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Forschungsprofil Dr. Janet Vonck


  • PhD, University of Groningen, The Netherlands, 1992
  • Postdoc, University of California at Berkeley, 1992-1995
  • Staff scientist, EMBL Heidelberg, 1996
  • At the institute since 1997

Electron microscopy of protein complexes in bioenergetics

Fig. 1: A model of the purple membrane, seen from the cytoplasm. The molecule in the center, labelled N, is shown in the N state where helices E, F and G have changed conformation.
Fig. 2: The proton path in bacteriorhodopsin.

A major function of biological membranes is the conversion of energy from food or light into a proton or ion gradient, which is utilised to form ATP, the energy currency of the cell. We study proteins and protein complexes involved in this process by electron crystallography of two-dimensional crystals and by single particle electron microscopy.

Halophilic archaea can utilize light energy directly using the light-driven proton pump bacteriorhodopsin. The structure of this 7-helix transmembrane protein is known at high resolution, but the mechanism of vectorial transport and the nature of conformational changes during the photocycle have long been elusive. Photocycle intermediates can be trapped in mutants and/or at low temperatures. We have studied a late intermediate, N, in the mutant F219L and succeeded in producing a three-dimensional difference map from diffraction patterns of illuminated (N) and unilluminated (ground state) crystals. The map clearly shows tilts of several helices, opening up a channel in the cytoplasmic half of the protein that enables the proton transfer in the second half of the photocycle.

Fig. 3: 3-D map of the c-rings from Ilyobacter tartaricus with 11-fold non-crystallographic symmetry applied. The hairpins are packed back-to-front, forming two concentric rings. The helices in the inner ring are very tightly packed and thus can be assigned as the glycine-rich N-terminal helices. The helices in the outer ring are kinked near the position of the conserved glutamate residue involved in Na+-translocation (Vonck et al., 2002).
Fig. 4: projection maps of c-ring crystals from Acetobacter woodii and Bacillus sp. TA2.A1 reveal 11 and 13 subunits per ring, respectively (Fritz et al., 2008, Matthies et al., 2009).

In bacteria, mitochondria and chloroplasts, a transmembrane proton or ion gradient drives the formation of ATP by F1Fo ATP synthases. Although the x-ray structure of the soluble F1 part has provided much insight into the mechanism of ATP synthesis, not much is known about the structure of the ion-translocating, membrane-embedded Fo part and the coupling of ion translocation to ATP synthesis. Fo in bacteria has a subunit composition of ab2c10-15. The c-subunits form a rotor ring attached to the γε central stalk in F1 and ab2 is part of the stator with the b subunits forming the peripheral stalk which connects the a subunit to the F1 head. Ion flow between the a subunit and the c-ring induces rotation of the γ subunit inside the F1 head by a still not fully understood mechanism.

In collaboration with the group of Thomas Meier, we study two-dimensional crystals of c-rings. We have calculated a 3-D map at 6 Å in-plane resolution of the 11-subunit c-rings from Ilyobacter tartaricus (Fig. 2). The relatively simple structure combined with a wealth of biochemical data about the rings made it possible to fit a C-α model into the density. This model was successfully used for phasing three-dimensional crystals of the rings, which yielded the first atomic model of a c-ring at 2.4 Å resolution (Meier et al., Science 308 [2005] 659-662).

The number of c-subunits per ring, and thus the number of ATPs formed per translocated ion, varies among different organisms. In an effort to understand the structural determinants of stoichiometry, we study 2-D crystals of c-rings from different organisms (fig. 4).

Figure 5: 3D EM map of archaeal ATP synthase with fitted subunits (Vonck et al., 2009)

Archaea and some bacteria contain A-type ATP synthases, which are distinct from the F1Fo found in mitochondria, chloroplasts and bacteria. By single-particle electron microscopy, we have determined the 3D structure of the enzyme from the hyperthermophilic archaeon Pyrococcus furiosus at a resolution of 23 Å. Using data from LILBID mass spectroscopy, SDS-PAGE and crystal structures from homologous subunits, we were able to deduce a model with locations for every subunit. The model has a subunit composition A3B3CDFE2H2ac10 and a molecular mass of 738 kDa (fig. 5).

Fig. 6: 3D-EM map of a 1.7 MDa supercomplex from bovine heart mitochondria containing one copy each of complex I and IV and a complex III dimer. Interpretation of the map: An EM map of complex I (yellow) and X-ray models of complex III (red) and complex IV (green) were fitted to the map. Cytochrome c is shown in grey and the putative ubiquinone binding sites are indicated as a grey box. The position of the membrane is indicated in blue. A proposed electron path within the supercomplex is indicated by arrows (Schäfer et al., 2007).

