Polypeptide Conformation and Interaction with Hsp70

本方案的目的是阐明递增蛋白N末端多肽的构象。 长度(属于全S螺旋单域模型蛋白的序列) 共翻译活性伴侣Hsp70。为了获得特定氨基酸分辨率的信息， 在同位素标记的多肽和未标记的伴侣存在的情况下，多维核磁共振将 受雇的。其他生物物理方法，如等温滴定量热法、尺寸排除法 还将使用层析和荧光光谱分析。该工作的重点是延长N- 脱脂蛋白衍生的末端多肽，是一种相对较小且特性极好的 该系统是研究全螺旋蛋白质的极佳模型。 拟议的调查将探索Hsp70是否仅仅通过以下方式阻止链间聚集 保持统计卷曲状态，或者它也通过诱导特定的多肽构象而起作用。 这项研究并不是要直接模拟细胞内的共翻译并立即 翻译后折叠事件。另一方面，它的目标是在体外提供一阶近似 Hsp70是如何影响伸长多肽的构象空间的。预期中的 特定细胞相关效应的影响，如多肽拴系(对核糖体出口通道)和 分子拥挤将在提案中讨论，并将通过单独的额外 实验。 关于Hsp70的主要共翻译活性的机制人们知之甚少 伴侣，影响蛋白质折叠的进程。然而，这一领域迫切需要取得进展，因为 共翻译伴侣蛋白的缺陷作用(或生物可利用量不足)与 不正确折叠的自相关物种的形成，例如涉及一些致命的 疾病。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，如亨廷顿氏症和阿尔茨海默氏症。实验以实现 包括(A)同位素标记的高分辨率二级结构作图 未标记Hsp70分子伴侣存在下的多肽的核磁共振研究；(B)氢/氢交换 脉冲标记法动力学实验研究结构形成的机理 HSP70；(C)在存在HSP40和HSP70-核苷酸交换因子的情况下的其他研究 辅护人。

The goal of this proposal is to elucidate the conformation of N-terminal polypeptides of increasing length (belonging to the sequence of an all-s-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-_ 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 carded 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.