The conformation of 24p3 protein purified from mouse uterine luminal fluid was studied by circular dichroism spectroscopy in 200-300 nm. At pH 7.4, the spectrum in the UV region appears as one negative band with a minimum mean residue ellipticity of -3,600 deg·cm2·dmole-1 at 217 nm, suggesting a very low or no helical content, but a considerable amount of β-form, β-turn, and unordered form in the protein molecule. This agrees with the predicted secondary structures consisting of only one α-helical segment of residues 150-163 and nine segments of residues 28-35, 50-60, 67-72, 78-86, 94-97, 106-114, 119-125, 136-140 and 166-172 in β-forms, which would construct two orthonormal β-sheets to form a less polar β-barrel. The environments around Trp-31 and Trp-81 of this protein were studied by intrinsic fluorescence and solute quenching. They give an emission peak at 332 nm, and only about 21% of them are accessible to quenching by acrylamide. This together with their low accessibility to either CsCl or KI suggests that they are located in the less polar β-barrel.
Hydrophobic compounds such as fatty acids, retinoids, and cholesteryl oleate in the protein solution diminish the protein fluorescence. Analysis of the fluorescence data suggests that the protein has a binding site for hydrophobic ligand. The association constants for the complex formation are 1.03 × 106M-1, 1.92 × 105M-1, 2.38 × 105M-1 or 1.25 × 105M-1 for cholesteryl oleate, oleic acid, retinol, or retinoic acid at pH 7.4. Analysis of the equilibrium binding data from binding assay using [H3]-retinol and [H3]-retinoic acid reveals a singular type of retinoid-binding site in the protein with the association constant of 4.92 × 105M-1 and 1.17 × 105M-1 for retinol and retinoic acid, respectively. Trp-31 or/and Trp-81 is in or very near the binding site and the gross conformation of protein changes considerably as the formation of protein-ligand complex.