Stepwise rotation of the γ-subunit of EFoF1-ATP synthase during ATP synthesis:: a single-molecule FRET approach

被引:35
作者
Börsch, M [1 ]
Diez, M [1 ]
Zimmermann, B [1 ]
Trost, M [1 ]
Steigmiller, S [1 ]
Gräber, P [1 ]
机构
[1] Univ Stuttgart, Inst Phys 3, D-70569 Stuttgart, Germany
来源
MANIPULATION AND ANALYSIS OF BIOMOLECULES, CELLS AND TISSUES | 2003年 / 4962卷
关键词
subunit rotation; ATP synthase; single-molecule spectroscopy; FRET; fluorescence labelling;
D O I
10.1117/12.479554
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
FoF1-ATP synthases couple proton translocation with the synthesis of ATP using two rotary motors within the enzyme. To monitor inter-subunit movements during catalysis, we selectively attached two fluorophores to the F-1 part, sulforhodamine B at one of three beta-subunits and Cy5 at the gamma-subunit. Reassembly with F-o parts embedded in liposomes yielded functional holoenzymes. Fluorescence resonance energy transfer (FRET) was investigated in photon bursts of freely diffusing liposomes with reconstituted ATP synthases using a confocal set-up for single-molecule detection. Incubation with AMPPNP resulted in stable intensity ratios within a burst and three different FRET efficiencies. Upon ATP addition, a repeating sequence of three distinct FRET efficiencies was observed, indicating the stepwise movement of the gamma-subunit during ATP hydrolysis. With this single-molecule FRET approach we detected a stepwise rotation of the gamma-subunit under conditions for ATP synthesis (i.e. energization of the proteoliposomes by an acid-base-transition). The direction of rotation is opposite to the direction observed during ATP hydrolysis.
引用
收藏
页码:11 / 21
页数:11
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