On-chip photoactivation of heterologously expressed rhodopsin allows kinetic analysis of G-protein signaling by surface plasmon resonance spectroscopy.
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.
Komolov
Konstantin E
KE
Biochemistry group, Faculty V, IBU, University of Oldenburg, 26111 Oldenburg, Germany.
Aguilà
Mònica
M
Toledo
Darwin
D
Manyosa
Joan
J
Garriga
Pere
P
Koch
Karl-Wilhelm
KW
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D055668:Photochemical Processes / Q000528:radiation effects; D011485:Protein Binding / Q000528:radiation effects; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000528:radiation effects; D020349:Surface Plasmon Resonance / Q000379:methods*
D012243:Rhodopsin; D019204:GTP-Binding Proteins