Principal Investigator
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P12 Josef Wachtveitl
Professor
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Institute of Physical and
Theoretical Chemistry
Goethe-University Frankfurt a.M.
Max-von-Laue-Str. 7
60438 Frankfurt am Main, Germany
Phone +49 (0)69 798-29351
Fax +49 (0)69 798-29708
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P12 Photoinitiated functional dynamics of retinal proteins and multidrug transporters
Our group is mainly concerned with time-resolved spectroscopy to study molecular dynamics and function of membrane proteins. We aim for a complete tracking of the hierarchy of reaction steps ranging from the fastest local structural changes (femtoseconds to picoseconds) to slower long range conformational changes (milliseconds to seconds).
The focus of this project is to observe photoinitiated functional dynamics of membrane proteins in real time with high temporal resolution over a wide spectral range. The molecular mechanisms of activation and substrate or signal transduction will be investigated for retinal proteins and multidrug transporters.
The current comparative analysis of primary reactions in various rhodopsins will be extended to processes succeeding photoactivation in sensory rhodopsin II / transducer and in rhodopsin / peptide complexes. For the SR II/Htr II complex these structural changes will be investigated by time-resolved anisotropy measurements, recording the orientation of the built-in chromophore retinal and a second ‘reporter’ chromophore at the transducer. The GPCR / G protein interaction will be investigated analyzing conformational changes in rhodopsin and specific model peptides upon illumination by comparing the dark state with different intermediates of the photocycle. The recent availability of sufficient amounts of protein will allow us to study the time-resolved dynamics of the light-gated cation-selective retinal protein channelrhodopsin II.
In addition, time resolved fluorescence studies on multi-drug transporters like TBsmr from M. tuberculosis will be performed. The functional dynamics can be recorded by a fast photoinitiation of the transport process. More specifically, this substrate/proton antiport shows a strong increase of ethidium bromide fluorescence quantum yield upon formation of a pH-gradient. This change in fluorescence is transport associated and can be studied in a time-resolved fashion, after a fast trigger mechanism, e.g. a pH jump assay is employed.
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- Fig. 1: Scheme of a femtosecond vis-pump/vis-probe spectrometer and transient absorbance changes of the NpSR II – Htr II Complex 1:1 (λexc = 500 nm, FWHM = 20 nm, 40 nJ; λprobe = 380 nm – 700 nm, CaF2 white light). Pump and probe pulse were polarized parallel with respect to each other.
- Fig. 2: Photocycle of channelrhodopsin II (ChR2) with intermediates (P0–P4) as deduced from the spectroscopic data and the functional states of the channel. P480, P400 and P520 denote the ground state, the blue- and red-shifted intermediates, respectively. ChR2L and ChR2D denote a putative light and dark-adapted ground state, respectively. The light reaction leads to an excited state (ChR2ex) before the channel opens (ChR2o) [modified after Bamann et al.(2008) J. Mol. Biol. 375, 686-694].
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Publications
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Hempelmann, F., Hölper, S., Verhoefen, M.-K., Woerner, A., Köhler, T., Fiedler, S.-A., Pfleger, N., Wachtveitl, J. and Glaubitz, C. (2011) The His75-Asp97 Cluster in Green Proteorhodopsin. J Am Chem Soc 133, 4645-4654.
Verhoefen, M.-K., Bamann, C., Blöcher, R., Förster, U., Bamberg, E. and Wachtveitl, J. (2010) The photocycle of Channelrhodopsin-2: Ultrafast reaction dynamics and subsequent reaction steps. ChemPhysChem 11, 3113-3122.
Gildenhoff, N., Amarie, S., Gundermann, K., Beer, A., Büchel, C. and Wachtveitl, J. (2010) Oligomerization and pigmentation dependent excitation energy transfer in fucoxanthin-chlorophyll proteins. BBA - Bioenerg 1797, 543-549.
Lörinczi, É., Verhoefen, M.-K., Wachtveitl, J., Woerner, A.C., Glaubitz, C., Engelhard, M., Bamberg, E. and Friedrich, T. (2009) Voltage- and pH-dependent Changes in Vectoriality of Photocurrents Mediated by Wild-type and Mutant Proteorhodopsins upon Expression in Xenopus Oocytes. J Mol Biol 393, 320-341.
Verhoefen, M.-K., Lenz, M.O., Amarie, S., KLare, J.P., Tittor, J., Oesterhelt, D., Engelhard, M. and Wachtveitl, J. (2009) Primary reaction dynamics of sensory rhodopsin II mutant D75N and the influence of azide. Biochemistry 48, 9677-9683.
Neumann, K., Verhoefen, M.-K., Weber, I., Glaubitz, C. and Wachtveitl, J. (2008) The initial reaction dynamics of proteorhodopsin observed by femtosecond infrared and visible spectroscopy. Biophys J 94, 4796-4807.