In mitochondria and bacteria, a proton gradient is created by the complexes of the electron transport chain. Increasing evidence in different organisms shows that the protein complexes do not float freely in the membrane, but form supercomplexes of different composition. Single particle electron microscopy (EM) is an ideal technique to study these very large protein assemblies. Eva Schäfer has determined the first 3D map of a respiratory chain supercomplex and interpreted the map by fitting x-ray and EM structures of the individual complexes (Fig. 6).

 

Figure 7. Cryo-EM map of yeast FAS at 6 Å resolution with fitted structure. Enzymatic domains are shown in different colors and connecting structural domains in light green (Gipson et al., 2010).

The fatty acid portions of the phospholipids and glycerides in biological membranes are synthesized by the enzyme system fatty acid synthase (FAS) from acetyl-coenzyme A (CoA) and malonyl CoA. In yeast and mammals, all enzymes involved in fatty acid synthesis are organised in a large multienzyme complex. Preeti Gipson has determined a cryo-EM map of the 2.6 MDa FAS complex from yeast at ~6 Å resolution (fig. 7). At this resolution, structural elements like α-helices are easily recognizable. The EM map shows significant differences to crystal structures of the same complex. Unlike the crystal structures the EM map reveals several positions for the mobile acyl carrier protein inside the barrel, which shuttles the substrate between the enzymatic sites.


Collaborations

Ph.D. theses

Preeti Kumari: High-resolution cryo-electron microscopy study of structure and dynamics of yeast fatty acid synthase by single particle analysis. Frankfurt am Main, 2010.

Eva Schäfer: Biochemische und strukturelle Untersuchungen von Superkomplexen der Atmungskette und von F0F1-ATP-Synthasen. Darmstadt, 2006.

Tassilo Krug von Nidda: Zweidimensionale Kristallisation und elektronenkristallographische Strukturbestimmung von Membranproteinen der Energieumwandlung. Frankfurt am Main, 2002.

Publications

Burkhardt, J., Vonck, J., Langer, J.D., Salzer, R., and Averhoff, B.: An unusual ααβαββα-fold of PilQ from T. thermophilus mediates ring formation and is essential for piliation.
J. Biol. Chem., in press; doi: 10.1074/jbc.M111.334912

Hizlan, D., Robson, A., Whitehouse, S., Gold, V.A., Vonck, J., Mills, D., Kühlbrandt, W., Collinson, I.: Structure of the SecY complex unlocked by a preprotein mimic.
Cell Reports 1, 21-28 (2012).

Matthies, D., Haberstock, S., Joos, F., Dötsch, V., Vonck, J., Bernhard, F., and Meier, T.: Cell-free expression and assembly of ATP synthase.
J. Mol. Biol. 413 593-603 (2011).

Burkhardt, J., Vonck, J., and Averhoff, B.: Structure and function of PilQ, a secretin of the DNA transporter from the thermophilic bacterium Thermus thermophilus HB27.
J. Biol. Chem. 286 (12) 9977-9984 (2011).

Goswami, P., Paulino, C., Hizlan, D., Vonck, J., Yildiz, Ö., and Kühlbrandt, W.: Structure of the archaeal Na+/H+ antiporter NhaP1 and functional role of transmembrane helix 1.
EMBO J. 30 439-449 (2011).

Kumar, J., Sreeramulu, S., Schmidt, T.L., Richter, C., Vonck, J., Heckel, A., Glaubitz, C., and Schwalbe, H.: Prion protein amyloid formation involves structural rearrangements in the C-terminal domain.
ChemBioChem 11 1208-1213 (2010).

Gipson, P., Mills, D.J., Wouts, R., Grininger, M., Vonck, J., and Kühlbrandt, W.: Direct structural insight into the substrate shuttling mechanism of yeast fatty acid synthase by electron cryo-microscopy.
Proc. Natl. Acad. Sci. USA 107 (20) 9164-9169 (2010). doi: 10.1073/pnas.0913547107

Rhinow, D., Vonck, J., Schranz, M., Beyer, A., Gölzhäuser, A., and Hampp, N.: Ultrathin carbon nanosheets as support films for structural analysis of biological specimens.
Phys. Chem. Chem. Phys. 12 4345-4350 (2010). doi: 10.1039/b923756a

Müller, V., Pisa, K.Y., and Vonck, J.: ATP-Synthase der Archäen - Neue Einblicke in die Struktur und Funktion eines Energiewandlers.
Biospektrum 16 (1) 19-21 (2010).

Matthies, D., Preiss, L., Klyszejko, A.L., Muller, D.J., Cook, G.M., Vonck, J., and Meier, T.: The c13 ring from a thermoalkaliphilic ATP synthase reveals an extended diameter due to a special structural region.
J. Mol. Biol. 388 (3) 611-618 (2009).

Seelert, H., Dani, D.N., Dante, S., Hauss, T., Krause, F., Schäfer, E., Frenzel, M., Poetsch, A., Rexroth, S., Schwassmann, H.J., Suhai, T., Vonck, J., and Dencher N.A.: From protons to OXPHOS supercomplexes and Alzheimer’s disease: structure-dynamics-function relationships of energy-transducing membranes.
Biochim. Biophys. Acta - Bioenergetics 1787 657-671 (2009).

