Mechanisms of Chaperonin-Mediated Protein Folding

伴侣蛋白介导的蛋白质折叠机制

• 批准号: 6891242
• 负责人: HAYS S RYE
• 金额: $ 27.6万
• 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/6891242
• 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; 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）：为什么某些蛋白质无法在没有分子伴侣的帮助的情况下表达在其氨基酸序列中编码的空间信息。然而，蛋白质错误折叠的基础是毁灭性的病理，例如囊性纤维化，thalassecnias和多种淀粉样神经病，例如阿尔茨海默氏病和亨廷顿氏病。该提案的长期目标是了解分子伴侣如何将蛋白质引导蛋白质的最终活性三维结构。为此，我们打算专注于一个分子伴侣的一个子类，即无处不在的环形复合物，称为伴侣蛋白。这种保守和必不可少的蛋白质家族的一个成员是细菌性凹槽 - 胶合体复合物。我们将使用凹槽 - 胶状系统作为模型，以及模型折叠底物，提出了旨在揭示依赖伴侣依赖蛋白折叠的一般原理的实验。为了研究凹槽伴侣蛋白的动力学及其与蛋白质折叠中间体的相互作用，我们正在开发荧光和快速混合方法。以前，我们成功使用荧光能量转移（FRET）遵循驱动凹槽反应周期的步骤序列。这种方法依赖于将半胱氨酸残基引入凹槽，凹槽和底物蛋白，然后用荧光探针标记。现在，我们将这种方法扩展到包括时间分辨的FRET测量值，以系统地映射凹槽结合的折叠中间体的形态。我们预计这种合并的方法将使我们能够确定为什么需要凹槽 - 伴侣蛋白来折叠某些蛋白质以及这些蛋白质，凹槽和凹槽之间的特定相互作用如何促进生产性折叠。我们的目的是：（1）开发一个FRET测定法，该测定方法可用于绘制凹槽结合的折叠中间体的形态，（2）应用此测定法以遵循折叠中间的结构变化，同时绑定到凹槽中以测试两种groel刺激的折叠折叠模型，并（3）确定groel依赖型蛋白质的折叠式折叠式的旋转式构图，以互补的构建构建构建，并构成了旋转的构建，并构成了互动的互动，并将其用于旋转。