Atomic force microscopy detects changes in the interaction forces between GroEL and substrate proteins.
The structure of the Escherichia coli chaperonin GroEL has been investigated by tapping-mode atomic force microscopy (AFM) under liquid. High-resolution images can be obtained, which show the up-right position of GroEL adsorbed on mica with the substrate-binding site on top. Because of this orientation, the interaction between GroEL and two substrate proteins, citrate synthase from Saccharomyces cerevisiae with a destabilizing Gly-->Ala mutation and RTEM beta-lactamase from Escherichia coli with two Cys-->Ala mutations, could be studied by force spectroscopy under different conditions. The results show that the interaction force decreases in the presence of ATP (but not of ATPgammaS) and that the force is smaller for native-like proteins than for the fully denatured ones. It also demonstrates that the interaction energy with GroEL increases with increasing molecular weight. By measuring the interaction force changes between the chaperonin and the two different substrate proteins, we could specifically detect GroEL conformational changes upon nucleotide binding.
Vinckier
A
A
Department of Biochemistry, Swiss Federal Institute of Technology, ETH Zentrum, Zürich. vinckier@anatomie.unizh.ch
Gervasoni
P
P
Zaugg
F
F
Ziegler
U
U
Lindner
P
P
Groscurth
P
P
Plückthun
A
A
Semenza
G
G
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000409:Alanine; D019943:Amino Acid Substitution; D018834:Chaperonin 60 / Q000378:metabolism* / Q000648:ultrastructure*; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D003545:Cysteine; D004926:Escherichia coli / Q000378:metabolism*; D018625:Microscopy, Atomic Force / Q000379:methods; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012680:Sensitivity and Specificity
D018834:Chaperonin 60; D011994:Recombinant Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D002950:Citrate (si)-Synthase; D003545:Cysteine; D000409:Alanine