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Principal Investigator

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P13  Harald Schwalbe
Professor

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Institute of Organic Chemistry
and Chemical Biology /
Center of Biomolecular
Magnetic Resonance
Goethe University Frankfurt a.M.
Max-von-Laue-Str. 7
60438 Frankfurt am Main, Germany

Phone +49 (0)69 798-29737
Fax +49 (0)69 798-29515

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P13 NMR investigations of retinal rhodopsin


Light triggers numerous signaling cascades, provides energy directly or indirectly for basically all biological systems and plays a major role in sensing environmental changes. An amazing variety of systems have evolved that interact with light. One extraordinary example of such a system is the class of membrane-embedded light sensing proteins. They can act as model systems for two of the most fundamental functions of membranes in Biology, i.e. active transport and sensory signaling. Our research interest focuses on investigating light-induced conformational changes and dynamics of light sensitive retinal-binding membrane proteins by light-triggered, time-resolved NMR spectros-copy. During the first funding period of CRC 807, combined research efforts in solution and solid-state NMR spectroscopy together with optical spectroscopy have culminated in the solution struc-ture of proteorhodopsin (Reckel et al., 2011). Further, we performed NMR studies to investigate light-induced changes in mammalian as well as microbial rhodopsins. We revealed the dynamic properties of rhodopsin and proteorhodopsin via time-resolved NMR spectroscopy using a unique setup that allows direct laser illumination within the NMR spectrometer. Our dynamic studies sug-gest that proteorhodopsin experiences only minor conformational changes as it undergoes its pho-tocycle, supporting the notion of a dynamically controlled activation process. By contrast, for bovine rhodopsin, we observed major structural rearrangements upon illumination indicating a different activation mechanism. We will extend our NMR studies of light cycle dynamics to another retinal-binding protein channelrhodopsin (ChR). We could establish expression systems (HEK293 cells, Pichia pastoris, E. coli) for these proteins and suitable constructs enabling isotopic labeling schemes necessary for large proteins amount for NMR measurements have been designed. For bovine visual rhodopsin, we will study its down-regulation of the photocascade by arrestin; studies of microbial rhodopsins will include time-resolved NMR studies of the light cycle of proteorhodopsin (PR) and channelrhodopsin (ChR).

 

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Publications

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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, 1-7.

Becker-Baldus J, Bamann C, Saxena K, 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. Proc Natl Acad Sci USA 112, 9896-901.

Danne L, Aktas M, Gleichenhagen J, Grund N, Wagner D, Schwalbe H, Hoffknecht B, Metzler-Nolte N, Narberhaus F (2015) Membrane binding mechanism of a bacterial phospholipid N-methyltransferase. Mol Microbiol 95, 313-31.

Zickermann V, Wirth C, Nasiri H, Siegmund K, Schwalbe H, Hunte C, Brandt U (2015) Mechanistic insight from the crystal structure of mitochondrial complex I. Science 347, 44-49.

Ardenkjaer-Larsen J-H., Boebinger GS., Comment A, Duckett S, Edison A, Engelke F, Griesinger C, Griffin RG, Hilty C, Maeda H, Parigi G, Prisner T, Ravera E, van Bentum J, Vega S, Webb A, Luchinat C*, Schwalbe H*, Frydman L* (2015) Facing and overcoming sensitivity challenges in biomolecular NMR spectroscopy. Angew Chem Int Ed 54, 9162-85.

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, 2078-84.

Herbert C, Schieborr U, Saxena K, Juraszek J, De Smet F, Alcouffe C, Bianciotto M, Saladino G, Sibrac D, Kudlinzki D, Sreeramulu S, Brown A, Rigon P, Herault JP, Lassalle G, Blundell TL,Rousseau F, Gils A, Schymkowitz J, Tompa P, Herbert JM, Carmeliet P, Gervasio FL*, Schwalbe H*, Bono F* (2013) Molecular mechanism of SSR128129E, an extracellularly acting, small molecule, allosteric inhibitor of FGF receptor signaling. Cancer Cell 23, 489-501.

Hiruma Y, Hass MAS, Kikui Y, Liu WM, Ölmez B, Skinner SP, Blok A, Klosterman A, Koteishi H, Löhr F, Schwalbe H, Nojiri M, Ubbink M (2013) The structure of the cytochrome P450cam-putidaredoxin complex determined by paramagnetic NMR spectroscopy and crystallography. J Mol Biol 425, 4353-4365.

Stehle J, Scholz F, Löhr F, Reckel S, Roos C, Blum M, Braun M, Glaubitz C, Dötsch V, Wachtveitl J, Schwalbe H (2012) Characterization of the ground state dynamics of proteorhodopsin by NMR and Optical Spectroscopies. J Biomol NMR 54, 401-13.

(* corresponding authors)

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Collaborations

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Dötsch/Bernhard (P02), Glaubitz (P06), Gottschalk (P11), Wachtveitl/Bamann (P12),
Ernst (P21)