多肽模型和一氧化氮合酶中局部结构的报道 特性，目标是扩展或开发新的方法 从蛋白质的红外光谱中提取蛋白质的结构和动力学信息 光谱。因为振动频率对大范围的 化学和静电相互作用，红外光谱是一种 关于蛋白质结构的详细信息的强大工具。 这一建议的第一个组成部分寻求使用酰胺I模式来获得 关于氨基酸残基和氨基酸之间相互作用的具体信息 他们的环境。因为跃迁偶极耦合导致了 所有氨基酸的酰胺I振动波函数的离域化 连接在二级结构元素中的残基，其频率 酰胺I模式对多肽骨架的构象敏感。 因此，使用红外光谱测量了酰胺I的频率 用于测定蛋白质的二级结构含量，也可用于 多肽链的全球动态运动。这种敏感度 整体的多肽构象是以失去特定的用途为代价的 作为局部探针的螺旋内特定残基的酰胺I带 环境相互作用。在这个项目中，纳入了特定的 同位素标记将被引入到模型螺旋肽中；酰胺I 来自C13标记的羰基的带将明显地与 由另一个(碳-12)残基产生的酰胺I带及其确切的 频率将同时反映它在螺旋中的位置和它的 与周围残留物的相互作用。这项技术将被用于 定量研究螺旋环境对酰胺I频率的影响及探讨 螺旋形成的热力学和动力学。 该项目的第二个组成部分涉及使用红外光谱来 获取关于一氧化氮的血红素活性部位的结构信息 合酶(NOS)，它催化一氧化氮的产生 L-精氨酸和双原子氧。这种酶是一种必不可少的酶 脑、血管内皮细胞和血管内皮细胞中一氧化氮的生物活性 免疫系统。然而，机制的细节或结构 一氧化氮合酶的血红素活性部位在很大程度上仍不清楚。现建议： 通过测量CO结合的一氧化氮合酶在存在和不存在情况下的红外光谱 底物和缓蚀剂的化学和静电性质 血红素口袋将被阐明。

DESCRIPTION: This proposal uses IR spectral analysis specific probes in peptide models and in nitric oxide synthase to report on local structure features, with the goal of expanding or developing new methodology for extracting structural and dynamic information on proteins from their IR spectra. Because vibrational frequencies are sensitive to a wide range of chemical and electrostatic interactions, infrared (IR) spectroscopy is a powerful tool for detailed information about protein structure. The first component of this proposal seeks to use the amide I modes to get specific information about interactions between amino acid residues and their environments. Because transition dipole coupling results in the delocalization of the amide I vibrational wavefunctions of all amino acid residues coupled in a secondary structural element, the frequency of the amide I mode is sensitive to the conformation of the peptide backbone. Thus, measurements of amide I frequencies using IR spectroscopy have been used to determine protein secondary structure content, as well as to follow global dynamic motions of the polypeptide chain. This sensitivity to overall peptide conformation comes at a loss of the use of a particular amide I band of a specific residue within a helix as a probe of local environmental interactions. In this project, incorporation of specific isotopic labels will be introduced into model helical peptides; the amide I band from C13 labeled carbonyls will be clearly distinguishable from the amide I band which arises from the other (carbon-12) residues, and its exact frequency will reflect both its position within the helix and its interactions with surrounding residues. This technique will be used to quantitate the effect of helix environment on amide I frequency and to probe thermodynamics and kinetics of helix formation. The second component of this project involves using the IR spectroscopy to elicit structural information about the heme active site of nitric oxide synthase (NOS), which catalyzes the production of nitric oxide from L-arginine and diatomic oxygen. This enzyme is an essential player in the biological activity of nitric oxide in the brain, the endothelium and the immune system. However, the details of the mechanism or the structure of the heme active site of NOS are still largely unknown. It is proposed that by measuring the IR spectra of CO-bound NOS, in the presence and the absence of substrate and inhibitors, the chemical and electrostatic nature of the heme pocket will be elucidated.