Fast Kinetic Studies of Protein Folding and Function

描述(申请人提供)：该项目应用快速时间分辨磁性圆二色谱(TRMCD)、天然圆二色谱(TRCD)和普通吸收光谱技术来研究血红素蛋白的功能和血红素蛋白和小肽的折叠。新的光学方法使用近零椭圆偏振法和旋光法来研究生物分子中的快速动力学过程(纳秒到秒)，这些生物分子包含磁光活性的生色团，如血红素和芳香族氨基酸，以及天然手性生色团，如蛋白质和多肽的酰胺基。这些技术将被用来识别和研究细胞色素c等血红素蛋白质折叠反应中最早的(亚毫秒)事件。一个主要目标是确定一个参数，该参数对于理解蛋白质折叠的性质是基本的：不同展开的构象相互转换的速度。如果这比折叠本身慢，那么理解蛋白质折叠将需要比用于典型化学反应的过渡态理论更复杂的理论(例如，能量图)。这种理解最终可能被证明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、帕金森氏症和克雅氏病。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协作，以更有效地运输氧气。最近关于这种协作性的假设(Ackers对称性规则)，最初基于热力学测量，在这个项目中通过动力学测量进行了验证。从热力学和动力学的这种联系中产生的一种新的血红蛋白变构模型，有望简化和系统化我们对血红蛋白的动力学及其在体内通过变构效应(如有机磷酸盐)控制的理解。此外，对芳香族氨基酸残基色氨酸[337]进行的TRMCD研究旨在进一步阐明血红蛋白亚基如何作为一台高效的“分子机器”一起工作，将氧气从肺部输送到组织。

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.