FG Prof. Dr. M. Engelhard
Transmembrane signal transduction
Involved: A. Goeppner, L. Lin, Y-J. Kim, S. Martell, N. Mennes
Archaebacterial photoreceptors mediate phototaxis by regulating cell motility through two-component signalling cascades (Figure 1). Homologs of this sensory pathway occur in all three kingdoms of life, most notably in enteric bacteria in which the corresponding chemotaxis signalling cascade has been extensively studied.
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Figure 1: The two-component signalling cascade. By activation of the transducer HtrII by SRII the signal is transferred to the cytoplasmic end of the molecule, where,in analogy with the bacterial chemotactic system, the homodimeric histidine kinase CheA is bound together with CheW. The next steps involved in this cascade happen through the response regulators/ aspartate kinases CheY and CheB,where phosphorylated CheY functions as a switch for the flagellar motor. The adaptation process is exhibited by the methylesterase CheB and the methyltransferase
Recent structural and functional studies on the sensory rhodopsin II/transducer (SR/Htr) complex mediating the photophobic response of Natronomonas pharaonis have yielded new insights into the mechanisms of signal transfer across the membrane. Electron paramagnetic resonance data and the atomic resolution structure of the receptor molecule in complex with the transmembrane segment of its cognate transducer provided a model for signal transfer from the receptor to the cytoplasmic side of the transducer (Figure 2): The light activated retinal isomerisation leads to specific conformational change within the receptor which consequently triggers a rotary motion of the C-terminal transmembrane helix of the transducer (TM2).
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Figure 2: Illustration of the light-induced conformational changes of the receptor helix F and the transducer helix TM2. The space filling model shows the tight interaction between F and TM2. The outward movement of helix F (straight arrow) clearly affects TM2 in the way of a clockwise rotary motion (bent arrow) Figure taken from Klare et al. 2004.
The main focus of the present research is directed towards an understanding how membrane proximal signalling events are triggered by extracellular and/or membrane associated ligand binding. Specifically there are three projects which are currently pursued.
Structural analysis of the linker region of the phototransducer in complex with sensory rhodopsin II.
In close collaboration with 
G. Büldt (Forschungszentrum Jülich) the X-ray structures of the photocycle intermediates K and late M (M2) were solved Moukhametzianov et al. 2006. These structures provide information about the evolution of the signal in the receptor after retinal isomerization and its transfer to the transducer in the complex allowing to propose a mechanism for the light induced activation of the signalling complex. Structural information of the linker region (Bordignon et al. 2005) and the elucidation of the mechanism of the signal transfer (Klare et al. 2004) in the cytoplasmic domain of the transducer are also obtained by Electron Spin Resonance in collaboration with H.-J. Steinhoff, Universität Osnabrück.
Solid state NMR-structural investigation of the sensory rhodopsin II-transducer complex and its intermediates:
A new research project (in collaboration with M. Baldus, Max Planck Institut für biophysikalische Chemie, Göttingen) is directed towards an understanding of how the rotary or screw like motion of the C-terminal transmembrane helix (TM2) of the transducer is transmitted to the signalling domain at the tip of the four helical bundle. In order to follow these small conformational changes into the cytoplasmic domain Solid State NMR experiments are performed on fragmentally isotope labelled transducer molecules in complex with its receptor.
Inducing transmembrane signal transduction by chemically engineered photoswitches
This research project is directed towards an understanding of transmembrane signal transduction. In order to simulate the extracellular conformational change a chemically engineered photoswitch is introduced into the extracellular loop region of the transducer NpHtrII between the N- and C-terminal transmembrane helices. Light activated isomerisation of the central double bond (or of the peptide bond) of the photoswitches should induce conformational changes of TM2 which can be analyzed by EPR spectroscopy of appropriate spin labelled peptides and by FTIR difference spectroscopy. Introducing a spin- or isotope label along the TM2 helix allows to follow the photoswitch induced perturbation across the membrane. This work (funded by the Deutsche Volkswagenstiftung) is a joined project between F. Siebert, Friedrich Albert Universität, H.-J. Steinhoff, Universität Osnabrück.
Funding: This work is funded by the Deutsche Forschungsgemeinschaft, Max Planck Society and the Volkswagenstiftung
Selected References
Bordignon, E., Klare, J. P., Doebber, M., Wegener, A. A., Martell, S., Engelhard, M. & Steinhoff, H. J. (2005). Structural Analysis of a HAMP Domain: The linker region of the phototransducer in complex with sensory rhodopsin II. J. Biol. Chem. 280, 38767-38775.
Gordeliy, V.
Hippler-Mreyen, S., Klare, J. P., Wegener, A. A., Seidel, R., Herrmann, C., Schmies, G., Nagel, G., Bamberg, E. & Engelhard, M. (2003). Probing the Sensory Rhodopsin II Binding Domain of its Cognate Transducer by Calorimetry and Electrophysiology. J. Mol. Biol. 330, 1203-1213.
