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 information regarding average structural properties of membrane films supported on planar solid substrates. Here we have used SPR spectroscopy for the first time to monitor the binding and activation of G-protein (transducin or Gt) by bovine rhodopsin incorporated into an egg phosphatidylcholine bilayer deposited on a silver film. Rhodopsin incorporation into the membrane, performed by dilution of a detergent solution of the protein, proceeds in a saturable manner. Before photolysis, the SPR data show that Gt binds tightly (Keq approximately equal to 60 nM) and with positive cooperativity to rhodopsin in the lipid layer to form a closely packed film. A simple multilayer model yields a calculated average thickness of about 57 A, in good agreement with the structure of Gt. The data also demonstrate that Gt binding saturates at a Gt/rhodopsin ratio of approximately 0.6. Moreover, upon visible light irradiation, characteristic changes occur in the SPR spectrum, which can be modeled by a 6 A 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 induced. These are interpreted as resulting from dissociation of the alpha-subunit of Gt, formation of new MII-Gt complexes, and possible conformational changes of Gt as a consequence of complex formation. The above results clearly demonstrate the ability of SPR spectroscopy to monitor interactions among the proteins associated with signal transduction in membrane-bound systems.
Salamon
Z
Z
Department of Biochemistry, University of Arizona, Tucson 85721, USA.
Wang
Y
Y
Soulages
J L
JL
Brown
M F
MF
Tollin
G
G
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002417:Cattle; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D008027:Light; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008567:Membranes, Artificial; D011485:Protein Binding; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D013057:Spectrum Analysis; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism
D008051:Lipid Bilayers; D008565:Membrane Proteins; D008567:Membranes, Artificial; D006160:Guanosine Triphosphate; D012243:Rhodopsin; D015293:Transducin