Mechanism of GroEL-assisted Protein Folding

GroEL 辅助蛋白质折叠机制

• 批准号: 7059912
• 负责人: LINGLING CHEN
• 金额: $ 28.53万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7059912
• 关键词: X ray crystallography; bioenergetics; chemical binding; fluorescence polarization; fluorescence spectrometry; hydrolysis; molecular assembly /self assembly; molecular chaperones; nuclear magnetic resonance spectroscopy; peptide library; protein folding; protein protein interaction; protein structure function; stereochemistry; structural biology

DESCRIPTION (provided by applicant): Molecular chaperones are involved in a wide range of essential cellular processes: protein synthesis, molecular assembly, translocation, degradation, and folding. The E. coli molecular chaperone GroEL, along with its co-chaperone GroES, increases the efficiency of protein folding in vivo, using an ATP-driven mechanism. In the GroEL-facilitated folding process, first GroEL sequesters the aggregation-prone nonnative forms of proteins from the complex cellular environment within its central cavity. Then with the actions of ATP binding/hydrolysis and GroES binding, the protein is allowed to carry out its initial folding events within an isolated "folding chamber" formed by GroEL/GroES. The long-term goals of this proposal are to elucidate the structural features of the interaction of GroEL and substrate proteins, to understand the mechanism of GroEL-facilitated protein folding in a structural context, and to further structural knowledge of molecular chaperones function in general. Various biochemical and biophysical techniques, including phage display, fluorescence spectroscopy/polarization and X-ray crystallography, will be used to determine how GroEL recognizes the substrate proteins, and to understand the energetics of ATP binding/hydrolysis. Three specific aims are to: 1) Select for small peptides that interact with the substrate binding domain of GroEL using a phage display method. 2) Study the interplay of GroEL-substrate by determining the structures of the substrate-trapped GroEL assemblies using X-ray protein crystallography and NMR, and carry out structure-guided mutational studies. 3) Create model polypeptide substrates for GroEL based on the peptides selected in 1) and use them to study the functional role of nucleotide binding/hydrolysis and the mechanism of GroEL-assisted protein folding. Knowledge of protein folding and the role of molecular chaperones in facilitating the folding process will contribute to a better understanding of folding-related human diseases, such as Cystic Fibrosis, Alzheimer's, Prion diseases and cataracts, at the molecular level, and could lead to the design of novel therapeutic approaches.

描述（由申请人提供）：分子伴侣参与广泛的基本细胞过程：蛋白质合成、分子组装、易位、降解和折叠。急诊大肠杆菌分子伴侣GroEL，沿着其辅助伴侣GroES，使用ATP驱动的机制增加体内蛋白质折叠的效率。在GroEL促进的折叠过程中，首先GroEL将易于聚集的非天然形式的蛋白质从其中心腔内的复杂细胞环境中隔离。然后通过ATP结合/水解和GroES结合的作用，允许蛋白质在由GroEL/GroES形成的隔离的“折叠室”内进行其初始折叠事件。该提案的长期目标是阐明GroEL和底物蛋白相互作用的结构特征，了解GroEL促进蛋白质折叠的结构背景下的机制，并进一步了解分子伴侣功能的结构知识。各种生物化学和生物物理技术，包括噬菌体展示，荧光光谱/偏振和X射线晶体学，将用于确定GroEL如何识别底物蛋白，并了解ATP结合/水解的能量学。三个具体目标是：1）使用噬菌体展示方法选择与GroEL的底物结合结构域相互作用的小肽。2)通过使用X射线蛋白质晶体学和NMR确定底物捕获的GroEL组装体的结构来研究GroEL-底物的相互作用，并进行结构指导的突变研究。3)基于1）中选择的肽创建GroEL的模型多肽底物，并使用它们来研究核苷酸结合/水解的功能作用和GroEL辅助蛋白质折叠的机制。蛋白质折叠的知识和分子伴侣在促进折叠过程中的作用将有助于在分子水平上更好地理解与折叠相关的人类疾病，如囊性纤维化、阿尔茨海默氏病、朊病毒病和白内障，并可能导致设计新的治疗方法。