Early Events in Protein Folding

蛋白质折叠的早期事件

• 批准号: 6599465
• 负责人: DENIS L. ROUSSEAU
• 金额: $ 32.58万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6599465
• 关键词: Raman spectrometry; binding proteins; biochemistry; conformation; cytochrome c; electron spin resonance spectroscopy; infrared spectrometry; myoglobin; protein folding; protein protein interaction; protein structure function; site directed mutagenesis; thermodynamics

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.

描述(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，这可能很难研究。有几条证据表明，蛋白质折叠的第一步涉及多肽链的崩溃。然而，尚不清楚坍塌是否与任何二级/三级结构的形成有关。如果构造元素是在褶皱过程的早期形成的，它们是类似自然的吗？或者它们是否含有可能延缓或加速随后的折叠事件的非自然(错误折叠)元素？这些过程对于每种蛋白质来说是不同的，还是对所有蛋白质都有共同的一般规则？几十年来，人们一直使用停流仪器研究折叠反应，在这种仪器中，典型的混合死时间约为几毫秒，在此期间可能会错过很大一部分反应。我们小组率先开发了亚毫秒混合器，用于研究早期的折叠事件。利用这项技术，我们首次能够在亚毫秒的时间域中观察到脂结合蛋白、细胞色素c和抹香鲸脱脂肌红蛋白的折叠。在这些研究的基础上，我们提出了一种双相机制，确保蛋白质高效、保真地折叠成其独特的天然构象。高效率是通过动力学控制的初始阶段实现的，其中构象空间通过多肽链缩合来减少；高保真是通过随后的热力学控制的平衡来实现的，在该平衡中，能量通过结构波动来最小化。我们将在关于脂结合蛋白、细胞色素c和肌红蛋白系统的新研究中进一步检验这一假说。将考察早期中间体的结构对整体折叠动力学的影响。定点突变将被用来改变参与折叠的关键元件。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，由硅制造的新型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。预计这种综合的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。