Polypeptide Conformation and Interaction with Hsp70

描述（由申请人提供）：本建议的目的是阐明在存在补体活性伴侣Hsp 70的情况下，长度增加的N-末端多肽（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。也将使用其他生物物理方法，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。 本研究不旨在直接模拟细胞内共翻译和翻译后即刻折叠事件。另一方面，它的目的是提供一个一级体外近似热休克蛋白70是如何能够影响的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。

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.