Subunit movement in individual H+-ATP synthases during ATP synthesis and hydrolysis revealed by fluorescence resonance energy transfer

2005 ◽  
Vol 33 (4) ◽  
pp. 878-882 ◽  
Author(s):  
M. Börsch ◽  
P. Gräber
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Single Molecule ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Atp Synthesis ◽  
Fluorescence Resonance ◽  
Atp Synthases

F-type H+-ATP synthases synthesize ATP from ADP and phosphate using the energy supplied by a transmembrane electrochemical potential difference of protons. Rotary subunit movements within the enzyme drive catalysis in either an ATP hydrolysis or an ATP synthesis direction respectively. To monitor these subunit movements and associated conformational changes in real time and with subnanometre resolution, a single-molecule FRET (fluorescence resonance energy transfer) approach has been developed using the double-labelled H+-ATP synthase from Escherichia coli. After reconstitution into a liposome, this enzyme was able to catalyse ATP synthesis when the membrane was energized.

Single-molecule fluorescence resonance energy transfer techniques on rotary ATP synthases

2011 ◽  
Vol 392 (1-2) ◽  
Author(s):  
Michael Börsch
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Molecule Fret ◽  
Intensity Changes ◽  
Fluorescence Resonance

Abstract Conformational changes of proteins can be monitored in real time by fluorescence resonance energy transfer (FRET). Two different fluorophores have to be attached to those protein domains which move during function. Distance fluctuations between the fluorophores are measured by relative fluorescence intensity changes or fluorescence lifetime changes. The rotary mechanics of the two motors of FoF1-ATP synthase have been studied in vitro by single-molecule FRET. The results are summarized and perspectives for other transport ATPases are discussed.

Structural rearrangements of the motor protein prestin revealed by fluorescence resonance energy transfer

2009 ◽  
Vol 297 (2) ◽  
pp. C290-C298 ◽  
Author(s):  
Kristin Rule Gleitsman ◽  
Michihiro Tateyama ◽  
Yoshihiro Kubo
Keyword(s):  
Energy Transfer ◽  
Membrane Potential ◽  
Fluorescence Resonance Energy Transfer ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Motor Protein ◽  
Resonance Energy ◽  
Outer Hair Cells ◽  
Nonlinear Capacitance ◽  
Fluorescence Resonance

Prestin is a membrane protein expressed in the outer hair cells (OHCs) in the cochlea that is essential for hearing. This unique motor protein transduces a change in membrane potential into a considerable mechanical force, which leads to a cell length change in the OHC. The nonlinear capacitance in cells expressing prestin is recognized to reflect the voltage-dependent conformational change of prestin, of which its precise nature remains unknown. In the present work, we aimed to detect the conformational changes of prestin by a fluorescence resonance energy transfer (FRET)-based technique. We heterologously expressed prestin labeled with fluorophores at the COOH- or NH2-terminus in human embryonic kidney-293T cells, and monitored FRET changes on depolarization-inducing high KCl application. We detected a significant decrease in intersubunit FRET both between the COOH-termini and between the COOH- and NH2-termini. A similar FRET decrease was observed when membrane potential was directly and precisely controlled by simultaneous patch clamp. Changes in FRET were suppressed by either of two treatments known to abolish nonlinear capacitance, V499G/Y501H mutation and sodium salicylate. Our results are consistent with significant movements in the COOH-terminal domain of prestin upon change in membrane potential, providing the first dynamic information on its molecular rearrangements.

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