Vonck, J., Pisa, K.Y., Morgner, N., Brutschy, B., and Müller, V.: Three-dimensional structure of A1Ao ATP synthase from the hyperthermophilic archaeon Pyrococcus furiosus by electron microscopy.
J. Biol. Chem. 284 (15) 10110-10119 (2009).

Vonck, J. and Schäfer, E.: Supramolecular organization of protein complexes in the mitochondrial inner membrane.
Biochim. Biophys. Acta - Molecular Cell Research 1793 117-124 (2009).

Bron, P. and Vonck, J.: Two-Dimensional Crystals.
in: Handbook of Cryo-Preparation Methods for Electron Microscopy, (Methods in Visualization Series), eds. Cavalier, A., Spehner, D., and Humbel, B. M.; CRC Press, Boca Raton (USA), Part II Chapt. 8 pp. 191-218 (2008).

Fritz, M., Klyszejko, A.L., Morgner, N., Vonck, J., Brutschy, B., Muller, D.J., Meier, T., and Müller, V.: An intermediate step in the evolution of ATPases - a hybrid F0-V0 rotor in a bacterial Na+ F1F0 ATP synthase.
FEBS J. 275 1999-2007 (2008).

Johansson, P., Wiltschi, B., Kumari, P., Kessler, B., Vonrhein, C., Vonck, J., Oesterhelt, D., and Grininger, M.: Inhibition of the fungal fatty acid synthase type I multienzyme complex.
Proc. Natl. Acad. Sci. USA 105 (35) 12803-12808 (2008).

Schäfer, E., Dencher, N. A., Vonck, J., and Parcej, D.N.: Three-dimensional structure of the respiratory chain supercomplex I1III2IV1 from bovine heart mitochondria.
Biochem. 46 12579 -12585 (2007).

Shastri, S., Vonck, J., Haase, W., Pfleger, N., Kühlbrandt, W.,  and Glaubitz, C.: Proteorhodopsin: characterisation of 2D crystals by electron microscopy and solid state NMR.
Biochim. Biophys. Acta – Biomembranes 1768 3012-3019 (2007).

Stocker, A., Keis, S., Vonck, J., Cook, G.M., and Dimroth, P.: The structural basis for unidirectional rotation of thermoalkaliphilic F1-ATPase.
Structure 15 904-914 (2007).

Meier, T., Ferguson, S. A., Cook, G. M., Dimroth, P., and Vonck, J.: Structural investigations of the membrane-embedded rotor ring of F-ATPase from Clostridium paradoxum.
J. Bacteriol. 188 7759-7764 (2006).

Schäfer, E., Seelert, H., Reifschneider, N.H., Krause, F., Dencher, N.A., and Vonck, J.: Architecture of active mammalian respiratory chain supercomplexes.
J. Biol. Chem. 281 15370-15375 (2006).

Pogoryelov, D., Yu, J., Meier, T., Vonck, J., Dimroth, P. and Müller, D.J.: The c15 ring of the Spirulina platensis F-ATP synthase: F1/F0 symmetry mismatch is not obligatory.
EMBO Rep. 6, 1045-1051 (2005).

Meier, T., Matthey, U., von Ballmoos, C., Vonck, J., Krug von Nidda, T., Kühlbrandt, W., and Dimroth, P.: Evidence for structural integrity in the undecameric c-rings isolated from sodium ATP synthases.
J. Mol. Biol. 325 389-397 (2003).

Vonck, J., Krug von Nidda, T., Meier, T., Matthey, U., Mills, D.J., Kühlbrandt, W., and Dimroth, P.: Molecular architecture of the undecameric rotor of a bacterial Na+-ATP synthase.
J. Mol. Biol. 321 307-316 (2002).

Behlau, M., Mills, D.J., Kühlbrandt, W., Quader, H. and Vonck, J.: Projection structure of the monomeric porin OmpG at 6 Å resolution.
J. Mol. Biol. 305 71-77 (2001).

Vonck, J.: Parameters affecting specimen flatness of two-dimensional crystals for electron crystallography.
Ultramicroscopy 85 123-129 (2000).

Vonck, J.: Structure of the bacteriorhodopsin mutant F219L N-intermediate revealed by electron crystallography.
EMBO J. 19 2152-2160 (2000).

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Kontaktinformationen:

Max-Planck-Institut für Biophysik

Dr. Janet Vonck
Department of Structural Biology

Tel.: +49 (0) 69 6303-3004
Fax:     +49 (0) 69 6303-3002

E-Mail:janet.vonck(at)biophys.mpg.de

Gruppenmitglieder:

  • Dr. Janet Vonck

PhD students:

  • Matteo Allegretti
  • Luciano Ciccarelli

Technical assistant:

  • Friederike Joos