Mechanical chaperonin : Mechanical folding of proteins at the single molecular level

本研究的目的是利用原子力显微镜（AFM）将一个蛋白质分子吸附在晶体基底和AFM针尖之间，然后进行延伸。我们选择碳酸脱氢酶作为模型酶，并将其衍生化，使其在N-和C-末端具有半胱氨酸残基。使用AFM延伸蛋白质揭示蛋白质被拉至约20 nm，并且进一步施加延伸力突然破坏蛋白质的3D结构。在3D结构断裂后，蛋白质表现为随机卷曲的链。该过程可被视为蛋白质分子的机械变性。尖端和基底之间的距离的收缩使蛋白质松弛以恢复某些结构元件，这些结构元件可以是或可以不是天然3D结构的一部分。该实验中的时间允许量为100 ms，我们将时间延长至10 s，以便观察到更多的结构形成。当从两端拉出时不会形成“结”结构的突变蛋白质揭示结的形成确实是表征野生型酶的机械行为的因素。我们还对在较低pH下形成α-螺旋的合成多肽进行了实验。

The purpose of the study is to use the atomic force microscope (AFM) to extend a single protein molecule after sandwiching it between the crystalline substrate and the tip of the AFM. We chose carbonic dehydratase as a model enzyme and derivatized it so that it had cysteine residues at its N- and C-termini. Extending the protein using the AFM revealed that the protein was pulled to about 20 nm and further application of the extending force abruptly broke the 3D structure of the protein. After the rupture of the 3D structure, the protein behaved as a randomly coiled chain. The process may be regarded as a mechanical denaturation of a protein molecule. Retraction of the distance between the tip and substrate relaxed the protein to resume certain structural elements which may or may not be parts of the native 3D structure. The time allowance in this experiment was in the order of 100 ms and we are extending the time to 10 s so that more structure formation should be observed. A mutant protein that would not form the "knot" structure when pulled from the two ends revealed that the knot formation was indeed a factor to characterize the mechanical behavior of the wild type enzyme. We also conducted experiments on a synthetic polypeptide that forms alpha-helix at a lower pH.