Mechanistic Studies of Complex Protein Folding Reactions

描述(申请人提供)：在过去的40年里，蛋白质折叠的机制研究主要集中在长度小于300个氨基酸的蛋白质上，折叠反应相当简单。然而，这些研究为所有蛋白质折叠的驱动力和机制提供了至关重要的见解，包括具有多结构域的大蛋白质和复杂的折叠反应。此外，人们越来越认识到，与蛋白质功能相关的构象变化在机制上类似于蛋白质折叠反应。我们研究的长期目标是了解如何使用我们基于简单系统生物物理研究的知识来推断更复杂的蛋白质的折叠机制，并在它们的功能和细胞环境的背景下这样做。具体地说，我们计划使用传统的动力学方法，如停流和温度跳跃荧光来测量RNaseP蛋白与配体，特别是其同源RNA结合时的折叠动力学。我们还计划扩展我们对蛋白A的B结构域(BdpA)的研究，包括A、C、D和E结构域，以了解金黄色葡萄球菌中这一重要致病因子的全球折叠。最后，我们开发了一种实验方法来研究生理条件下的未折叠蛋白质，并计划利用这种方法来研究单体、阻遏物和BdpA的未折叠形式。我们计划收集关于这些模型变性系综的各种光谱和生物物理数据，并将系综平均性质与每个系统的统计力学模型的性质进行比较。我们的目标是提供这些系综中显著填充的构象的更准确的图像，以便它们可以用于我们的折叠反应的机制模型。公共卫生相关性：这些研究很重要，因为人们观察到许多蛋白质结构域每秒都会对其展开状态进行数十次或数百次采样，使展开形式与折叠形式一样与功能相关。我们提议的研究的生物学意义在于重复和复杂的折叠反应与细胞中蛋白质的调节和功能的相关性。要详细了解蛋白质与配体的相互作用以及由此产生的各种生物现象，需要进行热力学和动力学描述。

DESCRIPTION (provided by applicant): Over the past forty years, mechanistic studies of protein folding have focused primarily on proteins fewer than 300 amino acids in length with quite simple folding reactions. Nevertheless, these studies have provided crucial insights into the driving forces and mechanisms of the folding of all proteins, including large proteins with many domains and complex folding reactions. In addition, there is growing recognition that conformational changes coupled to protein functions are mechanistically similar to protein folding reactions. The long term goal of our research is to understand how to use our knowledge based on biophysical studies of simple systems to deduce the folding mechanisms of much more complex proteins and to do so in the context of their function and cellular environment. Specifically, we plan to use conventional kinetic methods such as stopped-flow and temperature-jump fluorescence to measure the folding kinetics of RNase P protein when it binds ligands, particularly its cognate RNA. We also plan to extend our studies on the B domain of protein A (BdpA) to include the A, C, D and E domains so as to understand the global folding this important pathogenicity factor in the bacterium Staphylococcus aureus. Finally, we have developed an experimental approach to study unfolded proteins under physiological conditions and plan to exploit this method to study the unfolded forms of monomeric ; repressor and BdpA. We plan to collect a variety of spectroscopic and biophysical data on these model denatured ensembles and compare the ensemble-averaged properties with those of statistical mechanical models of each system. Our goal is to provide a more accurate picture of the significantly populated conformations in these ensembles so that they can be used in our mechanistic models of the folding reaction. PUBLIC HEALTH RELEVANCE: These studies are important because many protein domains have been observed to sample their unfolded states tens or hundreds of times every second, making the unfolded form as relevant to function as the folded form. The biological significance of our proposed studies rests on the relevance of recurrent and complex folding reactions to the regulation and function of proteins in the cell. A detailed understanding of protein-ligand interactions and the myriad biological phenomena that result from them requires a thermodynamic and kinetic description.