Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins (OBPs), using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ~100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ~200 nM for the silk moth pheromone bombykol and ~90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the pheromone receptor model proposed by Laughlin et al. (Cell 133: 1255–65, 2008) are discussed.

Forster resonance energy transfer (FRET) can be used as a spectroscopic ruler to measure nanometer-scale distances. The recovery of inter-dye distance depends on a calibration factor known as the Forster critical distance (R 0 ). This distance is currently estimated based on measurements of the quantum yield of the donor dye, the overlap integral between the donor and acceptor dyes, and assumptions about the index of refraction and the relative orientation of the donor and acceptor dye molecules.Here, we report a method to experimentally measure R 0 , using B-DNA as a structural reference. Fifteen donor (Cy3)-labeled oligonucleotides were generated, by placing donor-labeled Thymidines at positions 11, 14, /, 39. A single complementary strand was synthesized with acceptor (AlexaFluor647) at position 10. The strands were annealed, producing dsDNA consisting of a 30 base pair (bp) ruler with a 10 bp cap on each end. For each freely diffusing construct, the mean transfer efficiency (TE) was measured by singlepair FRET (sp-FRET) and ensemble (en-FRET). The TE's as a function of bp were fit to a reduced representation model of B-DNA that provided the absolute inter-dye distances. The reduced model was formulated based on an atomistic model of dye-labeled B-DNA, R 0 was recovered from the fit. We repeated our approach using three different donor/acceptor pairs, each with a different R 0 .

Luminescence resonance energy transfer (LRET) offers many advantages for accurate measurements of distances between specific sites in living cells, but progress in developing methodology for implementing this technique has been limited. We report here the design, expression, and characterization of a test protein for development of LRET methodology. The protein, which we call DAL, contains the following domains (from the N-terminus): E. coli dihydrofolate reductase (DHFR), the third and fourth ankyrin repeats of p16INK4a, a lanthanide-binding tag (LBT), and a hexahistidine tag. LBT binds Tb3+ with a sub-micromolar dissociation constant. LRET was measured from the Tb3+ site on LBT to transition metals bound to the hexa-His tag, and to fluorescein methotrexate bound to DHFR. The measured distances were consistent with a molecular model constructed from the known crystal structures of the constituent domains of DAL. The results indicate that the two C-terminal ankyrin domains of p16INK4a are stably folded when combined with other protein domains. We found that Tb3+ binds to DAL in the cytoplasm of live E. coli cells, and thus DAL is useful as an indicator for studies of metal transport. We also used DAL to measure LRET from Tb3+ to Cu2+ in the cytoplasm of live E. coli cells. The rates of Tb3+ and Cu2+ transport were not affected by a proton uncoupler or an ATP synthase inhibitor. Reversal of the membrane potential had a small inhibitory effect, and removal of lipopolysaccharide had a small accelerating affect on transport. Changing the external pH from 7 to 5 strongly inhibited the Tb3+ signal, suggesting that the Tb3+-LBT interaction is useful as a cytoplasmic pH indicator in the range of about pH 5-6.