Prof. Dr. Werner Kühlbrandt, Director
Membrane proteins are fascinating. Sitting in the membrane that surrounds every living cell and the compartments within it, they perform a vast range of different functions, many of which are central to life. None of these vital processes can be fully understood without knowing, ideally in atomic detail, what the proteins look like. Often, the structures of at least two different states are required, and this is especially true of membrane proteins, many of which undergo conformational changes in performing their function. Their special position in the lipid bilayer of the membrane means that their exterior is partly hydrophobic – the part that is embedded in the lipid bilayer – while the parts exposed to the aqueous cytoplasm or the surrounding medium are hydrophilic. This makes them difficult to study by the techniques of structural biology, but for many scientists, including us, this makes them all the more fascinating and attractive.
Currently, membrane proteins account for less than 0.5% of all entries in the protein data bank, whereas 20-30% of all genes in all organisms are thought to encode membrane proteins. Clearly, a great deal remains to be done. Although the situation has improved over the past few years, as we have learnt to produce and handle membrane proteins more successfully, there is still an immense need for more, and more detailed structures.
The Max Planck Institute of Biophysics is a well-known centre for studying all aspects of membrane proteins, and researchers at this institute have made ground-breaking contributions to this field. Our department of Structural Biology is especially interested in proteins and large complexes involved in membrane transport, biological energy conversion, and membrane biogenesis. We investigate the structure of these proteins by electron and X-ray crystallography and single-particle cryo-EM. In addition, we are studying the structure, interactions and distribution of the proteins in the membrane by electron tomography.
We produce most of the membrane proteins for structure determination within the department, but some are made by our collaborators. Expression, purification and crystallization of membrane proteins are demanding tasks that have to be re-optimized for each new protein. This accounts for a considerable part of our research effort. We attempt to grow both two-dimensional (2D) crystals for electron crystallography, as well as 3D crystals for X-ray crystallography, and then take the course that looks most promising. Often 2D crystals form first, but they are rarely well enough ordered for high-resolution structure determination. 3D crystallization of membrane proteins has become easier in recent years, largely due to the widespread use of special sparse matrix screens and crystallization robots. However, each protein presents a new challenge, and improving crystals from the first exciting hits to specimens suitable for high-resolution data collection usually takes years of dedicated, painstaking work.
Summary of research achievements
Structures of membrane proteins or membrane-related complexes that have been determined by members of our department include
In addition we have determined the high-resolution structures of several membrane-related proteins by X-ray crystallography, including
By electron cryo-tomography we have shown that
Facilities for X-ray and EM work
Our EM facility, brilliantly and congenially managed by Deryck Mills, has a 300 kV FEI Polara used mainly for electron cryo-tomography and single-particle work, and a helium-cooled JEOL SFF dedicated to electron crystallography. In July 2009 we took delivery of a prototype 200 kV electron microscope (PACEM) with a specially designed transfer lens for a phase plate and a Cs corrector for in-focus phase contrast microscopy of cryo specimens. This unique instrument has been developed with funds of the Frankfurt Cluster of Excellence “Macromolecular Complexes” jointly with Zeiss NTS. From late 2010 we will share a Titan Krios, purchased jointly with the Cluster of Excellence. Three other electron microscopes (Philips CM12, CM120, and EM208) are available for routine use. For image processing we have a high-resolution Zeiss SCAI scanner, and access to powerful computer clusters. For preparing specimens for cryo-EM and cryo-tomography we have a Vitrobot, a high-pressure freezer, a cryo microtome and a freeze-fracture apparatus.
For crystallization screens we use a Mosquito robot, and have access to a Rigaku protein crystallization facility. We are frequent users of the Max Planck beamline at the SLS, one of the best synchrotron sources in the world, for X-ray data collection.
