Polypeptide Conformation and Interaction with Hsp70

多肽构象及其与 Hsp70 的相互作用

• 批准号: 7455648
• 负责人: Silvia Cavagnero
• 金额: $ 3.54万
• 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/7455648
• 关键词: 3-Dimensional; Acids; Address; Affect; Alzheimer' s Disease; Amino Acids; Anabolism; Area; Binding; Bioavailable; Biochemical; Biological Models; Calorimetry; Cell-Free System; Cells; Class; Collection; Compatible; Complex; Condition; Crohn' s disease; Crowding; Cystic Fibrosis; Data; Deuterium; Development; Disease; Electrostatics; Ensure; Environment; Equilibrium; Event; Figs - dietary; Fluorescence; Fluorescence Spectroscopy; Future; Gel; Goals; Guanine Nucleotide Exchange Factors; Haiti; Hand; Heart Diseases; Huntington Disease; Hydrogen; In Vitro; Inflammatory; Investigation; Ionic Strengths; Kinetics; Label; Laboratories; Lead; Length; Life; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Methods; Modeling; Molecular; Molecular Biology; Molecular Chaperones; Molecular Conformation; Molecular Sieve Chromatography; N-terminal; NMR Spectroscopy; Nature; Neurodegenerative Disorders; Nuclear Magnetic Resonance; Nucleotides; Numbers; Oryctolagus cuniculus; Parkinson Disease; Pathway interactions; Peptide Fragments; Peptides; Personal Satisfaction; Physiologic pulse; Polar Regions; Positioning Attribute; Postdoctoral Fellow; Process; Protein Overexpression; Proteins; Publications; Pulse taking; Range; Rate; Research; Research Institute; Research Personnel; Resolution; Resources; Ribosomes; Role; Sampling; Scheme; Structure; System; Techniques; Tertiary Protein Structure; Testing; Time; Titrations; Translations; Universities; Wisconsin; Work; amyloid formation; apomyoglobin; experience; i(19); in vivo; instrumentation; interest; member; milligram; polypeptide; prevent; programs; protein folding; protein function; protein misfolding; research study; stopped-flow fluorescence

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.

该建议的目的是阐明N-末端多肽的构象， 长度（属于全S-螺旋单结构域模型蛋白的序列）， 免疫活性分子伴侣Hsp 70。为了获得氨基酸特异性分辨率的信息， 在同位素标记的肽和未标记的分子伴侣存在下的多维NMR将是 就业。其他生物物理方法，如等温滴定量热法、尺寸排阻法 也将使用色谱法和荧光光谱法。这项工作的重点是延长N- 来自脱辅基肌红蛋白的末端多肽，一种相对小且特征非常好的 系统，作为一个很好的模型，所有-_螺旋蛋白质。 拟议的研究将探索Hsp 70是否仅仅通过以下方式防止链间聚集： 保持统计卷曲状态，或者它也通过诱导特定的多肽构象起作用。 本研究不旨在直接模拟细胞内共翻译， 翻译后折叠事件另一方面，它的目的是提供一个一阶体外近似 热休克蛋白70如何能够影响延伸多肽的构象空间。预期 特异性细胞相关效应的影响，如多肽束缚（至核糖体出口通道）和 分子拥挤的建议中进行了讨论，并将通过单独的额外的 实验 目前对Hsp 70的作用机制知之甚少， 分子伴侣，影响蛋白质折叠的过程。然而，这一领域迫切需要取得进展，因为 共翻译分子伴侣的作用缺陷（或生物可利用量不足）与 错误折叠的自我关联物种的形成，例如参与许多致命的 疾病这些包括囊性纤维化，炎症性心脏病，克罗恩病，P53相关癌症， 和几种神经变性疾病如亨廷顿氏病和阿尔茨海默氏病。实验来 （a）同位素标记的高分辨率二级结构图谱 （B）在未标记的Hsp 70分子伴侣的存在下通过NMR测定多肽的分子量; 脉冲标记动力学实验，以检测在存在下的结构形成机制， Hsp 70;（c）在Hsp 40和Hsp 70-核苷酸交换因子存在下的额外研究 伴侣