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。一个中心问题在于澄清早期折叠事件，这可能是非常困难的研究。 几条证据表明，蛋白质折叠的初始步骤涉及多肽链的折叠。然而，目前还不清楚是否崩溃与任何二级/三级结构的形成。 如果结构元素在折叠过程的早期形成，它们是天然的还是包含非天然（错误折叠）的元素，这些元素可能会延迟或加速随后的折叠事件？这些过程对每种蛋白质都不同吗？还是所有蛋白质都有共同的一般规则？几十年来，折叠反应已经用停流仪器进行了研究，其中典型的混合停滞时间是几毫秒的量级，在此期间，大部分反应可能会错过。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。利用这种技术，我们已经能够观察到的脂质结合蛋白，细胞色素c和抹香鲸apo-myoglobin的亚毫秒时域的折叠的第一次。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。高效率是可能的动力学控制的新生阶段，其中的构象空间减少通过多肽链缩合;高保真度是通过随后的动力学控制的平衡，其中的能量被最小化的结构波动。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。将检查早期中间体的结构对整体折叠动力学的影响。定点诱变将用于在涉及折叠的关键元件中产生改变。为了启动折叠，我们将利用我们的良好特性的快速混频器与死区时间为100微秒。此外，由硅制造的新混合器已被开发用于具有50微秒死区时间的冷冻淬火应用。中间体的结构将通过可见光和UV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深入了解这些不同的蛋白质系统的折叠途径。

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.