Mechanisms of Chaperonin-Mediated Protein Folding

伴侣蛋白介导的蛋白质折叠机制

• 批准号: 7060721
• 负责人: HAYS S RYE
• 金额: $ 26.95万
• 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 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7060721
• 关键词: X ray crystallography; adenosine triphosphate; bacterial proteins; cryoelectron microscopy; electron microscopy; enzyme activity; fluorescence resonance energy transfer; mass spectrometry; method development; molecular chaperones; protein folding; protein protein interaction; protein 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.

描述（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无法表达其氨基酸序列中编码的空间信息尚不完全清楚。然而，蛋白质错误折叠是破坏性病理的基础，例如囊性纤维化、地中海贫血和各种淀粉样神经病，例如阿尔茨海默病和亨廷顿病。该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，我们打算重点关注分子伴侣的一个亚类，即普遍存在的环形复合物，称为伴侣蛋白。这个保守且重要的蛋白质家族的成员之一是细菌 GroEL-GroES 复合物。使用 GroEL-GroES 系统作为模型以及模型折叠底物，我们提出了旨在揭示伴侣依赖性蛋白质折叠的一般原理的实验。为了研究 GroEL 伴侣蛋白的动力学及其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。此前，我们成功地使用荧光能量转移 (FRET) 来跟踪驱动 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，然后用荧光探针进行标记。现在，我们扩展了这种方法，包括时间分辨 FRET 测量，以便系统地绘制 GroEL 结合折叠中间体的形态图。我们预计这种组合方法将使我们能够确定为什么需要 GroEL-GroES 伴侣蛋白来折叠某些蛋白质，以及这些蛋白质、GroEL 和 GroES 之间的特定相互作用如何促进高效折叠。我们的目标是：(1) 开发一种 FRET 测定，可用于绘制 GroEL 结合折叠中间体的形态图，(2) 应用该测定来跟踪折叠中间体在与 GroEL 结合时的结构变化，以便进行测试两个 GroEL 刺激折叠模型；(3) 通过测试 GroES 下底物封装的特定模型来确定如何触发 GroEL 依赖性蛋白质折叠。