Ligand-binding assays

Direct binding of fluorescent probes was monitored by adding aliquots of 1 mM methanol solution of the probe to a 2 μM solution of the protein in 50 mM Tris–HCl, pH 7.4 to final concentrations of 2 to 16 μM. The excitation wavelength was 337 nm and intensities were measured on the maximum of the peak, usually between around 410 and 420 nm. For VdesOBP1 and VdesOBP2, binding of ligands was evaluated by monitoring the intrinsic tryptophan quenching of the protein. Excitation wavelength was 295 nm and intensities were recorded around 335–340 nm. For VdesOBP4 and VdesOBP5, competitive binding experiments were performed by titrating a solution of protein and 1-NPN both at 2 μM in Tris–HCl buffer, pH 7.4 with 1 mM methanolic solutions of ligands to final concentrations of 2–16 μM. All measurements were performed with a FL 6500 spectrofluorometer (PerkinElmer); slits were set at 5 nm for both excitation and emission, and 1 cm path quartz cuvettes were used.

Dissociation constants for 1-NPN were evaluated using Prism software. Affinities of other ligands were calculated from the corresponding [IC]₅₀ values (the concentration of each ligand halving the initial value of fluorescence), from the equation:

where [1-NPN] is the concentration of free 1-NPN and K_NPN is the dissociation constant of the complex OBP/1-NPN.