POLYPEPTIDE CONFORMATION AND INTERACTION WITH HSP70

多肽构象以及与 HSP70 的相互作用

• 批准号: 7598687
• 负责人: Silvia Cavagnero
• 金额: $ 0.09万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598687
• 关键词: Address; Affect; Alzheimer' s Disease; Amino Acids; Area; Bioavailable; Cells; Computer Retrieval of Information on Scientific Projects Database; Crohn' s disease; Crowding; Cystic Fibrosis; Deuterium; Disease; Funding; Goals; Grant; Guanine Nucleotide Exchange Factors; Heart Diseases; Huntington Disease; Hydrogen; In Vitro; Inflammatory; Institution; Investigation; Kinetics; Label; Length; Link; Malignant Neoplasms; Maps; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; N-terminal; Neurodegenerative Disorders; Nucleotides; Numbers; Peptides; Physiologic pulse; Proteins; Pulse taking; Research; Research Personnel; Resolution; Resources; Ribosomes; Source; Structure; System; United States National Institutes of Health; Work; apomyoglobin; polypeptide; prevent; protein folding; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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. 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. 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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该建议的目标是阐明在存在协同活性伴侣Hsp 70的情况下，N-末端长度增加的多肽（属于全α-螺旋单结构域模型蛋白的序列）的构象。为了在氨基酸特异性分辨率下获得信息，将采用在同位素标记的肽和未标记的分子伴侣存在下的多维NMR。这项工作的重点是延长来自脱辅基肌红蛋白，一个相对较小的和非常好的表征系统，作为一个很好的模型，所有α-螺旋蛋白的N-末端多肽。拟议的调查将探讨是否热休克蛋白70仅仅通过保持统计线圈状态，或它也通过诱导特定的多肽构象防止链间聚集。它的目的是提供一个一阶的体外近似热休克蛋白70是如何能够影响构象空间的延伸多肽。特定的细胞相关的影响，如多肽拴系（到核糖体出口通道）和分子拥挤的预期影响进行了讨论的建议，并将通过单独的额外的实验来解决。Hsp 70是一种主要的免疫活性分子伴侣，其影响蛋白质折叠过程的机制目前还知之甚少。然而，这一领域迫切需要取得进展，因为共翻译分子伴侣的缺陷作用（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、炎性心脏病、克罗恩病、P53相关癌症和几种神经退行性疾病，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Hsp 70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作图;（B）氢/氘交换脉冲标记动力学实验，以检测在Hsp 70存在下的结构形成机制;（c）在Hsp 40和Hsp 70-核苷酸交换因子共伴侣存在下的额外研究。