How to orient the functional GroEL-SRI mutant for atomic force microscopy investigations

被引:8
作者
Schiener, J
Witt, S
Hayer-Hartl, M
Guckenberger, R
机构
[1] Max Planck Inst Biochem, Dept Mol Struct Biol, D-82152 Martinsried, Germany
[2] Max Planck Inst Biochem, Dept Cellular Biochem, D-82152 Martinsried, Germany
关键词
atomic force microscopy; GroEL; single-ring mutant; specific orientation; GroE cycle;
D O I
10.1016/j.bbrc.2005.01.005
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-chapcrone 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. (C) 2005 Elsevier Inc. All rights reserved.
引用
收藏
页码:477 / 483
页数:7
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