Research Profile Prof. Dr. Werner Kühlbrandt

Prof. Dr. Werner Kühlbrandt

PhD, MRC Laboratory of Molecular Biology, Cambridge, UK, 1981
Postdoc, ETH Zurich, Switzerland and Imperial College London, UK, 1981- 1986
Heisenberg Fellow 1986-1991
Visiting scientist, Imperial College London, 1986-1987
Lawrence Berkeley Laboratory, Berkeley, CA, 1988
Group leader and Senior Scientist, EMBL Heidelberg, 1988 – 1997
Habilitation (Biophysics), Heidelberg University, 1992
Director, Department of Structural Biology since 1997
Joint director, Cluster of Excellence Frankfurt Macromolecular Complexes, 2006 - present

Studies of membrane protein structure and function

: Fig. 1: The sodium-proton antiporters NhaA and NhaP. Projection maps of the NhaP (left) and NhaA dimer (right), as determined by electron crystallography of two-dimensional membrane crystals. pH-induced conformational changes in NhaP (red asterisk) are observed when the crystals are incubated at pH 4 or pH8 on the EM grid (Vinothkumar et al, EMBO J 2005).

We investigate the structure and organization of membrane proteins by electron cryo-microscopy and crystallography to find out how they work. Most of the proteins or membranes we examine are prepared within the group by molecular biology and biochemical techniques.
Long-standing research interests include secondary transporters. Examples are the carnitine transporter CaiT, studied by Sabrina Schulze, and the pH-activated sodium-proton antiporters that balance intracellular pH and ion concentration in all living organisms. NhaA and NhaP (Figure 1) represent two related classes of antiporters that show certain structural similarities, but striking differences in terms of pH activation and transport stoichiometry, which must reflect different transport mechanisms. We are especially interested in NhaP because it is similar to the mammalian NHE sodium-proton antiporters, which are important drug targets (Vinothkumar et al, EMBO J 2005; Appel et al, J Mol Biol 2009). We have just obtained a 3D map at 7 Å resolution of the low-pH state (Goswami et al, EMBO J 2011), and are now in the process of investigating its pH-induced conformational changes. This work is carried out by Cristina Paulino, together with Özkan Yildiz.

A recent 3D map of NhaP from Methanococcus jannaschii at 7 Å resolution (Figure 2) indicates that this antiporter has 13 trans-membrane helices, whereas NhaA has 12. The overall structure of the six helices that are thought to harbour the ion translocation site is similar, but the helices at the monomer interface in the NhaP dimer look very different from NhaA.
The current antiporter team includes Dilem Hizlan, Katja Kapotova, Cristina Paulino and Özkan Yildiz (more details...).

: Fig. 2: 3D map of the sodium-proton antiporter NhaP from Methanococcus jannaschii at 7 Å resolution, determined by electron crystallography of 2D crystals, with a fitted molecular model showing 13 membrane-spanning helices, labeled 0 to XII on the left (Goswami et al, under revision).

: Fig. 3: The outer membrane porin OmpG from E. coli. [»]High-resolution X-ray structure of OmpG in the open state at neutral pH (centre left), and in the closed state at low pH (centre right). Far left: ribbon diagram showing the 14 beta strands in rainbow colours. Right: volume-rendered representation showing surface charge (red, negative; blue, positive; white, neutral), and two rings of detergent molecules (green) around the hydrophobic perimeter, indicating the position of the outer membrane (Yildiz et al, EMBO J 2006).

The bacterial outer membrane porin OmpG from E. coli is a simple, robust membrane protein that nevertheless has fascinating properties. OmpG is an unusual porin in that it is pH- gated, and we have determined its high-resolution structure both in the open state at neutral pH, and in the closed state at low pH (Figure 3; Yildiz et al, EMBO J 2006). OmpG is an excellent template for engineering switchable nanopores with tailor-made properties. This is the project of Abhishek Acharya, Stefan Köster, and Özkan Yildiz.

Another long-standing research interest in the group is the structure and molecular mechanisms of the light-harvesting chlorophyll a/b protein complex, LHC-II. After determining the structure of this most abundant of all membrane proteins, first at 3.4 Å resolution by electron crystallography (Kühlbrandt et al, Nature 1994), and then at 2.5 Å resolution by X-ray crystallography (Figure 4; Standfuss et al, EMBO J 2005), we established by microspectroscopy that the crystal structure shows the active, energy transmitting state of the complex (Barros et al, EMBO J 2009; Barros and Kühlbrandt, BBA 2009). One remaining question related to LHC-II investigated by Laura Wilk and Nora Bluhme is the mechanism of controlled annihilation of excitation energy in the photosynthetic antenna of green plants.

