Distance measurements in the F0F1-ATP synthase from E. coli using smFRET and PELDOR spectroscopy
- 91 Downloads
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 distance is in agreement with the crystal structure. Two additional distance distributions are detected with smFRET and with PELDOR. These can be assigned by comparison with the structure to labels at endogenous cysteines. One additional distribution is detected only with PELDOR. The comparison indicates that under optimal conditions smFRET and PELDOR result in the same distance distributions. PELDOR has the advantage that different distributions can be obtained with ensemble measurements, whereas FRET requires single-molecule techniques.
KeywordsSingle-molecule spectroscopy smFRET EF0F1 PELDOR DEER Distance measurements
The authors thank the Deutsche Forschungsgemeinschaft for general support and SW thanks especially for the (Grant INST 39/928-1 FUGG) for financing, together with the “Struktur- und Innovationsfonds für die Forschung Baden-Württemberg” (SIBW), pulsed EPR instrumentation that is operated within the MagRes Center of the Albert-Ludwigs-Universität Freiburg.
- Landi G, Zama F (2006) The active-set method for nonnegative regularization of linear ill-posed problems. Appl Math Comput 175:715–729Google Scholar
- Tikhonov AN (1963) Solution of incorrectly formulated problems and the regularization method. Transl Sov Math 4:1035–1038 (151, 501-504) Google Scholar
- van der Meer BW, Coker GI, Chen S (1994) S-Y, resonance energy transfer: theory and data. VCH, New YorkGoogle Scholar