Early Events in Protein Folding

描述（由申请人提供）：蛋白质如何折叠成其天然结构是当今生物科学中最重要和最具挑战性的问题之一。一个中心问题在于对早期折叠事件的澄清，这可能是非常难以研究的。几条线索的证据表明，蛋白质折叠的第一步涉及多肽链的崩溃。然而，尚不清楚崩塌是否与任何二级/三级构造形成有关。如果结构元素是在折叠过程的早期形成的，那么它们是原生的还是包含可能延缓或加速后续折叠事件的非原生（错误折叠）元素？这些过程对于每种蛋白质是不同的，还是所有蛋白质都有共同的一般规则？几十年来，折叠反应一直是用停流仪器研究的，其中典型的混合死区时间在几毫秒左右，在此期间可能会错过大部分反应。我们的团队率先开发了亚毫秒混合器，用于研究早期折叠事件。利用该技术，我们首次能够在亚毫秒时间域内观察到脂质结合蛋白、细胞色素c和抹香鲸载脂蛋白肌红蛋白的折叠。基于这些研究，我们提出了一种双相机制，该机制保证了蛋白质以高效率和保真度折叠成其独特的天然构象。通过多肽链缩合减少构象空间的动力学控制的新生相使高效率成为可能；高保真度是通过随后的热力学控制平衡来实现的，其中能量被结构波动最小化。我们将在脂质结合蛋白、细胞色素c和肌红蛋白系统的新研究中进一步验证这一假设。早期中间体的结构对整体折叠动力学的影响将被检查。位点定向诱变将用于在涉及折叠的关键元素上产生改变。为了启动折叠，我们将利用我们的特性良好的快速混合器，死区时间为100微秒。此外，由硅制成的新型混合器已被开发用于冻结淬火应用，死亡时间为50微秒。中间体的结构将通过可见光和紫外共振拉曼散射、色氨酸荧光、红外光谱和自旋标记EPR光谱进行研究。预计这种综合方法将导致对这些不同蛋白质系统的折叠途径的深入了解。

DESCRIPTION (provided by applicant): How a protein folds into its native structure is one of the most important and challenging problems in biological science today. A central issue lies in clarification of the early folding events, which can be very difficult to study. Several lines of evidence suggest that the initial step in protein folding involves the collapse of a polypeptide chain. However, it is unclear whether the collapse is associated with any secondary / tertiary structure formation. If structural elements form early in the folding process, are they native-like or could they contain non-native (misfolded) elements that could retard or accelerate the subsequent folding events? Are these processes different for each protein or are there general rules that are common to all proteins? For decades, folding reactions have been studied with stopped-flow instrumentation in which the typical mixing dead time is on the order of a few milliseconds during which a large portion of the reaction may be missed. Our group pioneered the development of sub-millisecond mixers for studying the early folding events. With this technique, we have been able to observe the folding of a lipid binding protein, cytochrome c and sperm whale apo-myoglobin in the submillisecond time domain for the first time. Based on these studies, we proposed a biphasic mechanism, which guarantees that the protein folds into its unique native conformation with high efficiency and fidelity. The high efficiency is made possible by a kinetically controlled nascent phase, in which the conformational space is reduced through polypeptide chain condensation; the high fidelity is achieved through the subsequent thermodynamically controlled equilibrium, in which the energy is minimized by structural fluctuations. We will further test this hypothesis in new studies on the lipid binding protein, cytochrome c and myoglobin systems. The impact of the structure of the early intermediates on the overall folding kinetics will be examined. Site directed mutagenesis will be used to create alterations in key elements involved in folding. To initiate the folding, we will exploit our well-characterized rapid mixers with dead times of 100 microseconds. In addition, new mixers fabricated from silicon have been developed for a freeze quench application with a 50 microseconds dead time. The structures of the intermediates will be studied by visible and UV resonance Raman scattering, by tryptophan fluorescence, by infrared spectroscopy and by spin labeled EPR spectroscopy. It is anticipated that this integrated approach will lead to an in depth understanding of the folding pathways of these diverse protein systems.