The structure of large membrane protein complexes and related assemblies, such as the fatty acid synthase (FAS) is investigated by single-particle electron cryo-microscopy. The structure of mitochondrial respiratory chain complexes and supercomplexes is examined by Thorsten Althoff.

: Fig. 5: 3D map of the 2.6 MDa yeast fatty acid synthase, determined at 6 Å resolution by electron cryo-microscopy of non-crystalline single particles (Gipson et al, submitted).

Preeti Gipson and Janet Vonck have recently determined a 3D map of yeast FAS at ~6 Å resolution (Gipson et al, submitted; Figure 5) that shows interesting differences to the published X-ray structures. Janet Vonck uses this technique to look into the structure of ATP synthases in collaboration with Thomas Meier and his group in the department, and of other large membrane assemblies with other groups in Frankfurt and elsewhere.

: Fig. 6b: Tomograms of cristae vesicles from rat liver (left) and beef heart mitochondria (centre and right), with the membrane shown in grey, and ATP synthase dimers in yellow (Strauss et al, EMBO J 2008).

Electron tomography is a comparatively recent venture in the group. Because of our special interest in biological membranes, we apply this technique primarily to the study of the structure, interaction and distribution of large complexes in their native membrane environment. A striking example is the ATP synthase (Complex V) in the inner membrane of mitochondria. By electron cryo-tomography of membrane preparations or entire small mitochondria we discovered that the ATP synthase forms dimer ribbons along the highly curved edges of inner membrane cristae (Figure 6; Strauss et al, EMBO J 2008). Our growing cryo-tomography team, Karen Davies, Bertram Daum and Mike Strauss, is busy investigating mitochondrial membranes from various organisms to establish, amongst other things, a link between membrane organization and aging, which may be important for understanding mitochondria-related diseases such as Parkinson's. We also investigate other membrane systems and their molecular organization in national and international collaborations.

SELECTED PUBLICATIONS 1994-2009 (citations to July 2009 in brackets)

Kühlbrandt, W., Wang, D. N. & Fujiyoshi, Y. (1994).
Atomic model of plant light-harvesting complex by electron crystallography.
Nature 367, 614-621.(1,168)

Wang, D. N., Sarabia, V. E., Reithmeier, R. A. & Kühlbrandt, W. (1994).
Three-dimensional map of the dimeric membrane domain of the human erythrocyte anion exchanger, Band 3.
EMBO J 13, 3230-3235. (108)

Rhee, K.H., Morris, E.P., Zheleva, D., Hankamer, B., Kühlbrandt, W., Barber, J. (1997).
Two-dimensional structure of plant photosystem II at 8 Å resolution.
Nature 389, 522-526.(122)

Auer, M., Scarborough, G. A. & Kühlbrandt, W. (1998).
Three-dimensional map of the plasma membrane H+ ATPase in the open conformation.
Nature 392, 840-843.(155)

Rhee, K. H., Morris, E. P., Barber, J. & Kühlbrandt, W. (1998).
Three-dimensional structure of the plant photosystem II reaction centre at 8 Å resolution.
Nature 396, 283-286.(226)

Williams, K. A., Geldmacher-Kaufer, U., Padan, E., Schuldiner, S. & Kühlbrandt, W. (1999).
Projection structure of NhaA, a secondary transporter from Escherichia coli, at 4.0 Å resolution.
EMBO J 18, 3558-3563. (78)

Williams, K. A. (2000).
Three-dimensional structure of the ion-coupled transport protein NhaA.
Nature 403, 112-115.(144)

Vonck, J. (2000).
Structure of the bacteriorhodopsin mutant F219L N intermediate revealed by electron crystallography.
EMBO J 19, 2152-2160. (72)

Behlau, M., Mills, D. J., Quader, H., Kühlbrandt, W. & Vonck, J. (2001).
Projection structure of the monomeric porin OmpG at 6 Å resolution.
J Mol Biol 305, 71-77. (17)

Collinson, I., Breyton, C., Duong, F., Tziatzios, C., Schubert, D., Or, E., Rapaport, T. & Kühlbrandt, W. (2001).
Projection structure and oligomeric properties of a bacterial core protein translocase.
EMBO J 20, 2462-2471. (71)

Model, K., Prinz, T., Ruiz, T., Radermacher, M., Krimmer, T., Kühlbrandt, W., Pfanner, N. & Meisinger, C. (2002).
Protein translocase of the outer mitochondrial membrane: role of import receptors in the structural organization of the TOM complex.
J Mol Biol 316, 657-666. (48)

