How to orient the functional GroEL-SR1 mutant for atomic force microscopy investigations.
We present high-resolution atomic force microscopy (AFM) imaging of the single-ring mutant of the chaperonin GroEL (SR-EL) from Escherichia coli in buffer solution. The native GroEL is generally unsuitable for AFM scanning as it is easily being bisected by forces exerted by the AFM tip. The single-ring mutant of GroEL with its simplified composition, but unaltered capability of binding substrates and the co-chaperone GroES, is a more suited system for AFM studies. We worked out a scheme to systematically investigate both the apical and the equatorial faces of SR-EL, as it binds in a preferred orientation to hydrophilic mica and hydrophobic highly ordered pyrolytic graphite. High-resolution topographical imaging and the interaction of the co-chaperone GroES were used to assign the orientations of SR-EL in comparison with the physically bisected GroEL. The usage of SR-EL facilitates single molecule studies on the folding cycle of the GroE system using AFM.
Schiener
Jens
J
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Witt
Susanne
S
Hayer-Hartl
Manajit
M
Guckenberger
Reinhard
R
D000327:Adsorption; D018834:Chaperonin 60 / Q000032:analysis / Q000737:chemistry* / Q000648:ultrastructure*; D008846:Micromanipulation / Q000379:methods*; D018625:Microscopy, Atomic Force / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D013329:Structure-Activity Relationship
D018834:Chaperonin 60