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，本研究的目的有两个：（1）为了在微秒至毫秒的时间内监测球状蛋白质的重折叠动力学，我们开发了高压焦耳加热。 (2)为了在原子水平上描述蛋白质的折叠过程，我们进行了蛋白质在高温下的展开模拟（400-600 K），并将结果与已知的实验数据进行比较。得到以下结果：（1）本研究开发的高压跳温装置使我们能够通过紫外吸收和荧光来监测反应光谱，25℃时达到1,800 atm，-4℃时达到1,200 atm，因为许多蛋白质在-4℃和1,200℃时处于冷变性状态。 atm，将有可能通过温度跳跃方法在微秒时间内监测蛋白质的折叠反应。（2）我们研究了无脯氨酸的伪野生型葡萄球菌核酸酶的重折叠反应，该酶将用于作为温度跳跃测量中的模型蛋白质，结果发现该蛋白质从变性状态沿着多个平行反应途径重折叠，尽管该蛋白质不具有脯氨酸残基，这是第一个发现。其中清楚地显示了蛋白质折叠中多个平行途径的存在。(3) 实验表明，重组形式的山羊 α-乳清蛋白的展开速度比其真实形式快 100 倍。在这里，我们重现了实验结果。通过分子动力学展开模拟，由于重组蛋白的N末端存在额外的蛋氨酸残基，因此发现即使在室温下，N末端附近的结构也表现出显着的较大波动。温度。