Mou J, Sheng S, Ho R, Shao Z
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville 22908, USA.
Biophys J. 1996 Oct;71(4):2213-21. doi: 10.1016/S0006-3495(96)79422-5.
The Escherichia coli chaperonins, GroEL and GroES, as well as their complexes in the presence of a nonhydrolyzable nucleotide AMP-PNP, have been imaged with the atomic force microscope (AFM). We demonstrate that both GroEL and GroES that have been adsorbed to a mica surface can be resolved directly by the AFM in aqueous solution at room temperature. However, with glutaraldehyde fixation of already adsorbed molecules, the resolution of both GroEL and GroES was further improved, as all seven subunits were well resolved without any image processing. We also found that chemical fixation was necessary for the contact mode AFM to image GroEL/ES complexes, and in the AFM images. GroEL with GroES bound can be clearly distinguished from those without. The GroEL/ES complex was about 5 nm higher than GroEL alone, indicating a 2 nm upward movement of the apical domains of GroEL. Using a slightly larger probe force, unfixed GroEL could be dissected: the upper heptamer was removed to expose the contact surface of the two heptamers. These results clearly demonstrate the usefulness of cross-linking agents for the determination of molecular structures with the AFM. They also pave the way for using the AFM to study the structural basis for the function of GroE system and other molecular chaperones.
利用原子力显微镜（AFM）对大肠杆菌伴侣蛋白GroEL和GroES及其在存在不可水解核苷酸AMP-PNP时形成的复合物进行了成像。我们证明，吸附在云母表面的GroEL和GroES在室温下的水溶液中均可直接通过AFM分辨出来。然而，对已吸附分子进行戊二醛固定后，GroEL和GroES的分辨率进一步提高，因为所有七个亚基在未经任何图像处理的情况下都能清晰分辨。我们还发现，化学固定对于接触模式AFM成像GroEL/ES复合物是必要的，并且在AFM图像中，结合了GroES的GroEL与未结合的GroEL能够清晰区分。GroEL/ES复合物比单独的GroEL高约5 nm，这表明GroEL顶端结构域向上移动了2 nm。使用稍大的探针力，可以将未固定的GroEL剖析开：上层七聚体被移除，从而暴露出两个七聚体的接触面。这些结果清楚地证明了交联剂在利用AFM确定分子结构方面的有用性。它们也为利用AFM研究GroE系统及其他分子伴侣功能的结构基础铺平了道路。
