Fast Kinetic Studies of Protein Folding and Function

蛋白质折叠和功能的快速动力学研究

• 批准号: 6874802
• 负责人: DAVID S. KLIGER
• 金额: $ 32.59万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6874802
• 关键词: allosteric site; chemical kinetics; circular magnetic dichroism; conformation; cytochrome c; cytochrome oxidase; electron transport; hemoglobin; hemoprotein structure; myoglobin; optical rotation; photolysis; protein folding; protein structure function; structural biology; time resolved data; ultraviolet radiation

DESCRIPTION (provided by applicant): This project applies techniques for fast time-resolved magnetic circular dichroism (TRMCD), natural circular dichroism (TRCD, and ordinary absorption spectroscopies to the study of function in heme proteins and folding in heme proteins and small peptides. The novel optical methods employed use near-null ellipsometry and polarimetry to study rapid kinetic processes (nanosecond to seconds) in biomolecules that contain magneto-optically active chromophores, such as heme and the aromatic amino acids, and naturally chiral chromophores, such as the amide groups of proteins and peptides. These techniques will be used to identify and study the earliest (submillisecond) events in the folding reactions of heme proteins such as cytochrome c. A major goal is the determination of a parameter that is fundamental to understanding the nature of protein folding: the speed with which the different unfolded conformations interconvert with one another. If this is slow compared to folding itself, then understanding protein folding will require more complicated theories (e.g., energy landscape) than the transition state theory used for typical chemical reactions. Such understanding may ultimately prove helpful in developing therapies for the many diseases associated with protein misfolding, such as cystic fibrosis, type 2 diabetes, and Alzheimer's, Parkinson's, and Creutzfeldt- Jakob disease. A major goal of the functional studies is to understand how the four subunits that make up the hemoglobin molecule cooperate with each other to transport oxygen more efficiently. A recent hypothesis about this cooperativity (Ackers symmetry rule), based originally on thermodynamic measurements, is tested by kinetic measurements in this project. A novel model for hemoglobin allostery, emerging from this linkage of thermodynamics and kinetics, holds promise for simplifying and systematizing our understanding of hemoglobin's dynamics and its control in the body by allosteric effectors such as organic phosphates. In addition, TRMCD studies of the aromatic amino acid residue tryptophan [337, positioned at a site critical for cooperativity, are intended to further clarify how hemoglobin's subunits work together as an efficient "molecular machine" for transporting oxygen from the lungs to the tissues.

描述（由申请人提供）：本项目应用快速时间分辨磁性圆二色性（TRMCD）、天然圆二色性（TRCD）和普通吸收光谱技术研究血红素蛋白的功能以及血红素蛋白和小肽的折叠。采用的新的光学方法使用近零椭圆偏振法和旋光法研究快速的动力学过程（纳秒至秒），在生物分子中含有磁光活性发色团，如血红素和芳香族氨基酸，和天然手性发色团，如蛋白质和肽的酰胺基团。这些技术将用于识别和研究血红素蛋白质（如细胞色素c）折叠反应中的最早（亚毫秒）事件。一个主要的目标是确定一个参数，这是理解蛋白质折叠的性质的基础：不同的未折叠构象相互转化的速度。如果这比折叠本身慢，那么理解蛋白质折叠将需要更复杂的理论（例如，能量景观）比用于典型化学反应的过渡态理论。这种理解最终可能有助于开发与蛋白质错误折叠相关的许多疾病的治疗方法，例如囊性纤维化，2型糖尿病，阿尔茨海默氏症，帕金森氏症和Creutzfeldt- Jakob病。功能研究的一个主要目标是了解组成血红蛋白分子的四个亚基如何相互合作以更有效地运输氧气。最近的一个假设，这种协同性（阿克斯对称规则），最初基于热力学测量，在这个项目中的动力学测量进行测试。从热力学和动力学的这种联系中出现的血红蛋白变构的一种新模型，有望简化和系统化我们对血红蛋白的动力学及其在体内由变构效应物如有机磷酸盐控制的理解。此外，对位于协同性关键位点的芳香族氨基酸残基色氨酸[337]的TRMCD研究旨在进一步阐明血红蛋白的亚基如何作为有效的“分子机器”一起工作，将氧气从肺输送到组织。