Breyton, C., Haase, W., Rapoport, T.A., Kühlbrandt, W. and Collinson, I. (2002).
Three-dimensional structure of the bacterial protein-translocation complex SecYEG.
Nature 418, 662-665.(126)

Vonck, J., Krug von Nidda, T., Meier, T., Matthey, U., Mills, D. J., Kühlbrandt, W. & Dimroth, P. (2002).
Molecular architecture of the undecameric rotor of a bacterial Na+-ATP synthase.
J Mol Biol 321, 307-316. (44)

Kühlbrandt W., Zeelen, J. and Dietrich J. (2002).
Structure, mechanism and regulation of the Neurospora plasma membrane H+-ATPase.
Science 297, 1692-1696. (45)

Schleiff, E., Soll, J., Kuchler, M., Kühlbrandt, W., Harrer,R. (2003).
Characterization of the translocon of the outer envelope of chloroplasts.
J Cell Biol 160, 541-551. (77)

Rehling, P., Model, K., Brandner, K., Kovermann, P., Sickmann, A., Meyer, H.E., Kühlbrandt, W., Wagner, R., Truscott, K.N., Pfanner, N. (2003).
Protein insertion into the mitochondrial inner membrane by a twin-pore translocase.
Science 299, 1747-1751. (86)

Standfuss, J., Terwisscha van Scheltinga, A., Lamborghini, M., Kühlbrandt, W. (2005).
Mechanisms of photoprotection and non-photochemical quenching in pea LHC-II at 2.5 Å resolution.
EMBO J 24, 919-928.(19)

Vinothkumar, K.R., Smits, S.H.J., Kühlbrandt, W. (2005).
pH-induced structural change in a sodium/proton antiporter from Methanococcus jannaschii.
EMBO J 24, 2720-2729. (21)

Hiller, M., Krabben, L., Vinothkumar, K.R., Castellani, F., Van Rossum, B., Kühlbrandt, W., Oschkinat, H. (2005).
Solid-state magic-angle spinning NMR of outer-membrane protein G from Escherichia coli.
Chem Bio Chem 6, 1679-1684. (31)

Yildiz, Ö., Vinothkumar, K.R., Goswami, P., Kühlbrandt, W. (2006).
Structure of the monomeric outer-membrane porin OmpG in the open and closed conformation.
EMBO J 25, 3702-3713. (25)

Yildiz, Ö., Kalthoff, C., Raunser, S., Kühlbrandt, W. (2007).
Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake.
EMBO J 26, 589-599.(19)

Strauss, M., Hofhaus, G., Schröder, R.R., Kühlbrandt, W. (2008).
Dimer ribbons of ATP synthase shape the inner mitochondrial membrane.
EMBO J 27, 1154-1160. (28)

Model, K., Meisinger, C., Kühlbrandt, W. (2008).
Cryo-Electron Microscopy Structure of a Yeast Mitochondrial Preprotein
Translocase.
J Mol Biol 383, 1049-1057. (2)

Barros, T., Royant, A., Standfuss, J., Dreuw, A. and Kühlbrandt, W. (2009).
Crystal structure of plant light-harvesting complex shows the active, energy-transmitting state.
EMBO J 28, 298-306.(0)

Appel, M., Hizlan, D., Vinothkumar, K.R., Ziegler, C. and Kühlbrandt, W. (2009):
Conformations of NhaA, the Na/H exchanger from Escherichia coli, in the pH-activated and ion-translocating states.
J Mol Biol 386, 351-365. (2)

Köster, S., Wehner, M., Herrmann, C., Kühlbrandt, W. & Yildiz, Ö (2009):
Structure and function of the FeoB G-domain from Methanococcus jannaschii.
J Mol Biol, in press (2009).

Ph. D. THESES

Bastian Barton: Development and Application of In-Focus Phase Contrast TEM for Materials and Life Sciences.
Ruprecht-Karls-Universität Heidelberg, 2008.

Fuensanta Martinez Rucobo: Overexpression, Biochemical Characterization and Crystallization of Chitin Synthase 2 from Saccharomyces cerevisae. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2008.

Mike Strauss: Electron Tomography of Membrane Proteins. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2008.

Ching-Ju Tsai: Three-dimensional structure of the glycine-betaine transporter BetP by cryo electron crystallography. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2008.

Mirko Lotz: Studies on the protein translocation in Escherichia coli: anaysis of the integral membrane proteins SecYEG and YidC employing biochemical and crystallographic methods.
Johann Wolfgang Goethe-Universität Frankfurt am Main, 2007.

