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 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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abrahams JP, Leslie AGW, Lutter R, Walker JE (1994) Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria. Nature 370:621–628
Aggeler R, Capaldi RA (1992) Cross-linking of the gamma subunit of the Escherichia coli ATPase (ECF1) via cysteines introduced by site-directed mutagenesis. J Biol Chem 267:21355–21359
Bienert R, Zimmermann B, Rombach-Riegraf V, Gräber P (2011) Time-dependent FRET with single enzymes: domain motions and catalysis in H+-ATP synthases. ChemPhysChem 12:510–517
Bordignon E, Steinhoff HJ (2007) Membrane Protein Structure and Dynamics Studied by Site-Directed Spin-Labeling ESR. In: Hemminga MA, Berliner LJ (eds) ESR spectroscopy in membrane biophysics, vol 217. Kluwer Academic/Plenum Publ, New York, pp 129–164
Börsch M, Diez M, Zimmermann B, Reuter R, Gräber P (2002) Stepwise rotation of the γ-subunit of EF0F1-ATP synthase observed by intramolecular single-molecule fluorescence resonance energy transfer. FEBS Lett 527:147–152
Boyer PD (1993) The binding change mechanism for ATP synthase—some probabilities and possibilities. Biochim Biophys Acta 1140:215–250
Chiang Y-W, Borbat PP, Freed JH (2005) The determination of pair distance distributions by pulsed ESR using Tikhonov regularization. J Magn Reson 172:279–295
Cingolani G, Duncan TM (2011) Structure of the ATP synthase catalytic complex (F1) from Escherichia coli in an autoinhibited conformation. Nat Struct Mol Biol 18:701–707
Diez M, Zimmermann B, Börsch M, König M, Schweinberger E, Steigmiller S, Reuter R, Felekyan S, Kudryavtsev V, Seidel CAM, Gräber P (2004) Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase. Nat Struct Mol Biol 11:135–141
Feniouk BA, Suzuki T, Yoshida M (2006) The role of subunit epsilon in the catalysis and regulation of FOF1-ATP synthase. Biochim Biophys Acta 1757:326–338
Fischer S, Gräber P (1999) Comparison of ΔpH- and Δφ-Driven ATP Synthesis Catalyzed by the H+-ATPases from Escherichia coli or chloroplasts reconstituted into Liposomes. FEBS Lett 457:327–332
Förster T (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann Phys Berl 437:55–75
Grohmann D, Klose D, Klare JP, Kay CWM, Steinhoff H-J, Werner F (2010) RNA-binding to archaeal RNA polymerase subunits F/E: a DEER and FRET study. J Am Chem Soc 132:5954–5955
Ha T, Enderle T, Ogletree DF, Chemla DS, Selvin PR, Weiss S (1996) Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc Natl Acad Sci USA 93:6264–6268
Hellenkamp B et al (2017) Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study. Nat Methods 14:3597–3619
Henzler-Wildman K, Kern D (2007) Dynamic personalities of proteins. Nature 450:964–972
Jeschke G (2012) DEER distance measurements on proteins. Annu Rev Phys Chem 63:419–446
Jeschke G (2016) Dipolar spectroscopy1double-resonance methods. eMagRes 5:1459–1475
Jeschke G, Chechik V, Ionita P, Godt A, Zimmermann H, Banham J, Timmel CR, Hilger D, Jung H (2006) DeerAnalysis2006—a comprehensive software package for analyzing PELDOR data. Appl Magn Reson 30:473–498
Klare JP, Steinhoff HJ (2009) Spin labeling EPR. Photosynth Res 102:377–390
Klose D, Klare JP, Grohmann D, Kay CWM, Werner F, Steinhoff H-J (2012) Simulation vs. reality: a comparison of in silico distance predictions with DEER and FRET measurements. PLoS One 7:e39492
Landi G, Zama F (2006) The active-set method for nonnegative regularization of linear ill-posed problems. Appl Math Comput 175:715–729
