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On-chip photoactivation of heterologously expressed rhodopsin allows kinetic analysis of G-protein signaling by surface plasmon resonance spectroscopy

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Abstract

Surface plasmon resonance spectroscopy allows the study of protein interaction dynamics in real-time. Application of this technique to G-protein coupled receptors, the largest family of receptors involved in signal transduction, has been complicated by their low level of expression and the critical dependence of their native conformation on the hydrophobic transmembrane lipid environment. Here, we investigate and compare three different strategies to immobilize rhodopsin, a prototypical G-protein coupled receptor on a sensor chip surface using antibodies and a lectin for receptor capturing. By further probing of different experimental conditions (pH, detergent type) we identified the optimal factors to maintain rhodopsin in a functional conformation and extended this approach to recombinant rhodopsin that was heterologously expressed in COS cells. Functional operation of rhodopsin on the sensor chip surface was proven by its activation and subsequent light-stimulated G-protein coupling. The influence of these experimental parameters on the association and dissociation kinetics of G-protein receptor coupling was determined. Thereby, we found that the kinetics of Gt interaction were not changed by the strategy of immobilization or the type of detergent. Regeneration of opsin directly on a chip allowed recycling of the immobilized native and recombinant receptor. Thus, the approach provides an experimental framework for choosing the most suitable conditions for the solubilization, immobilization, and for functional tests of rhodopsin on a biosensor surface.

Light-triggered binding of the G-protein transducin to recombinant rhodopsin on a biosensor surface. Rhodopsin is immobilized via a specific antibodies, interaction is monitored by surface plasmon resonance

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Acknowledgments

We thank Dr. Paul Hargrave and Dr. Clay Smith for providing us with an anti-rhodopsin antibody. This work has been supported by grants SAF2005-08148-C04-02 and Acciones Integradas Hispano-Alemanas (HA2006-0130) from the Spanish Ministry of Science (to PG). Support was also from a grant of the EWE Stiftung (to KWK) and from the Deutscher Akademischer Austauschdienst DAAD within the program Acciones Integradas Hispano-Alemanas (to KWK). MA is the recipient of a predoctoral FPI fellowship from the Spanish Ministry of Science and DT is the recipient of a predoctoral fellowship from Universitat Politècnica de Catalunya (UPC).

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Authors and Affiliations

  1. Biochemistry group, Faculty V, IBU, University of Oldenburg, 26111, Oldenburg, Germany

    Konstantin E. Komolov & Karl-Wilhelm Koch

  2. Departament d’Enginyeria Química, Centre de Biotecnologia Molecular, Universitat Politècnica de Catalunya, Colom 1, 08222, Terrassa, Spain

    Mònica Aguilà, Darwin Toledo & Pere Garriga

  3. Universitat Autònoma de Barcelona, Departament de Bioquímica i de Biologia Molecular, Unitat de Biofisica, and Centre d’Estudis en Biofísica, 08193, Bellaterra, Spain

    Joan Manyosa

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  1. Konstantin E. Komolov
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  2. Mònica Aguilà
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  3. Darwin Toledo
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  4. Joan Manyosa
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  5. Pere Garriga
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Correspondence to Pere Garriga or Karl-Wilhelm Koch.

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Komolov, K.E., Aguilà, M., Toledo, D. et al. On-chip photoactivation of heterologously expressed rhodopsin allows kinetic analysis of G-protein signaling by surface plasmon resonance spectroscopy. Anal Bioanal Chem 397, 2967–2976 (2010). https://doi.org/10.1007/s00216-010-3876-4

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