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理，本研究已经通过两个目标进行。（1）为了监测在微秒至毫秒至毫秒的时间状态下，球状蛋白重折叠的动力学，我们开发了高压焦耳加热温度悬挂装置，测试了该设备并提高了其功能。 (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 abuse and fluorescence spectroscopy, and the pressure achieved was 1,800 atm在25℃，-4℃时为1,200 atm。因为许多蛋白质处于-4℃和1200 atm处的冷变性态，因此可以通过温度悬而未决的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无蛋白pseudo pseudo野生型葡萄球菌核酸酶的重新折叠反应，将其用于模型 - 蛋白酶蛋白酶蛋白酶蛋白酶蛋白酶蛋白酶蛋白J. pottect te Protine just protjy protjust the the protjy thejuct the protjy the protjy the protjy the pot the protjust art the potsect。结果，尽管该蛋白质没有脯氨酸残留物，但已经发现该蛋白从变性状态重新散发了从变性状态的多个平行反应途径。发现是第一种情况，即清楚地显示了蛋白质折叠中多个平行途径的存在。（3）在实验上已经知道，山羊α-乳脂蛋白的重组形式比其正宗形式快100倍。在这里，我们通过通过分子动力学进行模拟来重现实验结果。由于重组蛋白的N末端存在额外的甲二居住地，因此已发现N末端附近的结构即使在室温下也显示出明显的波动。