Polypeptide Conformation and Interaction with Hsp70

多肽构象及其与 Hsp70 的相互作用

• 批准号: 7047781
• 负责人: Silvia Cavagnero
• 金额: $ 22.4万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7047781
• 关键词: bacterial proteins; biophysics; calorimetry; chemical kinetics; chromatography; conformation; deuterium; fluorescence spectrometry; heat shock proteins; molecular chaperones; nuclear magnetic resonance spectroscopy; peptide structure; protein binding; protein folding; protein protein interaction; protein structure function; stoichiometry

DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the conformation of N-terminal polypeptides of increasing length (belonging to the sequence of an all-alpha-helical single domain model protein) in the presence of the cotranslationally active chaperone Hsp70. In order to obtain information at amino-acid specific resolution, multidimensional NMR in the presence of isotopically labeled peptide and unlabeled chaperone will be employed. Other biophysical methods such as isothermal titration calorimetry, size exclusion chromatography and fluorescence spectroscopy will also be used. The work focuses on elongating N-terminal polypeptides derived from apomyoglobin, a relatively small and extremely well characterized system which serves as an excellent model for all-alpha-helical proteins. The proposed investigations will explore whether Hsp70 merely prevents interchain aggregation by holding a statistical coil status, or it also acts by inducing specific polypeptide conformations. This study is not intended to directly mimic intracellular cotranslational and immediately posttranslational folding events. On the other hand, it aims at providing a first order in vitro approximation to how Hsp70 is able to affect the conformational space of elongating polypeptides. The expected influence of specific cell-related effects such as polypeptide tethering (to the ribosome exit channel) and molecular crowding are discussed in the proposal and will be addressed by separate additional experiments. Very little is known about the mechanisms by which Hsp70, the main cotranslationally active chaperone, influences the course of protein folding. Yet, progress is urgently needed in this area since defective action (or insufficient bioavailable amount) of cotranslational chaperones has been linked to the formation of incorrectly folded self-associated species such as those involved in a number of deadly diseases. These include cystic fibrosis, inflammatory heart disease, Crohn disease, P53-related cancers, and several neurodegenerative disorders such as Huntington's and Alzheimer's disease. Experiments to be carried out include (a) high resolution secondary structure mapping of isotopically labeled polypeptides by NMR in the presence of unlabeled Hsp70 chaperone; (b) hydrogen/deuterium exchange pulse labeling kinetic experiments to detect the mechanisms of structure formation in the presence of Hsp70; (c) additional studies in the presence of the Hsp40 and Hsp70-nucleotide exchange factor cochaperones.

描述（由申请人提供）：该提案的目的是阐明在共翻译活性伴侣 Hsp70 存在的情况下长度增加的 N 端多肽（属于全α螺旋单域模型蛋白的序列）的构象。为了获得氨基酸特异性分辨率的信息，将在同位素标记的肽和未标记的分子伴侣存在下采用多维核磁共振。还将使用其他生物物理方法，例如等温滴定量热法、尺寸排阻色谱法和荧光光谱法。这项工作的重点是延长源自脱辅基肌红蛋白的 N 端多肽，脱辅基肌红蛋白是一个相对较小且特征极其明确的系统，可作为全 α 螺旋蛋白的出色模型。 拟议的研究将探讨 Hsp70 是否仅仅通过保持统计线圈状态来防止链间聚集，或者它还通过诱导特定的多肽构象来发挥作用。 这项研究并非旨在直接模拟细胞内共翻译和翻译后立即折叠事件。另一方面，它的目的是提供 Hsp70 如何影响延伸多肽的构象空间的一级体外近似。该提案讨论了特定细胞相关效应的预期影响，例如多肽束缚（到核糖体出口通道）和分子拥挤，并将通过单独的附加实验来解决。 关于 Hsp70（主要的共翻译活性伴侣）影响蛋白质折叠过程的机制知之甚少。然而，这一领域迫切需要取得进展，因为共翻译伴侣的作用缺陷（或生物可利用量不足）与错误折叠的自相关物种的形成有关，例如与许多致命疾病有关的物种。这些疾病包括囊性纤维化、炎症性心脏病、克罗恩病、P53 相关癌症以及几种神经退行性疾病，例如亨廷顿病和阿尔茨海默病。待进行的实验包括 (a) 在未标记的 Hsp70 分子伴侣存在下，通过 NMR 对同位素标记的多肽进行高分辨率二级结构图谱； (b) 氢/氘交换脉冲标记动力学实验，以检测 Hsp70 存在下结构形成的机制； (c) 在 Hsp40 和 Hsp70 核苷酸交换因子共伴侣存在下的其他研究。