Mechanisms of Chaperonin-Mediated Protein Folding

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，这一点尚不完全清楚。然而，蛋白质错误折叠是破坏性疾病的基础，如囊性纤维化、地中海贫血和各种淀粉样神经病变，如阿尔茨海默氏症和亨廷顿氏病。这项提议的长期目标是了解分子伴侣如何引导蛋白质进入其最终的、活跃的三维结构。为此，我们打算将重点放在分子伴侣蛋白的一个亚类上，即普遍存在的环状复合物伴侣蛋白。细菌GroEL-GroES复合体是这个保守而重要的蛋白质家族的成员之一。使用GroEL-GroES系统作为模型，以及模型折叠底物，我们提出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，我们成功地使用荧光能量转移（FRET）来遵循驱动GroEL反应周期的步骤序列。该方法依赖于将半胱氨酸残基引入GroEL、GroES和底物蛋白中，然后用荧光探针对其进行标记。我们现在将这种方法扩展到包括时间分辨FRET测量，以便系统地绘制groel束缚折叠中间体的形态。我们预计，这种结合的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生产性折叠。我们的目标是：(1)开发一种FRET试验，可用于绘制GroEL结合折叠中间体的形态图；(2)应用该试验跟踪与GroEL结合时折叠中间体的结构变化，以测试GroEL刺激折叠的两种模型；(3)通过测试GroES下底物封装的特定模型，确定GroEL依赖性蛋白质折叠是如何触发的。

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.