Mechanistic Studies of Complex Protein Folding Reactions

描述（由申请人提供）：在过去的四十年中，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，这些蛋白质的折叠反应非常简单。尽管如此，这些研究为所有蛋白质的折叠驱动力和机制提供了重要的见解，包括具有许多结构域和复杂折叠反应的大蛋白质。此外，越来越多的人认识到，与蛋白质功能耦合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利用我们基于简单系统的生物物理研究的知识来推断更复杂蛋白质的折叠机制，并在其功能和细胞环境的背景下这样做。具体来说，我们计划使用常规的动力学方法，如停止流动和温度跳变荧光，来测量RNase P蛋白结合配体时的折叠动力学，特别是它的同源RNA。我们还计划扩大对蛋白A （BdpA）的B结构域的研究，包括A、C、D和E结构域，以了解这一重要致病性因子在金黄色葡萄球菌中的全局折叠。最后，我们开发了一种在生理条件下研究未折叠蛋白的实验方法，并计划利用这种方法研究单体的未折叠形式；抑制因子和BdpA。我们计划收集这些模型变性系系的各种光谱和生物物理数据，并将每个系统的系系平均性质与统计力学模型的性质进行比较。我们的目标是提供一个更准确的图像，在这些集成显著填充构象，使他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，因为许多蛋白质结构域被观察到每秒数十或数百次采样其未折叠状态，使未折叠形式与折叠形式一样具有相关功能。我们提出的研究的生物学意义在于反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。详细了解蛋白质-配体相互作用以及由此产生的无数生物现象需要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.