Peptide and Protein Conformations

描述(申请人提供)：大量生物的全基因组测序和确定所有表达的蛋白质的身份的主要工作正在进行中，然而将蛋白质的初级序列与其三维结构联系起来的基本原理仍然不完全清楚。我们建议通过实验来阐明细胞内脂质结合蛋白(ILBP)家族的代表-细胞维甲酸结合蛋白I(CRABP I)采用其天然折叠的机制，目的是确定这些β-桶蛋白如何成功折叠并避免竞争聚集过程。我们还试图从这一重要家族的行为中提取关于贝塔折叠和贝塔桶折叠的一般原则。ILBP家族广泛存在于真核细胞中，参与调节能量代谢、信号转导和转录调控等重要功能。除了为CRABP I的体外折叠提供一个完整的能量图景之外，我们还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。这一新的研究方向试图填补目前对细胞内折叠机制的理解中的显著空白。在下一个项目期间，我们将扩展我们对Hsp70分子伴侣作用机制的研究。这个无处不在的伴侣家族在细胞中执行几个相关的功能，所有这些都基于它们以核苷酸依赖的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，拆解分子复合体，蛋白质跨膜转移，蛋白质降解，以及对热休克等应激反应。我们将检验我们的新假说，即配体诱导的Hsp70蛋白的域间变构依赖于这两个结构域蛋白的动力学和域内稳定性的变化。我们将详细绘制E.coliHsp70，DNAK的构象信号转导途径。我们还将比较两种Hsp70与底物的结合方式：来自大肠杆菌的DNAK和来自真核内质网的Bip。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)，对两者的深入了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.