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Structural and biochemical characterization of novel tools in optogenetics

Schertler    Prof. Dr.
Gebhard F. X. Schertler
Paul Scherrer Institut
CH-5232 Villigen PSI
Laboratory of Biomolecular Research, PSI
+41 56 310 4265 
Project starts   1.1.2016
Project ends    30.6.2018
Goals   Optogenetics has revolutionized neurobiology by allowing researchers to non-invasively control neuronal activity in live animals. It currently relies primarily on light-sensitive proteins (photopigments) that can be used to modulate ionic conductance of cell membranes and is therefore restricted to cell types which can be stimulated with ion fluxes such as neurons and myocytes.

The goal of our study is to expand the reach of optogenetics by developing new tools that would allow for a light- triggered control of any G-Protein Coupled Receptor (GPCR) - dependent cellular signaling pathway. The GPCR protein family is one of the most important families of membrane receptors in all eukaryotes and mostly facilitates optical, chemical and hormone induced signaling. GPCR signaling primarily operates by activating heterotrimeric G proteins (Gαβγ) and arrestins. The cellular response upon receptor activation is mostly dependent on the identity of the Gα protein to which the receptor couples and GPCRs influence a wide array of cellular processes such as vison, muscle contraction, glucose/glycogen concentration and many more. Being able to photoactivate any GPCR signaling pathway will dramatically increase the scope and possibilities of optogenetics, and will allow for a precise spatial and temporal activation of defined signaling pathways in a living organism. In order to reach our goal we identified so-called bistable pigments, mostly from invertebrates, as being the most suitable base for our project, since they can photochemically self-regenerate in any tissue.
Elena Lesca
Paul Scherrer Institut
5232 VIlligen-PSI
+4156 310 4032
Gregor Cicchetti
Paul Scherrer Institut
5232 VIlligen-PSI
+4156 310 4469
Gebhard Schertler
Paul Scherrer Institut
CH-5232 Villigen PSI
+41 56 310 4265  
Abstract    We aim to engineer new optogenetic tools by fusing bistable pigments with functional intracellular domains of different GPCRs to obtain control over divers GPCR signaling pathways. Using cutting edge methods in structure determination and advanced computational modeling will enable us to design new receptor variants with modified spectral sensitivity and targeting specific G protein dependent and MAP Kinase signaling pathways.
To reach our objective, we need a much better knowledge of the structural properties and dynamics of bistable pigments. Due to the very fast nature of the photoactivation process, bistable pigments crystals will be ideal samples for pump-probe experiments of time-resolved crystallography at X-ray femtosecond (X-FEL) beamlines. For an overview see Neutze et al. “Membrane protein structural biology using X-ray free electron lasers”, Curr Opin Struct Biol. 2015 Aug; 33:115-25. These experiments will be performed at the SwissFEL facility at the Paul Scherrer Institute. A typical experiment would be crystallizing a bistable pigment in a dark state into a viscous solution (i.e. lipidic cubic phase, LCP). Then, a continuous stream of microcrystals is injected across the X-FEL beam by a microjet and X-ray diffraction patterns are recorded. Before injection, the crystal proteins are activated through illumination by a femtosecond laser of the appropriate wavelength with a defined time delay allowing for the snapshot recording of reaction intermediates. Since Retinal isomerization take from femtoseconds to several picoseconds and femtosecond laser pulses are required to allow for a sufficient short time delay to measure/catch this ultrafast process. Using molecular dynamics and molecular modeling we can exploit these results on receptor dynamics to guide the protein engineering.

Fig. 1. Schematic drawing of pump–probe time-resolved serial femtosecond crystallography experimental setup for retinal protein dynamics study. The figure depicts the overall setup containing the optical pump laser (yellow), the X-ray FEL pulse (blue) and the detectors with an diffraction pattern (white). The high X-FEL intensities used in the “diffract before destroy” regime of SFX require a constant delivery of crystals at room temperature, precisely aligned with the path of the pump laser activating the retinal protein at t1. After a defined time-delay t2, the X-FEL pulse is delivered resulting in a diffraction pattern specic for the conformational arrangement of the protein at t2. Varying t2 results in a series of intermediate “snapshots” of the light-induced conformational change. Currently, two sample delivery modes are available for SFX, a fixed target wafers with arrays of windows with painted 2D or 3D crystals in a glucose-containing solution, and a microjet system delivering a liquid stream of submicron crystals. Figure adapted from Panneels et al. “Time-resolved structural studies with serial crystallography: A new light on retinal proteins.” Struct Dyn. 2015 Jun 29;2(4):041718.
  • Lesca, E., V. Panneels and G. F. X. Schertler (2018). The role of water molecules in phototransduction of retinal proteins and G protein-coupled receptors. Faraday Disc. 207: 27-37. (10.1039/c7fd00207f)
  • Nango, E., A. Royant, M. Kubo, T. Nakane, C. Wickstrand, T. Kimura, T. Tanaka, K. Tono, C. Song, R. Tanaka, T. Arima, A. Yamashita, J. Kobayashi, T. Hosaka, E. Mizohata, P. Nogly, M. Sugahara, D. Nam, T. Nomura, T. Shimamura, D. Im, T. Fujiwara, Y. Yamanaka, B. Jeon, T. Nishizawa, K. Oda, M. Fukuda, R. Andersson, P. Båth, R. Dods, J. Davidsson, S. Matsuoka, S. Kawatake, M. Murata, O. Nureki, S. Owada, T. Kameshima, T. Hatsui, Y. Joti, G. Schertler, M. Yabashi, A.-N. Bondar, J. Standfuss, R. Neutze and S. Iwata (2016). A three-dimensional movie of structural changes in bacteriorhodopsin. Science 354: 1552. (10.1126/science.aah3497)
  • Nogly, P., T. Weinert, D. James, S. Carbajo, D. Ozerov, A. Furrer, D. Gashi, V. Borin, P. Skopintsev, K. Jaeger, K. Nass, P. Båth, R. Bosman, J. Koglin, M. Seaberg, T. Lane, D. Kekilli, S. Brünle, T. Tanaka, W. Wu, C. Milne, T. White, A. Barty, U. Weierstall, V. Panneels, E. Nango, S. Iwata, M. Hunter, I. Schapiro, G. Schertler, R. Neutze and J. Standfuss (2018). Retinal isomerization in bacteriorhodopsin captured by a femtosecond x-ray laser. Science. (10.1126/science.aat0094)
  • Tsai, C. J., F. Pamula, R. Nehme, J. Muhle, T. Weinert, T. Flock, P. Nogly, P. C. Edwards, B. Carpenter, T. Gruhl, P. Ma, X. Deupi, J. Standfuss, C. G. Tate and G. F. X. Schertler (2018). Crystal structure of rhodopsin in complex with a mini-G(o) sheds light on the principles of G protein selectivity. Sci. Adv. 4. (10.1126/sciadv.aat7052)
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