Elucidating Protein Folding Mechanisms

描述（由申请人提供）：蛋白质折叠是细胞通路的最后一步，将DNA序列中存储的信息翻译成具有结构定义功能的异聚分子，对细胞存活至关重要，质量控制机制已经发展到确保天然结构形成，错误折叠的蛋白质被修复或靶向处理。正如人们所预料的那样，对于这样一个至关重要的过程，不正确和低效的蛋白质折叠与大量疾病有关。治疗蛋白质构象疾病的新药可用于修复或挽救错误折叠的蛋白质。这些化合物的合理设计取决于对折叠和错误折叠机制的基本理解。本研究计划的目的是阐明将无序多肽转化为正确折叠的功能蛋白质的构象变化。四个具体目标涉及实验研究，旨在首先确定，然后调节，一组螺旋束蛋白的折叠机制。在Aim 1中，将使用荧光能量转移方法来定义螺旋束血红素蛋白折叠时对残基之间的距离分布。这些分布将提供螺旋束细胞色素形成天然结构时的低分辨率结构图。目的2涉及对酰基辅酶A结合蛋白折叠机制的联合实验/计算研究。放置在整个蛋白质中的多个供体-受体对的距离分布将直接与计算结果进行比较。来自实验的数据将改进计算模型，模拟的输出将通过指导探针的放置来帮助实验设计。将氟化非极性氨基酸掺入蛋白质中有可能显著改变重折叠途径。Aim 3的工作涉及在四螺旋束蛋白的选定位置放置氟化亮氨酸、异亮氨酸和缬氨酸残基，目的是优化和重定向天然折叠途径。在Aim 4中，将使用能量和电子联合转移方法来评估非天然多肽结构中的扩散动力学。目的是确定拓扑相似蛋白质的折叠率差异是否源于扩散动力学的差异。为蛋白质构象疾病设计新的治疗剂需要了解蛋白质如何折叠和错误折叠。对多肽构象运动动力学的清晰理解是通往新分子途径的第一步，新分子可以帮助蛋白质避免或逃离导致疾病的不正确结构。

DESCRIPTION (provided by applicant): Protein folding, the final step in the cellular pathway that translates the information stored in a DNA sequence into a heteropolymeric molecule with a structure-defined function, is of such paramount importance for cell survival that quality-control mechanisms have evolved to ensure native structures form and that misfolded proteins are repaired or targeted for disposal. As might be expected for such a vital process, incorrect and inefficient protein folding are associated with a large number of diseases. Novel drugs for protein conformational diseases could be directed at repairing or rescuing misfolded proteins. Rational design of these compounds depends on fundamental understanding of folding and misfolding mechanisms. The objective of this research program is the elucidation of the conformational changes that transform disordered polypeptides into properly folded functional proteins. Four specific aims involve experimental studies intended first to identify, and then to modulate, the folding mechanisms in a set of helical-bundle proteins. Fluorescence energy transfer methods will be used in Aim 1 to define distributions of distances between pairs of residues in helical-bundle-heme proteins as they fold. These distributions will provide low-resolution structural maps of helical-bundle cytochromes as they form native structures. Aim 2 involves joint experimental/computational studies of the folding mechanism of acyl coenzyme A binding protein. Distance distributions for multiple donor-acceptor pairs placed throughout the protein will be compared directly to computational results. Data from experiments will refine computational models, and output from simulations will aid experimental designs by directing probe placement. The incorporation of fluorinated nonpolar amino acids into proteins has the potential to dramatically alter refolding pathways. Work on Aim 3 involves placement of fluorinated leucine, isoleucine, and valine residues at selected locations in four-helix-bundle proteins with the aim of optimizing and redirecting native folding pathways. Combined energy- and electron-transfer methods will be used in Aim 4 to evaluate the diffusive dynamics in nonnative polypeptide structures. The objective is to determine whether differences in folding rates of topologically similar proteins arise from differences in diffusion dynamics. Designing new therapeutic agents for protein conformational diseases requires knowledge of how proteins fold and misfold. A clear understanding of the dynamics of polypeptide conformational motions is the first step on the pathway to new molecules that can help proteins avoid or escape from the incorrect structures that lead to disease.