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

这项研究的目的是利用原子力显微镜(AFM)将单个蛋白质分子夹在晶体底物和AFM的尖端之间，然后将其延伸。我们选择碳酸脱水酶作为模型酶，并对其进行衍生化，使其在N-端和C-端都有半胱氨酸残基。利用AFM对蛋白质进行拉伸，发现蛋白质被拉到约20 nm处，进一步施加拉伸力突然破坏了蛋白质的三维结构。在3D结构断裂后，蛋白质表现为随机卷曲的链。这个过程可以看作是蛋白质分子的机械性变性。尖端和底物之间距离的回缩使蛋白质松弛，恢复了某些结构元素，这些结构元素可能是也可能不是天然3D结构的一部分。这个实验中的时间大约是100ms，我们将时间延长到10个S，以便观察到更多的结构形成。一个突变的蛋白质，当从两端拉出时不会形成“结”结构，这表明结的形成确实是表征野生型酶机械行为的一个因素。我们还对一种在较低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.