POLYPEPTIDE CONFORMATION AND INTERACTION WITH HSP70

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 该提议的目的是阐明在共翻译活性伴侣蛋白Hsp70存在下，长度不断增加的N末端多肽(属于全α-螺旋单域模型蛋白的序列)的构象。为了获得氨基酸特定分辨率的信息，将使用同位素标记的多肽和未标记的伴侣存在的多维核磁共振。这项工作的重点是延长来自无肌红蛋白的N-末端多肽，这是一个相对较小且特征非常好的系统，是研究全α-螺旋蛋白质的极佳模型。拟议的研究将探索Hsp70是否仅仅通过保持统计卷曲状态来防止链间聚集，或者它也通过诱导特定的多肽构象来起作用。它的目的是在体外提供一个一级近似的热休克蛋白70如何能够影响延长多肽的构象空间。特定细胞相关效应的预期影响，如多肽拴系(核糖体出口通道)和分子拥挤，将在提案中讨论，并将通过单独的额外实验加以解决。对主要的共翻译活性伴侣蛋白Hsp70影响蛋白质折叠过程的机制知之甚少。然而，在这一领域迫切需要取得进展，因为共翻译伴侣的作用有缺陷(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，例如涉及一些致命疾病的物种。这些疾病包括囊性纤维化、炎症性心脏病、克罗恩病、与P53相关的癌症，以及几种神经退行性疾病，如亨廷顿病和阿尔茨海默病。将进行的实验包括：(A)在未标记的Hsp70伴侣的存在下，用核磁共振对同位素标记的多肽进行高分辨率的二级结构作图；(B)氢/氢交换脉冲标记动力学实验，以检测在Hsp70存在下的结构形成机制；(C)在Hsp40和Hsp70-核苷酸交换因子辅助伴侣存在下的其他研究。

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.