COUPLED PLASMON-WAVE-GUIDE RESONANCE SPECTROSCOPY STUDIES OF THE CYTOCHROME B(6)F PLASTOCYANIN SYSTEM IN SUPPORTED LIPID BILAYER-MEMBRANES/

The incorporation of cytochrome (cyt) b(6)f into a solid-supported pla nar egg phosphatidylcholine (PC) bilayer membrane and complex formatio n with plastocyanin have been studied by a variant of surface plasmon resonance called coupled plasmon-waveguide resonance(CPWR) spectroscop y, developed in our laboratory. CPWR combines greatly enhanced sensiti vity and spectral resolution with direct measurement of anisotropies i n refractive index and optical extinction coefficient, and can therefo re probe structural properties' of lipid-protein and protein-protein i nteractions. Cyt b(6)f incorporation into the membrane proceeds in two stages. The first occurs at low protein concentration and is characte rized by an increase in total proteolipid mass without significant cha nges in the molecular order of the system, as demonstrated by shifts o f the resonance position to larger incident angles without changing th e refractive index anisotropy. The second stage; occurring at higher p rotein concentrations, results in a decrease in both the mass density and the molecular order of the system, evidenced by shifts of the reso nance position to smaller incident angles and a large decrease in the membrane refractive index anisotropy. Plastocyanin can bind to such a proteolipid system in three different ways. First, the addition of pla stocyanin before the second stage of b(6)f incorporation begins result s in complex formation:between the two proteins with a K-D of similar to 10 mu M and induces structural changes in the membrane that are sim ilar to those occurring during the second stage of complex incorporati on. The addition of larger amounts of plastocyanin under these conditi ons leads to nonspecific binding to the lipid phase with a K-D of simi lar to 180 mu M. Finally,;the addition of plastocyanin after the compl etion of the second phase of b(6)f incorporation results in tighter bi nding between the two proteins (K-D approximate to 1 mu M) Quantitatio n of the binding stoichiometry indicates that two plastocyanin molecul es bind tightly to the dimeric form of the cyt b(6)f complex, assuming random insertion of the cytochrome into the bilayer. The structural b asis for these results and formation of the proteolipid membrane are d iscussed.