A molecular model for singlet/singlet energy transfer of monovalent ligand/receptor interactions

A stochastic model is described that predicts the degree of singlet/singlet energy transfer in complexes formed between monovalent ligands and monovalent receptors. The modeling approach is intended to serve as an analytical tool for approximating the level of fluorescence quenching that can be expected to occur in fluorescently labeled monovalent ligands and receptors that are bound together in complexes. This approach has utility in areas such as modeling protein/protein interactions and designing fluorescence energy transfer assays.Using the crystallographic data for papain (monovalent ligand ) and concanavalin A (monovalent receptor ) along with a molecular graphics computational package the ligand and receptor were docked together to form a ligand/receptor complex. The intermolecular distances between the lysine resides of the ligand and receptor were then estimated, receptor complex was calculated assuming a value for the characteristic length R(0) of the donor/acceptor pair. Results from the stochastic model were used to calculate the level of fluorescence quenching one would expect for a resonance energy transfer competition assay based on the monovalent ligand/pair.Three key assumptions were made during the model development. First, all lysine resides for the ligand and receptor were equally reactive with the dye molecules so the stoichiometry of the donor and acceptor chromophores was governed by a binomial distribution. Second, the dye molecules were located at the alpha-carbon position for each reactive lysine residue. Finally, in the energy transfer competition assay, it was assumed that equilibrium existed between the ligand, receptor, and competing hapten at all times. Based on these assumptions, results are presented that indicate the maximum energy transfer for the monovalent papain/concanavalin. A complex is strongly dependent on the number of acceptor chromophores and on the value of R(0). Results are also presented on the approximate level of fluorescence quenching that may occur in a competition assay based on the papain/pConA complex. Lastly, a strategy is discussed for maximizing the dynamic range and linearity of energy transfer assays by optimizing several key design variables.