Peptide and Protein Conformations

描述（由申请人提供）：主要的努力正在进行中，以测序广泛的生物体的全基因组，并确定所有表达的蛋白质的身份，但有关蛋白质的一级序列与其三维结构的基本原理仍然没有完全理解。我们提出的实验，以阐明细胞内脂质结合蛋白（iLBP）家族的代表，细胞视黄酸结合蛋白I（CRABP I），采用其天然的倍，确定这些β桶蛋白如何成功地折叠，避免竞争聚集过程的目标的机制。我们还试图从这个重要家族的行为中提取关于β折叠和折叠β桶的一般原则。iLBP家族广泛存在于真核细胞中，并介导关键功能，如能量代谢、信号传导和分化的转录调节。除了在体外开发CRABP I折叠的能量景观的全貌，我们提出了新的实验来探索CRABP I在其生物合成过程中如何折叠。这一新的研究方向旨在填补目前对细胞折叠机制的理解中的空白。在下一个项目期间，我们将扩大对Hsp70分子伴侣作用机制的研究。这种普遍存在的伴侣蛋白家族在细胞中执行几种相关功能，所有这些功能都基于它们以核苷酸依赖性方式结合多肽链疏水区域的能力。它们的功能包括促进蛋白质折叠、分子复合物的分解、蛋白质跨膜移位、蛋白质降解和对诸如热休克的应激的响应。我们将测试我们新兴的假设，即配体诱导的域间变构热休克蛋白70蛋白依赖于在这两个域蛋白的动力学和域内稳定性的变化。我们将详细绘制E. coli Hsp70、DnaK.我们还将比较两种Hsp70的底物结合模式：来自E.大肠杆菌，和BiP从真核内质网。越来越多的疾病与蛋白质折叠错误或伴侣功能不足有关（例如，基于p53的癌症，阿尔茨海默氏症，亨廷顿氏症，帕金森氏症，囊性纤维化，BSE），以及对两者的增强理解将有助于治疗策略的设计。

DESCRIPTION (provided by applicant): Major efforts are underway to sequence whole genomes of a wide array of organisms and to determine the identities of all expressed proteins, yet the fundamental principles relating primary sequences of proteins to their three-dimensional structures remain incompletely understood. We propose experiments to elucidate the mechanism by which a representative of the intracellular lipid-binding protein (iLBP) family, cellular retinoic acid-binding protein I (CRABP I), adopts its native fold, with a goal of determining how these beta-barrel proteins successfully fold and avoid competing aggregation processes. We seek, as well, to extract general principles about beta-sheet folding and folding beta-barrels from the behavior of this important family. The iLBP family is widespread in eukaryotic cells and mediates critical functions, such as energy metabolism, signaling, and transcriptional regulation of differentiation. In addition to developing a full picture of the energy landscape for the folding of CRABP I in vitro, we propose new experiments to explore how CRABP I folds during its biosynthesis. This new direction of research seeks to fill the remarkable void in current understanding of the mechanism of folding in the cell. In the second major focus of this grant for the next project period, we will extend our studies on the mechanism of action of Hsp70 molecular chaperones. This ubiquitous family of chaperones carries out several related functions in the cell, all based on their ability to bind hydrophobic regions of polypeptide chains in a nucleotide-dependent manner. Their functions include facilitation of protein folding, disassembly of molecular complexes, protein translocation across membranes, protein degradation, and responses to stresses such as heat shock. We will test our emerging hypothesis that ligand-induced interdomain allostery in Hsp70 proteins relies on changes in the dynamics and intradomain stability in these two domain proteins. We will map in detail the conformational signal transduction pathway of the E. coli Hsp70, DnaK. We will also compare the modes of substrate binding by the two Hsp70s: DnaK from E. coli, and BiP from the eukaryotic endoplasmic reticulum. An increasing number of diseases has been associated with mistakes in protein folding or inadequacies of chaperone function (e.g., p53-based cancers, Alzheimer's, Huntington's, Parkinson's, cystic fibrosis, BSE), and enhanced understanding of both will aid design of therapeutic strategies.