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不能表达其氨基酸序列中编码的空间信息，这一点还没有完全理解。然而，蛋白质的错误折叠导致了毁灭性的病理，如囊性纤维化、地中海贫血，以及各种淀粉样神经病，如阿尔茨海默氏症和亨廷顿病。这一提议的长期目标是了解分子伴侣如何引导蛋白质到达其最终的、活跃的三维结构。为此，我们打算专注于分子伴侣的一个子类，即普遍存在的环状复合体，称为伴侣蛋白。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。此前，我们成功地使用了荧光能量转移(FRET)来跟踪驱动GroEL反应周期的一系列步骤。这种方法依赖于在GroEL、GroES和底物蛋白中引入半胱氨酸残基，然后用荧光探针标记。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结合的折叠中间体的形态图。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高效折叠。我们的目标是：(1)建立一种FRET方法，可用于绘制GroEL结合的折叠中间体的形态图；(2)应用该方法跟踪与GroEL结合的折叠中间体的结构变化，以测试两种GroEL刺激的折叠模型；以及(3)通过测试GroEL下底物封装的特定模型，确定GroEL依赖的蛋白质折叠是如何被触发的。

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.