McInnes EJL, Collison D (2016) EPR interactions1coupled spins. eMagRes 5:1445–1458
Moerner WE, Fromm DP (2003) Methods of single-molecule fluorescence spectroscopy and microscopy. Rev Sci Instrum 74:3597–3619
Noji H, Yasuda R, Yoshida M, Kinosita K (1997) Direct observation of the rotation of F1-ATPase. Nature 386:299–302
Pannier M, Veit S, Godt A, Jeschke G, Spiess HW (2000) Dead-time free measurement of dipole-dipole interactions between electron spins. J Magn Reson 142:331–340
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF chimera-A visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612
Phillips DL (1962) A technique for the numerical solution of certain integral equations of the first kind. J Assoc Comput Mach 9:84–96
Rein S, Lewe P, Andrade SL, Kacprzak S, Weber S (2018) Global analysis of complex PELDOR time traces. J Magn Reson 295:17–26
Rodgers AJW, Wilce MCJ (2000) Structure of the γ–ε complex of ATP synthase. Nat Struct Mol Biol 7:1051–1054
Schiemann O, Prisner TF (2007) Long-range distance determinations in biomacromolecules by epr spectroscopy. Q Rev Biophys 40:1–53
Schmidt T, Wälti MA, Baber JL, Hustedt EJ, Clore GM (2016) Long distance measurements up to 160 Å in the GroEL tetradecamer using Q-band DEER EPR spectroscopy. Angew Chem Ind Ed 55:15905–15909
Sielaff H, Duncan TM, Börsch M (2018) The regulatory subunit ε in Escherichia coli FOF1-ATP synthase. Biochim Biophys Acta 1859:775–788
Sobti M, Smits C, Wong AS, Ishmukhametov R, Stock D, Sandin S, Stewart AG (2016) Cryo-EM structures of the autoinhibited E. coli ATP synthase in three rotational states. eLife 5:e21598
Sobti M, Ishmukhametov R, Bouwer JC, Ayer A, Suarna C, Smith NJ, Stocker R, Duncan TM, Stewart AG (2019) Cryo-EM reveals distinct conformations of E. coli ATP synthase on exposure to ATP. eLife 8:e43864
Steigmiller S, Börsch M, Gräber P, Huber M (2005) Distances between the b-subunits in the tether domain of F0F1-ATP synthase from E. coli. Biochim Biophys Acta 1708:143–153
Stryer L, Haugland RP (1967) Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci USA 58:719–726
Tikhonov AN (1963) Solution of incorrectly formulated problems and the regularization method. Transl Sov Math 4:1035–1038 (151, 501-504)
Uhlin U, Cox GB, Guss JM (1997) Crystal structure of the ε subunit of the proton-translocating ATP synthase from Escherichia coli. Structure 5:1219–1230
Vámosi G, Gohlke C, Clegg RM (1996) Fluorescence characteristics of 5-Carboxytetramethylrhodamine linked covalently to the 5′ end of oligonucleotides: multiple conformers of single-stranded and double-stranded Dye-DNA complexes. Biophys J 71:972–994
van der Meer BW, Coker GI, Chen S (1994) S-Y, resonance energy transfer: theory and data. VCH, New York
Zarrabi N, Zimmermann B, Diez M, Gräber P, Wrachtrup J, Börsch M (2005) Asymmetry of rotational catalysis of single membrane-bound F0F1-ATP synthase. Proc SPIE 5699:175–188
Zimmermann B, Diez M, Zarrabi N, Gräber P, Börsch M (2005) Movements of the ɛ-subunit during catalysis and activation in single membrane-bound H+-ATP synthase. EMBO J 24:2053–2063
Acknowledgements
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.
Author information
Authors and Affiliations
Corresponding author
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Burger, M., Rein, S., Weber, S. et al. Distance measurements in the F0F1-ATP synthase from E. coli using smFRET and PELDOR spectroscopy. Eur Biophys J 49, 1–10 (2020). https://doi.org/10.1007/s00249-019-01408-w
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00249-019-01408-w