title: The hydrophobic pocket of 24p3 protein from mouse uterine luminal fluid: fatty acid and retinol binding activity and predicted structural similarity to lipocalins.
abstract: 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 beta-form, beta-turn, and unordered form in the protein molecule. This agrees with the predicted secondary structures consisting of only one a-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 beta-forms, which would construct two orthonormal beta-sheets to form a less polar beta-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 CsCI or KI suggests that they are located in the less polar beta-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 x 10(6) MM(-1), 1.92 x 10(5) M(-1), 2.38 x 10(5) M(-1) or 1.25 x 10(5) M(-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 x 10(5) M(-1) and 1.17 x 10(5) M(-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.
authors.affiliation: Institute of Biological Chemistry, Academica Sinica, National Taiwan University, Taipei, Republic of China. stc316@gate.sinica.edu.tw
authors.forename: S T
authors.initials: ST
authors.lastname: Chu
authors.forename: H J
authors.initials: HJ
authors.lastname: Lin
authors.forename: H L
authors.initials: HL
authors.lastname: Huang
authors.forename: Y H
authors.initials: YH
authors.lastname: Chen
mesh_terms: D000209:Acute-Phase Proteins / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D005260:Female; D000071068:Lipocalin-2; D054834:Lipocalins; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D014364:Tryptophan; D014599:Uterus / Q000378:metabolism; D014801:Vitamin A / Q000378:metabolism*
chemical_list: D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D005227:Fatty Acids; D000071068:Lipocalin-2; D054834:Lipocalins; D015513:Oncogene Proteins; D014801:Vitamin A; C071332:Lcn2 protein, mouse; D014364:Tryptophan