Infrared spectroelectrochemical detection of protein - Protein interactions involved in phototransduction process in a model membrane embedding rhodopsin.

A sparsely tethered membrane containing uniformly oriented rhodopsin was fabricated on a gold electrode surface by bicelles spreading. This model membrane system served to investigate a cascade of reactions involved in G-protein signaling of the phototransduction process. Electrochemical impedance spectroscopy results showed a large decrease in the capacitance of the spacer layer and an increase in the capacitance of the membrane upon binding of the G-protein (Gt), indicating membrane swelling. In situ polarization modulation infrared reflection absorption spectroscopy experiments, we observed an increase in the tilt of the helical bundle in photoactivated rhodopsin by ca. 10° towards the membrane plane. Association of Gt was connected with a thinning of the membrane, a process that accompanies the bilayer swelling detected as electrochemical changes of the membrane and spacer layer capacitance and conformational/hydration changes in the helical fragments of the interacting proteins. After the addition of guanosine 5'-O-[gamma-thio]-triphosphate, nucleotide exchange, and splitting of the Gtα and Gtβγ subunits, the environment on the helical bundle of opsin is restored, indicating a dissociation of the protein from opsin and partially from the membrane. This transition of our membrane model system resembles the trigger steps of the phototransduction process by maintaining membrane integrity and function.