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B. J. Tobe, T. G. Wensel; Monoclonal Antibody Binding Stoichiometry Reveals Rhodopsin Dimers in Native Membranes. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4668.
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To measure the dimerization equilibrium of rhodopsin in disk membranes.
The concentrations of rhodopsin and a monoclonal antibody, 1D4, that binds its carboxyl terminus can be measured with good accuracy and precision. In proposed structures of rhodopsin dimers, the carboxyl termini are positioned so that there is only room for one antibody Fab fragments to bind to each dimer. Therefore measurements of antibody binding stoichiometry were used to probe the dimerization equilibrium.
We have determined that in disk membranes of bovine rod outer segments, a single 1D4 full length bivalent antibody binds per 4 rhodopsins, and that a single monovalent 1D4 Fab binds per 2 rhodopsins. Very high antibody concentrations, long incubation times and sonication of the membranes do not change these stoichiometries. At least 98% of detergent extracted native membrane rhodopsin binds to immobilized 1D4, indicating the carboxyl-terminal epitopes are intact. Virtually all native membrane rhodopsin C-termini are accessible to an added protein, endoproteinase Asp-N. Prebinding of native membrane rhodopsin with excess 1D4 effectively inhibits phosphorylation of the entire population of photoactivated rhodopsins and also inhibits the activation of transducin.
The simplest explanation for the 1:2 binding stoichiometry is that neighboring rhodopsins in each dimer within the membrane are in such close proximity, that the binding of one antibody to rhodopsin prevents another rhodopsin from being bound. The measured affinity of 1D4 for rhodopsin is high affinity (Kd<100 nM) and because rhodopsin is known to be very mobile in the fluid disk membranes, these results indicate not only that dimers can form, but that they are very stable, with a dissociation constant more than three orders of magnitude lower than the density of rhodopsin in the membranes. The position of the carboxyl terminus in photoactivated rhodopsin, R*, is such that 1D4 binding inhibits both rhodopsin phosphorylation and transducin activation, suggesting close proximity to the cytoplasmic loops that interact with transducin.
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