Conformational Substates of Calmodulin Revealed by Single-Pair Fluorescence Resonance Energy Transfer:  Influence of Solution Conditions and Oxidative Modification†

Biochemistry ◽  
2005 ◽  
Vol 44 (10) ◽  
pp. 3694-3707 ◽  
Author(s):  
Brian D. Slaughter ◽  
Jay R. Unruh ◽  
Michael W. Allen ◽  
Ramona J. Bieber Urbauer ◽  
Carey K. Johnson
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Oxidative Modification ◽  
Single Pair ◽  
Conformational Substates ◽  
Fluorescence Resonance

Quantum dots for single-pair fluorescence resonance energy transfer in membrane- integrated EFoF1

2008 ◽  
Vol 36 (5) ◽  
pp. 1017-1021 ◽  
Author(s):  
Eva Galvez ◽  
Monika Düser ◽  
Michael Börsch ◽  
Jörg Wrachtrup ◽  
Peter Gräber
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Covalent Binding ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Range ◽  
Single Pair ◽  
Subunit C ◽  
Organic Fluorophores ◽  
Fluorescence Resonance

spFRET (single-pair fluorescence resonance energy transfer) with organic fluorophores has been used to demonstrate rotation of the subunits γ and ε in membrane-integrated FoF1 during proton transport-coupled ATP synthesis. Owing to the high light intensities used in single-molecule spectroscopy, organic fluorophores show a high probability for photobleaching. Luminescent CdSe/ZnS nanocrystals with a hydrophilic shell have been covalently bound to FoF1 either to the stator subunit b or to the rotor subunit c. TIRFM (total internal reflection microscopy) shows that covalent binding of the QD (quantum dot) via cysteine to FoF1 leads to a significant decrease in the blinking probability in the microsecond-to-second time range. This effect allows the observation of subunit movements in an extended time range. If the QD is bound to the rotor subunit c, the fluorescence anisotropy shows fluctuations in the presence of ATP, in contrast with the constant anisotropy observed in the absence of ATP.

Sign in / Sign up

Export Citation Format

Share Document