Chaperonins GroEL and GroES: views from atomic force microscopy.
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.
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville 22908, USA.
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Sheng
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Shao
D000266:Adenylyl Imidodiphosphate; D000818:Animals; D018835:Chaperonin 10 / Q000378:metabolism / Q000648:ultrastructure*; D018834:Chaperonin 60 / Q000378:metabolism / Q000648:ultrastructure*; D004926:Escherichia coli / Q000378:metabolism; D008274:Magnesium; D051379:Mice; D018625:Microscopy, Atomic Force / Q000379:methods; D008961:Models, Structural; D011485:Protein Binding; D011487:Protein Conformation; D012996:Solutions
D018835:Chaperonin 10; D018834:Chaperonin 60; D012996:Solutions; D000266:Adenylyl Imidodiphosphate; D008274:Magnesium