Surface plasmon resonance spectroscopy studies of membrane proteins: Transducin binding and activation by rhodopsin monitored in thin membrane films

Z. Salamon, Y. Wang, J. L. Soulages, M. F. Brown, G. Tollin

Research output: Contribution to journalArticle

79 Scopus citations

Abstract

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 G(t)) 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 G(t) binds tightly (K(eq) ≃ 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 .Å, in good agreement with the structure 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 light irradiation, characteristic changes occur in the SPR spectrum, which can be modeled by a 6 Å 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 α- subunit of G(t), formation of new MII-G(t) complexes, and possible conformational changes of G(t) 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.

Original languageEnglish (US)
Pages (from-to)283-294
Number of pages12
JournalBiophysical Journal
Volume71
Issue number1
DOIs
StatePublished - Jul 1996

ASJC Scopus subject areas

  • Biophysics

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