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Studies on Protein Folding by the High-Pressure Temperature-Jump Method and Computer Simulations

高压跳温法和计算机模拟研究蛋白质折叠

基本信息

批准号：
12480197
负责人：
KUWAJIMA Kunihiro
金额：
$ 9.41万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

To elucidate the folding mechanism of proteins, the present study has been carried out with two objectives. (1) For the purpose of monitoring refolding kinetics of globular proteins in a time regime of microsecond to millisecond, we have developed a high-pressure Joule heating temperature-jump apparatus, tested the apparatus and improved its capability. (2) For the purpose of describing the folding process of proteins at an atomic level, we have carried out unfolding simulations of a protein at a high temperature (400〜600 K), and compared the results with known experimental data.The following results were obtained.(1) The high-pressure temperature-jump apparatus developed in the present study allows us to monitor the reactions by both ultraviolet absorption and fluorescence spectroscopy, and the pressure achieved was 1,800 atm at 25℃ and 1,200 atm at -4℃. Because many proteins are in the cold denatured state at -4℃ and 1,200 atm, it will be possible to monitor the folding reaction of the proteins in a microsecond time regime by the temperature-jump method.(2) We have studied the refolding reaction of proline-free pseudo wild-type staphylococcal nuclease, which will be used as a model protein in the temperature-jump measurements. As a result, this protein has been found to refold along multiple parallel reaction pathways from the denatured state although the protein does not have proline residues. This finding is the first case in which the presence of multiple parallel pathways in protein folding is clearly shown.(3) It has been known experimentally that a recombinant form of goat α-lactalbumin unfolds 100-fold faster than its authentic form. Here, we have reproduced the experimental results by unfolding simulations by molecular dynamics. Because of the presence of an additional methione residue at the N terminus in the recombinant protein, the structure near the N-terminus has been found to show significantly large fluctuations even at room temperature.

为了阐明蛋白质的折叠机制，本研究有两个目的。(1)为了在微秒到毫秒的时间范围内监测球形蛋白质的复性动力学，我们研制了一种高压焦耳加热温度跃变装置，并对该装置进行了测试和改进。(2)为了在原子水平上描述蛋白质的折叠过程，我们对蛋白质在高温（400 ~ 600 K）下的去折叠进行了模拟，并将模拟结果与已知的实验数据进行了比较，得到了以下结果。(1)在本研究中开发的高压温度跳跃装置允许我们通过紫外吸收和荧光光谱来监测反应，并且在25℃和-4℃下达到的压力分别为1，800 atm和1，200 atm。由于许多蛋白质在-4℃和1，200 atm下处于冷变性状态，因此可以通过温度跳跃方法在微秒时间范围内监测蛋白质的折叠反应。(2)我们研究了无脯氨酸的假野生型葡萄球菌核酸酶的复性反应，它将被用作温度跳跃测量中的模型蛋白。结果，发现该蛋白质虽然不具有脯氨酸残基，但从变性状态沿着沿着多个反应途径再折叠。这一发现是第一个明确显示蛋白质折叠中存在多个平行途径的案例。(3)实验表明，重组形式的山羊α-乳白蛋白的展开速度比其真实形式快100倍。在这里，我们再现了实验结果，展开模拟的分子动力学。由于在重组蛋白的N末端存在另外的甲硫酮残基，已发现N末端附近的结构即使在室温下也显示出显著大的波动。