Single Molecule Protein and Biopolymer Folding Dynamics

单分子蛋白质和生物聚合物折叠动力学

• 批准号: 6637254
• 负责人: SHIMON WEISS
• 金额: $ 34.42万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6637254
• 关键词: DNA; chemical kinetics; chemical stability; chemical structure function; chemical synthesis; chymotrypsin inhibitor; computer program /software; computer system design /evaluation; data collection methodology /evaluation; fluorescence resonance energy transfer; gel; method development; model design /development; molecular dynamics; molecular site; molecular size; nanotechnology; physical model; protein denaturation; protein folding; solvents; structural biology; temperature; thermodynamics; time resolved data

The objective of this proposal is to study protein folding using single-molecule fluorescence methodology. Understanding protein folding is fundamental to deciphering how the genetic code is translated into functional protein units. Improper protein folding is related to complex aggregation phenomena that cause diseases such to the prion family of diseases and Alzheimer's disease. We will develop single-molecule fluorescence methodology to follow conformational dynamics of individual fluorescently-labeled proteins and other biopolymers. In contrast to ensemble methods, single-molecule methods provide information on fluctuations, distributions and time-trajectories of observables that are obscured in ensemble measurements. In particular, single-molecule methods are free from synchronization requirements that are impossible to achieve with ensemble methods. We will use single-pair fluorescence resonance energy transfer (spFRET) as reporter of the distance between two amino acid residues in the polypeptide chain, or the site-to-site distance of a fluctuating biopolymer. The recovered distance information will be used as a reaction coordinate of protein folding, and as a reporter of conformational dynamics. We will emphasize early events in the folding pathway, especially focusing on the "fast-collapse" of the polypeptide chain when exposed to specific solvent conditions. We will address both equilibrium and non-equilibrium reaction conditions, and study molecules that are (i) diffusing in solution, or (ii) immobilized on surfaces and in gels. We will develop single-molecule, continuous flow, fast-mixing methods (for diffusing molecules) and rapid liquid-exchange methods (for immobilized molecules). Our measurements will combine fluorescence-intensity ratiometric methods with fluorescence-lifetime methods to provide unprecedented sensitivity and temporal resolution that cover many orders of magnitude. Advanced data acquisition, data analysis and signal processing algorithms will be developed for a variety of samples, experimental formats and reaction conditions. A theoretical framework will be constructed to accurately describe the results of our experiments. We will: (i) develop single-molecule methods with spatial and temporal resolution relevant to protein folding; and (ii) use homopolymeric single-stranded DNA (ssDNA) as a model system for method development. We will combine the proposed methodology with existing single-molecule fluorescence methodology to study the folding of protein chymotrypsin inhibitor 2 (CI2). In particular, we will address: (1) early events of the folding pathway, such as fast collapse; (2) late events of the folding pathway, such as formation of native protein contacts; and (3) effects of amino acid substitutions on early and late events of the folding pathway.

该提案的目的是使用单分子荧光方法研究蛋白质折叠。了解蛋白质折叠对于破译遗传密码如何转化为功能性蛋白质单元至关重要。 不正确的蛋白质折叠与复杂的聚集现象有关，这些现象会导致朊病毒家族疾病和阿尔茨海默病等疾病。 我们将开发单分子荧光方法来跟踪单个荧光标记蛋白质和其他生物聚合物的构象动力学。 与系综方法相比，单分子方法提供了在系综测量中模糊的可观测值的波动、分布和时间轨迹信息。 特别是，单分子方法没有同步要求，这是集成方法无法实现的。 我们将使用单对荧光共振能量转移（spFRET）作为多肽链中两个氨基酸残基之间距离的报告器，或波动生物聚合物的位点到位点距离。 恢复的距离信息将用作蛋白质折叠的反应坐标，并作为构象动力学的报告器。 我们将强调折叠途径中的早期事件，尤其关注暴露于特定溶剂条件时多肽链的“快速折叠”。我们将解决平衡和非平衡反应条件，并研究 (i) 在溶液中扩散，或 (ii) 固定在表面和凝胶中的分子。 我们将开发单分子、连续流动、快速混合方法（用于扩散分子）和快速液体交换方法（用于固定分子）。 我们的测量将荧光强度比率测量方法与荧光寿命方法相结合，以提供前所未有的灵敏度和覆盖多个数量级的时间分辨率。 将为各种样品、实验形式和反应条件开发先进的数据采集、数据分析和信号处理算法。 将构建一个理论框架来准确描述我们的实验结果。 我们将：（i）开发具有与蛋白质折叠相关的空间和时间分辨率的单分子方法； (ii) 使用同聚单链 DNA (ssDNA) 作为方法开发的模型系统。 我们将把所提出的方法与现有的单分子荧光方法相结合，研究蛋白质糜蛋白酶抑制剂 2 (CI2) 的折叠。 我们将特别讨论：（1）折叠路径的早期事件，例如快速塌陷； (2)折叠途径的晚期事件，例如天然蛋白质接触的形成； (3)氨基酸取代对折叠途径早期和晚期事件的影响。