SURFACE-PLASMON RESONANCE SPECTROSCOPY STUDIES OF MEMBRANE-PROTEINS -TRANSDUCIN BINDING AND ACTIVATION BY RHODOPSIN MONITORED IN THIN MEMBRANE FILMS

Surface plasmon resonance (SPR) spectroscopy can provide useful inform ation regarding average structural properties of membrane films suppor ted on planar solid substrates. Here we have used SPR spectroscopy for the first time to monitor the binding and activation of G-protein (tr ansducin or G(t)) by bovine rhodopsin incorporated into an egg phospha tidylcholine bilayer deposited on a silver film. Rhodopsin incorporati on into the membrane, performed by dilution of a detergent solution of the protein, proceeds in a saturable manner. Before photolysis, the S PR data show that G(t) binds tightly (K-eq approximate to 60 nM) and w ith positive cooperativity to rhodopsin In the lipid layer to form a c losely packed film. A simple multilayer model yields a calculated aver age thickness of about 57 Angstrom, in good agreement with the structu re of G(t). The data also demonstrate that G(t) binding saturates at a G(t)/rhodopsin ratio of approximately 0.6. Moreover, upon visible lig ht irradiation, characteristic changes occur in the SPR spectrum, whic h can be modeled by a 6 Angstrom increase in the average thickness of the lipid/protein film caused by formation of metarhodopsin II (MII). Upon subsequent addition of GTP, further SPR spectral changes are indu ced. These are interpreted as resulting from dissociation of the alpha -subunit of G(t), formation of new MII-G(t) complexes, and possible co nformational changes of G(t) as a consequence of complex formation. Th e above results clearly demonstrate the ability of SPR spectroscopy to monitor interactions among the proteins associated with signal transd uction in membrane-bound systems.