title: How to orient the functional GroEL-SR1 mutant for atomic force microscopy investigations.
abstract: 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.
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Schiener
authors.forename: Susanne
authors.initials: S
authors.lastname: Witt
authors.forename: Manajit
authors.initials: M
authors.lastname: Hayer-Hartl
authors.forename: Reinhard
authors.initials: R
authors.lastname: Guckenberger
mesh_terms: 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
chemical_list: D018834:Chaperonin 60