To complement our structural studies, we investigate the function of LHC-II, BetP, OmpG, and NhaP1 by a wide range of biophysical techniques, including uptake studies and electrophysiology, microspectroscopy (in collaboration with A. Royant , ESRF Grenoble, France), FTIR (in collaboration with W. Mäntele at Frankfurt University), and AFM (in collaboration with Daniel Müller, TU Dresden). The subunit stoichiometry of ATP synthase c-rings is characterized by LILBID mass spectrometry in collaboration with B. Brutschy (University of Frankfurt)
For a more detailed description of the research pursued in the Department, please refer to the web sites of the individual group leaders and staff scientists. Group leaders have obtained research grants to support their own projects, and supervise postdocs and students.
The main interests in Werner Kühlbrandt’s research group is electron and X-ray crystallography of membrane proteins in biological energy conversion and secondary transporters, and the native arrangement of large complexes in the membrane, studied by electron tomography. The group has determined the structure of the plant light-harvesting complex LHC-II (Kühlbrandt et al, Nature 1994; Standfuss et al, EMBO J 2005). The structures of the dimeric sodium/proton antiporter NhaA (Williams et al, Nature 2000), and the bacterial protein translocase SecYEG (Breyton et al, Nature 2002) in the membrane have been determined at 6-8Å resolution by electron crystallography. These projects are being continued. Together with Janet Vonck, we work on the structure of large macromolecular assemblies, such as the fatty acid synthase, by single particle EM.
Two comparatively recent research themes in the group are electron cryo-tomography of biological membranes, and the development of a new phase contrast electron microscope for cryo-EM. Electron tomography has revealed the arrangement of the ATP synthase in rows of dimes in the mitochondrial inner membrane, and the chloroplast ATP synthase in thylakoid membranes. In collaboration with H. Osiewacz at Frankfurt University we study the effects of aging on mitochondrial ultrastructure by cryo-tomography. Bastian Barton and Mike Strauss are working with the new phase contrast electron microscope, and its applications to new biological questions.
The group of Thomas Meier is devoted to investigating the structure and function of the ATP synthase, in particular the Fo part in the membrane that drives rotary synthesis of ATP in the F1 head using a proton or (in special cases) a Na+ gradient. Thomas Meier has determined the first structure of a c-ring rotor (Meier et al, Science 2009) in the group of P. Dimroth at the ETH Zürich before he joined the department. Since then, he has extended his studies to c-rings from a number of organisms, which have different subunit stoichiometries, and has characterized them by electron crystallography of 2D crystals, AFM in collaboration with the Müller group in Dresden, and by LILBID mass spectrometry in collaboration with Brutschy in Frankfurt.
Eva Schäfer rejoined the department recently, after a 3 year postdoc abroad. Her research interests include single-particle cryo-EM of large membrane protein complexes, especially the mitochondrial supercomplexes of the respiratory chain (Schäfer et al, Biochemistry 2007).
Özkan Yildiz specializes in X-ray crystallography of membrane and membrane-associated proteins. He has determined the structure of the E. coli outer membrane porin OmpG in the open and closed conformation (Yildiz et al, EMBO J 2006), and the structure of the protein GlnK1 that regulates nitrogen uptake in bacteria and archaea in three different functional states (Yildiz et al, EMBO J 2007). Most recently he and his group have solved the structure of the the GTP-binding domain of the ferrous iron uptake protein FeoB, the only known membrane-bound G-protein in prokaryotes (Köster et al, J. Mol. Biol.2009).
Christine Ziegler and her group focus on membrane transporters that maintain the osmotic pressure inside cells. BetP from the soil bacterium Corynebacterium glutamicum has the amazing ability of both sensing the internal osmotic pressure, and balancing it by specific uptake of glycine betaine, a small, inert molecule that acts as a compatible solute. They determined the structure of the protein at 3.4 Å resolution by single-anomalous dispersion X-ray crystallography (Ressl et al, Nature 2009), which showed for the first a transporter in the occluded state. Surprisingly, the arrangement of membrane spanning helices was very similar to that found in other Na-dependent solute transporters, even though there was no sequence homology. Other osmolyte transporters by Christine Ziegler include the TeaABC system, and stress-induced solute channels.
Prof. Dr. Werner Kühlbrandt, Director
Department of Structural Biology
Secretary: Monika Hobrack
Phone: +49 (0) 69 6303-3001
Fax: +49 (0) 69 6303-3002