P12 Josef Wachtveitl
Professor
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 Christian Bamann
Group Leader
Principle Investigator
07/2016 - 06/2019
Former member of
Biophysical Chemistry
Max Planck Institute of Biophysics
Frankfurt am Main, Germany
The project addresses the functional dynamics of rhodopsins and the coupling of photoreaction to ion translocation. It entails the time-resolved study by various spectroscopic techniques covering several orders of magnitude in time, rhodopsin sample preparation and the electrophysiological assessment of their ion transport properties. The combined orthogonal approach yields a detailed description of the complete photocycle.
In the last funding period, we provided a structural and biophysical basis for color-tuning mecha-nisms of microbial retinal proteins and investigated the consequences for quantum yield, reaction dynamics and structural changes. In a joint time-resolved optical and NMR study we revealed the influence of arrestin on the photodecay of bovine rhodopsin and its role for retinal homeostasis in rod outer segments. We will continue our functional studies on microbial rhodopsins. Here, we address the central question, how in channelrhodopsin (ChR2) photoisomerization, protonation changes and channel opening and closing are connected. To identify obligatory events, we will create a local perturbation at the chromophore site with different retinal analogues, use functionally defect mutants or monitor global conformational changes with fluorescent probes. We will combine visible and mid-IR spectroscopy to elucidate the essential steps of this unique photocycle on a molecular level. Global changes and functional phenotypes are described by voltage-clamp fluorometry and patch-clamp recordings. We will extend our studies to Chrimson, a new channelrhodopsin with a red-shifted spectrum, and to KR2, a light-driven sodium pump from marine bacteria. Furthermore, we will provide a full kinetic analysis of the blue light quenching effect in different systems (PR and ChR2) and the formation of highly fluorescent, voltage-sensitive photointermediates (Arch) in multiphoton experiments.
Fig. 1: Overview of the spectroscopic methods for time-resolved experiments.
Fig. 3: Light-induced currents from Channelrhodopsin-2 from patch-clamp recordings.
Becker-Baldus J, Bamann C, Saxena K, Gustmann H, Brown LJ, Brown RCD, Reiter C, Bamberg E, Wachtveitl J, Schwalbe H, Glaubitz C (2015) Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy. Proc Nat Acad Sci USA 112, 9896-901.
Bühl E, Braun M, Lakatos A, Glaubitz C, Wachtveitl J (2015) Fluorescence and excited state dynamics of the deprotonated Schiff base retinal in proteorhodopsin. Biol Chem 396, 1109.
Chatterjee D, Eckert CE, Slavov C, Saxena K, Fürtig B, Sanders CR, Gurevich VV, Wachtveitl J, Schwalbe H (2015) Influence of arrestin on the photodecay of bovine rhodopsin. Angew Chem Int Ed 54, 13555-60.
Mehler M, Eckert CE, Busche A, Kulhei J, Michaelis J, Becker-Baldus J, Wachtveitl J, Dötsch V, Glaubitz C (2015) Assembling a correctly folded and functional heptahelical membrane protein by protein trans-splicing. J Biol Chem 290, 27712-22.
Mao J, Do N-N, Scholz F, Reggie L, Mehler M, Lakatos A, Ong Y-S, Ullrich SJ, Brown LJ, Brown RCD, Becker-Baldus J, Wachtveitl J, Glaubitz C (2014) Structural basis of the green–blue color switching in proteorhodopsin as determined by NMR spectroscopy. J Am Chem Soc 136, 17578-17590.
Stehle J, Silvers R, Werner K, Chatterjee D, Gande S, Scholz F, Dutta A, Wachtveitl J, Klein-Seetharaman J, Schwalbe H (2014) Characterization of the simultaneous decay kinetics of metarhodopsin states II and III in rhodopsin by solution-state NMR spectroscopy. Angew Chem Int Ed 53, 2078-2084.
Janke C, Scholz F, Becker-Baldus J, Glaubitz C, Wood PG, Bamberg E, Wachtveitl J, Bamann C (2013) Photocycle and vectorial proton transfer in a rhodopsin from the eukaryote Oxyrrhis marina. Biochemistry 52, 2750-63.
Mehler M, Scholz F, Ullrich Sandra J, Mao J, Braun M, Brown Lynda J, Brown Richard CD, Fiedler Sarah A, Becker-Baldus J, Wachtveitl J, Glaubitz C (2013) The EF loop in green proteorhodopsin affects conformation and photocycle dynamics. Biophys J 105, 385-97.
Neumann-Verhoefen M-K, Neumann K, Bamann C, Radu I, Heberle J, Bamberg E, Wachtveitl J (2013) Ultrafast infrared spectroscopy on channelrhodopsin-2 reveals efficient energy transfer from the retinal chromophore to the protein. J Am Chem Soc 135, 6968-76.
Bamann C, Bamberg E, Wachtveitl J, Glaubitz C (2013) Proteorhodopsin (Review). BBA-Bioenergetics 1837, 614-625.
Mörs K, Roos C, Scholz F, Wachtveitl J, Dötsch V, Bernhard F, Glaubitz C (2013) Modified lipid and protein dynamics in nanodiscs. BBA-Biomembranes 1828, 1222-1229.
Herz J, Verhoefen M-K, Weber I, Bamann C, Glaubitz C, Wachtveitl J (2012) Critical role of Asp227 in the photocycle of proteorhodopsin. Biochemistry 51, 5589-600.