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

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 G(t) 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.

Analytical methods for kinetic studies of biological interactions: A review

“…Validation of SPR with reference methods is ideally required when this system is used to study what is normally a solution-phase interaction. However, if the immobilization method and conditions are properly selected, the results obtained by SPR can give good agreement with those seen in solution and by other techniques [89,97]. …”

“…One recent report examined the choice of suitable conditions for the kinetic analysis of G-protein signaling, based on the use of immobilized native rhodopsin (Rho, a G-protein coupled receptor) and transducin [89]. A number of antibody-antigen interactions have been examined by using SPR [90–92], and this technique has been applied in studying the DNA-protein interactions [93,94].…”

“…For small molecules, the number and types of functional groups that are present may limit the options for immobilization. If the coupling conditions are not properly selected, whether it is for a large or small molecule, this can lead to some changes in the binding properties of the immobilized agent [3,5,89]. Validation of SPR with reference methods is ideally required when this system is used to study what is normally a solution-phase interaction.…”

Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization

“…SPR experiments were performed using Biacore 2000 (GE healthcare, Germany). Two flow cells of the CMD 50 d SPR sensorchip (Xantec Bioanalytics, Germany) were activated as previously described [21]. Each flow cell was then coated with Rho-1D4 as the primary antibody for the working surface and IgG1 (Sigma, Germany) for a negative control surface, both at a concentration of 0.1mg/ml in 10mM sodium acetate buffer pH 5.…”

“…We have been able to successfully characterize the rhodopsin activation process by means of this technique [21]. In spite of the many advantages of this technique like high sensitivity and reliability, its application to GPCR analysis is still under development [39].…”