Measurement of Changes in Denatured State Energetics

描述(申请人提供)：蛋白质变性状态仍然知之甚少，然而，作为蛋白质折叠的起点，了解作用于变性状态的构象限制对于解决蛋白质如何有效折叠的问题至关重要。错误折叠的疾病，如阿尔茨海默氏症和帕金森氏症，是主要的健康问题，其病因被认为是蛋白质的非天然和变性状态。因此，对变性蛋白质的基础研究对于新的认识这些疾病状态的发生是至关重要的。该实验室开发了一种新的策略来探索未折叠蛋白质的构象和热力学性质。通过组氨酸-血红素环平衡来评估形成不同大小环的倾向。在细胞色素c中已经产生了单表面组氨酸变体，允许在变性条件下测量9到83个氨基酸的环的环平衡。当蛋白质折叠时，在结构增长的最早阶段需要形成闭合环。这个系统已经对蛋白质变性状态与随机卷曲行为的偏差产生了重要的见解。该系统将用于研究未折叠蛋白质的几个关键性质。为了了解链硬度和残基结构如何影响变性蛋白质中的接触概率，将用平衡和动力学方法研究变性剂浓度对环形成的依赖关系(特定目标1)。这些研究将与有关选定变体变性状态的核磁共振结构数据相辅相成。拓扑和排除体积在限制变性蛋白质的构象空间中的作用将通过理论晶格模型和细胞色素c变体来探索(特定目标2)，细胞色素c变体在变性状态的组氨酸-血红素环中包含短的二硫键。为了阐明氨基酸序列和折叠早期结构偏好之间的关系，将开展序列组成对环平衡的影响的基础研究(具体目标3)，重点是甘氨酸和脯氨酸如何调节变性状态的构象性质。总之，提出的实验使用了一种新的热力学方法来探索蛋白质变性状态的关键参数，这些参数有望成为蛋白质折叠早期事件的主要调节因素。

DESCRIPTION (provided by applicant): Protein denatured states remain poorly understood and yet, as the starting point for protein folding, an understanding of the conformational constraints acting upon the denatured state is critical to solving the problem of how a protein folds efficiently. Misfolding diseases, such as Alzheimer's and Parkinson's diseases, are major health problems, where the causative agents are believed to be non-native and denatured states of proteins. Thus, fundamental research on denatured proteins is essential to new insight into the genesis of these disease states. This laboratory has developed a novel strategy to probe the conformational and thermodynamic properties of unfolded proteins. The propensity for forming loops of different sizes is assessed through histidine-heme loop equilibria. Single surface histidine variants have been produced in cytochrome c, allowing loop equilibria for loops of 9 to 83 amino acids to be measured under denaturing conditions. Formation of closed loops are required in the earliest stages of structure accretion when a protein folds. This system has already yielded important insights into the deviation of protein denatured states from random coil behavior. Several key properties of unfolded proteins will be probed with this system. To understand how chain stiffness and residual structure affect contact probability in a denatured protein, the dependence of loop formation on denaturant concentration will be studied by both equilibrium and kinetic methods (specific aim 1). These studies will be complemented with NMR structural data on the denatured states of selected variants. The role of topology and excluded volume in limiting the conformational space of a denatured protein will be explored (specific aim 2) with both theoretical lattice models and cytochrome c variants which contain short disulfide bridges within the denatured state histidine-heme loop. To clarify the relationship between amino acid sequence and structural preferences early in folding, fundamental studies on the effects of sequence composition on loop equilibria (specific aim 3) will be carried out, with an emphasis on how glycine and proline modulate denatured state conformational properties. In sum, the proposed experiments use a novel thermodynamic approach to probe key parameters of protein denatured states expected to be principal modulators of early events in protein folding.