Klare, J. P., Gordeliy, V. I., Labahn, J., Büldt, G., Steinhoff, H.-J. & Engelhard, M. (2004). The archaeal sensory rhodopsin II/transducer complex: a model for transmembrane signal transfer. FEBS Letters 564, 219-224.
Klare, J. P., Bordignon, E., Doebber, M., Fitter, J., Kriegsmann, J., Chizhov, I., Steinhoff, H. J. & Engelhard, M. (2006). Effects of Solubilization on the Structure and Function of the Sensory Rhodopsin II/Transducer Complex. J. Mol. Biol. 356, 1207-1221.
Moukhametzianov, R., Klare, J. P., Efremov, R., Baeken, C., Göppner, A., Labahn, J., Engelhard, M., Büldt, G. & Gordeliy, V.
Schmies, G., Engelhard, M., Wood, P. G., Nagel, G. & Bamberg, E. (2001). Electrophysiological characterization of specific interactions between bacterial sensory rhodopsins and their transducers. Proc. Natl. Acad. Sci. USA 98, 1555-1559.
Wegener, A. A., Klare, J. P., Engelhard, M. & Steinhoff, H. J. (2001). Structural insights into the early steps of receptor-transducer signal transfer in archaeal phototaxis. EMBO J. 20, 5312-5319.
Probing the Molecular Basis of Protein Function through Chemistry
Involved: K. Lausecker, J. Milic, J. Sauermann, M. Schumacher
The ability to produce proteins in the laboratory and to change their structures and therefore their properties in a controlled fashion is of crucial importance in basic biological research, in biotechnology and increasingly in medical applications. Recent developments have led to a substantial expansion of the spectrum of methods available for the production of proteins that have extended the semi-classical approaches of overexpression on the one hand and peptide synthesis on the other hand so that the limitations of these methods no longer dictate the availability of desired protein (or protein analog) structures. Presently, several projects are pursued which afford the semisynthesis of proteins.
The role of membrane anchoring in the folding of PrPC and the formation of PrPSc: in vivo and in vitro studies
In this project a site specifically labeled prion protein (PrP) with a cleavable C-terminal membrane anchor (PrP-lipid) has been generated in order to study folding of PrP and PrP-lipid attached to liposomes. In further experiments it is intended to elucidate the formation of infectious PrP-lipid (PrPSc-lipid) in vitro and in scrapie-infected cells. This work is a joined project with J. Tatzelt (Ludwig Maximilian Universität, München) and supported in the frame of a MPG inter-institute fund.
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Figure 1: Schematic representation of the ligation product: PrP90-231 with a lipidated peptide fused to its C-terminus.
Active site modification of Ras
In the second project the active site of Ras will be site specifically modified by unnatural amino acids in order to elucidate the mechanism of the intrinsic and GAP catalysed GTP hydrolysis. A couple of artificial Boc-amino acids have been synthesized including e.g. BocCF3Thr or a caged spin label amino acids) which have been introduced into position 16, 35, and 61 (Figure 1). The work is a joint project between 
R. Goody, and R. Seidel (all Max Planck Institut für Molekulare Physiologie,
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Figure 1: Structure of Ras:GppNHp. The GTP analog GppNHp is shown in grey and the amino acids that will be modified in order to study the influence of these residues on GTP hydrolysis are highlighted: Lys16 (blue), Thr35 (yellow) and Gln61 (magenta).
Selected References
Becker, C. F., Lausecker, K., Balog, M., Kalai, T., Hideg, K., Steinhoff, H. J. & Engelhard, M. (2005). Incorporation of spin-labelled amino acids into proteins. Magn Reson. Chem 43 Spec no., S34-S39.
Becker, C. F., Seidel, R., Jahnz, M., Bacia, K., Niederhausen, T., Alexandrov, K., Schwille, P., Goody, R. S. & Engelhard, M. (2006). C-Terminal Fluorescence Labeling of Proteins for Interaction Studies on the Single-Molecule Level. Chembiochem.
Becker, C. F. W., Wacker, R., Bouschen, W., Seidel, R., Kolaric, B., Lang, P., Schroeder, H., Müller, O., Niemeyer, C. M., Spengler, B., Goody, R. S. & Engelhard, M. (2005). Direct Readout of Protein-Protein Interactions by Mass Spectrometry from Protein-DNA Microarrays. Angew. Chem. (Engl) 44, 7635-7639.
Becker, C. F. W., Hunter, C. L., Seidel, R., Kent, S. B. H., Goody, R. S. & Engelhard, M. (2003). Total chemical synthesis of a functional interacting protein pair: The protooncogene H-Ras and the Ras-binding domain of its effector c-Raf1. PNAS 100, 5075-5080.
Lovrinovic, M., Seidel, R., Wacker, R., Schroeder, H., Seitz, O., Engelhard, M., Goody, R. S. & Niemeyer, C. M. (2003). Synthesis of protein-nucleic acid conjugates by expressed protein ligation. Chemical Communications 822-823.