RESIDUE & DOMAIN SPECIFIC IR PROBES OF PROTEIN STRUCTURE

残留物

• 批准号: 2806811
• 负责人: Sean M. Decatur
• 金额: $ 1.08万
• 依托单位: MOUNT HOLYOKE COLLEGE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2806811
• 关键词: active sites; amides; chemical kinetics; conformation; enzyme structure; heme; infrared spectrometry; interferometry; ionic bond; method development; molecular site; nitric oxide synthase; protein structure; structural biology; thermodynamics

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.

描述：本提案使用IR光谱分析专用探针， 肽模型和一氧化氮合酶，以报告局部结构 功能，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 谱 因为振动频率对大范围的 化学和静电相互作用，红外（IR）光谱是一种 这是一个强大的工具，可以提供有关蛋白质结构的详细信息。 该提案的第一部分寻求使用酰胺I模式来获得 关于氨基酸残基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 所有氨基酸的酰胺I振动波函数的离域 在二级结构元件中偶联的残基， 酰胺I模式对肽骨架的构象敏感。 因此，使用IR光谱法测量酰胺I频率已被证明是可行的。 用于测定蛋白质二级结构含量，以及 多肽链的全局动态运动。 这种敏感性 整体肽构象的损失是由于使用了特定的 螺旋内特定残基的酰胺I带，作为局部 环境相互作用。 在这个项目中，具体的 同位素标记将被引入到模型螺旋肽中;酰胺I 来自C13标记的羰基的条带将与来自C13标记的羰基的条带清楚地区分开。 酰胺I带，其产生于其他（碳-12）残基，以及其确切的 频率将反映其在螺旋内的位置以及其 与周围残基的相互作用。 这项技术将用于 定量螺旋环境对酰胺I频率的影响， 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， 引出一氧化氮血红素活性部位的结构信息 一氧化氮合酶（NOS），它催化一氧化氮的产生， 精氨酸和双原子氧。 这种酶是一个重要的球员， 一氧化氮在脑、内皮和脑内的生物活性 免疫系统 然而，该机制或结构的细节 NOS的血红素活性部位仍是未知的。 拟将 通过测量CO结合NOS的IR光谱，在存在和不存在 底物和抑制剂的化学和静电性质， 血红素口袋将被阐明。

项目成果

Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution.

• DOI: 10.1073/pnas.140161997
• 发表时间: 2000-07
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: R. A. Silva;J. Kubelka;P. Bouř;S. Decatur;T. Keiderling