Mechanisms of Chaperonin-Mediated Protein Folding

伴侣蛋白介导的蛋白质折叠机制

• 批准号: 7228251
• 负责人: HAYS S RYE
• 金额: $ 26.17万
• 依托单位: PRINCETON 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 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7228251
• 关键词: ATP Hydrolysis; Address; Alzheimer' s Disease; Amino Acid Sequence; Amyloid Neuropathies; Binding; Biological Assay; Cells; Class; Complex; Cryoelectron Microscopy; Cysteine; Cystic Fibrosis; DNA Sequence Rearrangement; Energy Transfer; Family member; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Goals; GroEL Protein; GroES Protein; Huntington Disease; Label; Link; Location; Maps; Measurement; Measures; Mediating; Methods; Modeling; Molecular Chaperones; Molecular Conformation; Morphology; Pathology; Population; Positioning Attribute; Protein Family; Proteins; Reaction; Relative (related person); Rhodospirillum rubrum; Ribulose-Bisphosphate Carboxylase; Route; Series; Shapes; Speed; Structure; System; Testing; Thalassemia; Time; Variant; X-Ray Crystallography; analog; chaperonin; member; protein folding; protein misfolding; research study; three dimensional structure

DESCRIPTION (provided by applicant): Why certain proteins are unable to express the spatial information encoded in their amino acid sequences without the aid of molecular chaperones is not fully understood. Yet protein misfolding underlies devastating pathologies, such as cystic fibrosis, thalassemias, and a variety of amyloid neuropathies such as Alzheimer's and Huntington's disease. The long-term goal of this proposal is to understand how molecular chaperones guide proteins to their final, active three dimensional structures. Toward this end, we intend to focus on one subclass of the molecular chaperones, the ubiquitous, ring-shaped complexes known as chaperonins. One member of this conserved and essential family of proteins is the bacterial GroEL-GroES complex. Using the GroEL-GroES system as a model, along with model folding substrates, we propose experiments intended to uncover general principles for chaperone-dependent protein folding. In order to study the dynamics of the GroEL chaperonin and how it interacts with protein folding intermediates, we are developing fluorescence and rapid mixing methods. Previously, we successfully used fluorescence energy transfer (FRET) to follow the sequence of steps that drive the GroEL reaction cycle. This method relies on the introduction of cysteine residues into GroEL, GroES and substrate protein, which are then labeled with fluorescent probes. We now extend this approach to include time-resolved FRET measurements, in order to systematically map the morphology of a GroEL-bound folding intermediate. We anticipate that this combined approach will allow us to determine why the GroEL-GroES chaperonin is required to fold certain proteins and how specific interactions between these proteins, GroEL, and GroES facilitate productive folding. Our aims are: (1) to develop a FRET assay which can be used to map the morphology of a GroEL bound folding intermediate, (2) to apply this assay to follow structural changes in a folding intermediate while bound to GroEL in order to test two models of GroEL-stimulated folding and (3) to determine how GroEL-dependent protein folding is triggered, by testing specific models of substrate encapsulation beneath GroES.

说明（由申请人提供）：为什么某些蛋白质在没有分子伴侣的帮助下不能表达其氨基酸序列中编码的空间信息，这一点尚未完全理解。然而，蛋白质错误折叠是破坏性病理的基础，如囊性纤维化、地中海贫血和各种淀粉样神经病，如阿尔茨海默病和亨廷顿病。这项提案的长期目标是了解分子伴侣如何引导蛋白质形成最终的活性三维结构。为此，我们打算集中在分子伴侣的一个子类，无处不在，环形复合物称为伴侣蛋白。这种保守的和必需的蛋白质家族的一个成员是细菌GroEL-GroES复合物。使用GroEL-GroES系统作为模型，沿着与模型折叠底物，我们提出的实验旨在揭示分子伴侣依赖性蛋白质折叠的一般原则。为了研究GroEL伴侣蛋白的动力学以及它如何与蛋白质折叠中间体相互作用，我们正在开发荧光和快速混合方法。以前，我们成功地使用荧光能量转移（FRET）来遵循驱动GroEL反应循环的步骤序列。该方法依赖于将半胱氨酸残基引入GroEL、GroES和底物蛋白中，然后用荧光探针标记。我们现在扩展这种方法，包括时间分辨FRET测量，以系统地映射GroEL结合折叠中间体的形态。我们预计，这种组合的方法将使我们能够确定为什么需要GroEL-GroES伴侣蛋白折叠某些蛋白质，以及这些蛋白质，GroEL和GroES之间的特定相互作用如何促进生产性折叠。我们的目标是：（1）开发可用于绘制GroEL结合的折叠中间体的形态的FRET测定，（2）应用该测定来跟踪与GroEL结合时折叠中间体的结构变化，以测试GroEL刺激的折叠的两种模型，和（3）通过测试GroES下方的底物包封的特定模型来确定GroEL依赖性蛋白质折叠是如何被触发的。