Early Events in Protein Folding

蛋白质折叠的早期事件

• 批准号: 9507280
• 负责人: Anthony Fink
• 金额: $ 26.94万
• 依托单位: University of California-Santa Cruz
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9507280&HistoricalAwards=false
• 关键词: Early; Events; Protein; Folding

9507280 Fink The goal of this research is to measure the rate at which an unfolded protein becomes compact, when placed under native conditions, and by comparison with the rates of secondary and tertiary structure formation, to develop a more complete picture of the folding process. Protein folding is currently believed to be a process in which the unfolded protein initially collapses rapidly to a species with unstable secondary structure, followed by transformation into intermediates with stable secondary structure and ultimately the tightly-packed native state. The specific questions we plan to answer are: How fast does the unfolded protein initially become compact, and how does the compactness change as folding progresses from the initial intermediates to the native state? We will measure the rate at which an unfolded protein becomes compact during the refolding process using two techniques: small-angle x-ray scattering and fluorescence energy transfer; these are among the few techniques available for the measurement of the size of proteins in solution. Recent technical improvements in small-angle X-ray scattering instrumentatimn have now made it feasible to carry out the proposed experiments. The proteins to be investigated initially are cytochrome c, staphylococcal nuclease and DnaK. Clearly, knowledge of the rate at which the protein collapses is very important and will help distinguish competing models for folding, and should significantly increase our understanding of the process of protein folding and thus benefit many fields involving proteins. %%% The question of now proteins fold into their native three-dimensional structures is an important one from may perspectives. This research is aimed at answering one aspect of the overall protein folding problem, namely, how fast does the protein become compact, and how does the rate of collapse compare to the other structural events occurring? We propose to use small-angle X-ray scattering and fluorescence energy transfer to mo nitor the size of a folding protein as a function of time using stopped-flow rapid mixing. Small-angle X-ray scattering provides information about the overall size and shape of the molecule; in particular, we will use mostly the radius of gyration to monitor the size changes. The fluorescence energy transfer will involve donor and acceptor groups in distant regions of the protein sequence, so that in the unfolded sate they are far apart and little energy transfer occurs, whereas as the molecule becomes more compact during folding, they become closer together and the fluorescence energy transfer efficiency increases. The proteins to be investigated initially are cytochrome c, staphylococcal nuclease and DnaK. ***

9507280 Fink本研究的目的是测量未折叠蛋白质在天然条件下变得紧密的速率，并与二级和三级结构形成的速率进行比较，以更完整地了解折叠过程。 目前认为蛋白质折叠是这样一个过程，其中未折叠的蛋白质最初迅速折叠成具有不稳定二级结构的物质，随后转化成具有稳定二级结构的中间体，并最终形成紧密堆积的天然状态。 我们计划回答的具体问题是：未折叠的蛋白质最初变得紧凑的速度有多快，以及当折叠从最初的中间体发展到天然状态时，紧凑性如何变化？ 我们将使用两种技术测量未折叠蛋白质在重折叠过程中变得紧凑的速率：小角度X射线散射和荧光能量转移;这些是少数几种可用于测量溶液中蛋白质大小的技术。 最近在小角X射线散射仪器方面的技术改进现在已经使进行所提出的实验变得可行。 最初要研究的蛋白质是细胞色素c、葡萄球菌核酸酶和DnaK。 很明显，了解蛋白质折叠的速率是非常重要的，这将有助于区分折叠的竞争模型，并将大大增加我们对蛋白质折叠过程的理解，从而使涉及蛋白质的许多领域受益。 从许多方面来看，蛋白质折叠成它们天然的三维结构是一个重要的问题。 这项研究旨在回答整个蛋白质折叠问题的一个方面，即蛋白质变得紧凑的速度有多快，以及与其他结构事件相比，折叠的速度如何？ 我们建议使用小角X射线散射和荧光能量转移来监测折叠蛋白质的大小作为时间的函数，使用停流快速混合。 小角X射线散射提供了关于分子的整体大小和形状的信息;特别是，我们将主要使用回转半径来监测尺寸变化。 荧光能量转移将涉及蛋白质序列的远距离区域中的供体和受体基团，使得在未折叠状态下它们远离并且几乎不发生能量转移，而随着分子在折叠期间变得更紧凑，它们变得更靠近在一起并且荧光能量转移效率增加。 最初要研究的蛋白质是细胞色素c、葡萄球菌核酸酶和DnaK。 ***