Structural Variability of Nucleosomes Detected by Single-Pair Förster Resonance Energy Transfer: Histone Acetylation, Sequence Variation, and Salt Effects†

2009 ◽  
Vol 113 (9) ◽  
pp. 2604-2613 ◽  
Author(s):  
Alex Gansen ◽  
Katalin Tóth ◽  
Nathalie Schwarz ◽  
Jörg Langowski
Keyword(s):  
Energy Transfer ◽  
Histone Acetylation ◽  
Sequence Variation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Pair ◽  
Salt Effects ◽  
Structural Variability

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.

EXPLORING THE FOLDING FREE ENERGY LANDSCAPE OF SMALL RNA MOLECULES BY SINGLE-PAIR FÖRSTER RESONANCE ENERGY TRANSFER

2008 ◽  
Vol 03 (04) ◽  
pp. 439-457 ◽  
Author(s):  
ANDREI YU. KOBITSKI ◽  
ALEXANDER NIERTH ◽  
MARTIN HENGESBACH ◽  
ANDRES JÄSCHKE ◽  
MARK HELM ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Small Rnas ◽  
Energy Landscape ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Single Pair ◽  
Rna Molecules ◽  
Förster Resonance

Proteins and RNA are biological macromolecules built from linear polymers. The process by which they fold into compact, well-defined, three-dimensional architectures to perform their functional tasks is still not well understood. It can be visualized by Brownian motion of an ensemble of molecules through a rugged energy landscape in search of an energy minimum corresponding to the native state. To explore the conformational energy landscape of small RNAs, single pair Förster resonance energy transfer (spFRET) experiments on solutions as well as on surface-immobilized samples have provided new insights. In this review, we focus on our recent work on two FRET-labeled small RNAs, the Diels-Alderase (DAse) ribozyme and the human mitochondrial tRNA Lys . For both RNAs, three different conformational states can be distinguished, and the associated mean FRET efficiencies provide clues about their structural properties. The systematic variation of their free energies with the concentration of Mg 2+ counterions was analyzed quantitatively by using a thermodynamic model that separates conformational changes from Mg 2+ binding. Furthermore, time-resolved spFRET studies on immobilized DAse reveal slow interconversions between intermediate and folded states on the time scale of ~ 100 ms. The quantitative data obtained from spFRET experiments may likely assist in the further development of theories and models addressing the folding dynamics and (counterion-dependent) energetics of RNA molecules.

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