Distance measurements in the F0F1-ATP synthase from E. coli using smFRET and PELDOR spectroscopy.

Distance measurements in the F0F1-ATP synthase from E. coli using smFRET and PELDOR spectroscopy. Eur Biophys J. 2019 Nov 08;: Authors: Burger M, Rein S, Weber S, Gräber P, Kacprzak S Abstract Fluorescence resonance energy transfer in single enzyme molecules (smFRET, single-molecule measurement) allows the measurement of multicomponent distance distributions in complex biomolecules similar to pulsed electron-electron double resonance (PELDOR, ensemble measurement). Both methods use reporter groups: FRET exploits the distance dependence of the electric interaction between electronic transition dipole moments of the attached fluorophores, whereas PELDOR spectroscopy uses the distance dependence of the interaction between the magnetic dipole moments of attached spin labels. Such labels can be incorporated easily to cysteine residues in the protein. Comparison of distance distributions obtained with both methods was carried out with the H+-ATPase from Escherichia coli (EF0F1). The crystal structure of this enzyme is known. It contains endogenous cysteines, and as an internal reference two additional cysteines were introduced (EF0F1-γT106C-εH56C). These positions were chosen to allow application of both methods under optimal conditions. Both methods yield very similar multicomponent distance distributions. The dominating distance distribution (> 50%) is due to the two cysteines introduced by site-directed mutagenesis and the dista...
Source: European Biophysics Journal : EBJ - Category: Physics Authors: Tags: Eur Biophys J Source Type: research