Sivaram Chandra Chintalapati: Expression and Characterization of P-type ATPases for Structural Studies. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2007.

Matthias Appel: Proteinproduktion und Strukturuntersuchungen von Natrium/Protonen-Austauschern. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2006.

Vinothkumar Kutti Ragunath: Structure and function of membrane transport proteins. Universität Johann Wolfgang Goethe-Universität Frankfurt am Main, 2006.

Jörg Standfuß: Struktur und Funktion des LHC-II: Röntgenkristallographische Strukturaufklärung und funktionelle Charakterisierung der drei Isoformen. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2005.

Mihnea Bostina: Determination of the structure of complex I of Yarrowia lipolytica by single particle analysis. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2004.

Luana Licata: High level production, characterization, and structural analysis of neuronal calcium-activated potassium channels. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2004.

Stefan Raunser: Überproduktion, Aufreinigung, Funktions- und Strukturanalyse und intramembrane Lokalisierung der Glutamat-Transporter GltP aus E. coli und GLT-1 aus Rattenhirn. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2004.

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

Matteo Lamborghini: Three dimensional structure of the light-harvesting chlorophyll a/b protein complex from plant chloroplasts. Frankfurt am Main, 2002.

Jens Dietrich: Biochemische und elektronenkristallographische Untersuchungen an Membranproteinen (Biochemical and electron crystallographic studies of membrane proteins). Frankfurt am Main, 2001.

Hans Rogl: Struktur und Funktion des Lichtsammlerkomplexes LHC-II höherer Pflanzen (Structure and function of the plant light-harvesting complex, LHC-II). Frankfurt am Main, 2000.

DIPLOMA THESES

Bertram Daum: Tomographische Studien an Thylakoidmembranen. Universität Kassel, 2008.

Eva-Maria Heller: Untersuchungen zur Stöchiometrie des membrangebundenen Rotor-Rings aus bakteriellen F-ATP-Synthasen. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2008.

Doreen Matthies: Untersuchungen zum Fo-Subkomplex der F-Typ ATP-Synthase aus Bacillus sp. strain TA2.A1. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2008.

Eva Schweikhard: Untersuchung von Funktion und Struktur des universellen Stressproteins TeaD aus dem halotoleranten Bakterium Halomonas elongata. Johannes Gutenberg-Universität Mainz, 2008.

Laura Wilk: Structure and Function of Plants' Minor Light-Harvesting Complexes. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2007.

Jonna Hakulinen: Funktionelle und strukturelle Untersuchung von N-terminal verkürzten und hinsichtlich des Betain-Transports gehemmten Mutanten von BetP aus Corynebacterium glutamicum. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2006.

Susanne Ressl: Röntgenstrukturelle und elektronenmikroskopische Untersuchungen zweier Mutanten des Glycinbetain Transportproteins BetP aus Corynebacterium glutamicum. Johannes Gutenberg-Universität Mainz, 2006.

Franz Weiss: Kristallisation von Mutanten eines Membrantransportproteins. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2006.

Sonja Kuhlmann: Untersuchung von TeaBC, der Transporteinheit des osmoregulatorischen TRAP Transporters TeaABC von Halomonas elongata: Expression in E. coli und Aufreinigung, für Strukturuntersuchungen. Johannes Gutenberg-Universität Mainz, 2005.

Daniel Rhinow: Biologische cryo-Elektronenmikroskopie mit neuartigen TiSi-Trägerfolien und strukturelle Untersuchungen an 2D-Kristallen des Na+/H+ Antiporters aus Methanococcus Jannaschii. Johann Wolfgang Goethe-Universität Frankfurt am Main, 2004.

Christian Meesters: Identifizierung der Extramembrandomänen im Na+/H+-Antiporter NhaA von E. coli durch Markierung mit Goldpartikel und Antikörperfragmenten an 2D-Kristallen (Localisation of extra-membraneous domains of the Na+/H+-antiporter NhaA by gold and antibody labelling). Frankfurt am Main, 2001.

Jörg Standfuß: Untersuchungen zur Stabilisierung des Trimers von rekombinatem Light-Harvesting Complex II (Structure and stability of recombinant light-harvesting complex, LHC-II). Frankfurt am Main, 2000.

Tassilo Krug v. Nidda: 2D-Kristallisation und Strukturuntersuchung am Photosystem-II Reaktionszentrum (2D crystallisation of the photosystem II reaction centre). Frankfurt am Main, 1999.