The location and depth of each residue of lung pulmonary surfactant protein B (SP-B(1-25)) in a phospholipid bilayer (PB) was determined by fluorescence quenching using synthesized single-residue-substituted peptides that were reconstituted into 1,2-dipalmitoyl phosphatidylcholine (DPPC)-enriched liposomes. The single-residue substitutions in peptides were either aspartate or tryptophan. The aspartate was subsequently labeled with the N-cyclohexyl-N'-(4-(dimethylamino)naphthyl)carbodiimide (NCD-4) fluorophore, whereas tryptophan is autofluorescent. Spin-labeled compounds, 5-doxylstearic acid (5-DSA), 7-doxylstearic acid (7-DSA), 12-doxylstearic acid (12-DSA), 4-(N,N-dimethyl-N-hexadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT-16), and 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxy iodide (CAT-1), were used in the quenching experiments. The effective quenching order is determined by the accessibility of the quencher to a fluorescent group on the peptide. The order of quenching efficiency provides information about the relative locations of individual residues in the PB. Our data indicate that residues Phe1-Pro6 are located at the surface of PB, residues Tyr7-Trp9 are embedded in PB, and residues Leu10-Ile22 are involved in an amphipathic alpha-helix with its axis parallel to the surface of PB; residues Pro23-Gly25 reside at the surface. The effects of intermolecular disulfide bond formation in the SP-B(1-25) dimer were also investigated. The experiments suggest that the SP-B helix A has to rotate at an angle to form a disulfide bond with the neighboring cysteine, which makes the hydrophobic sides of the amphipathic helices face each other, thus forming a hydrophobic domain. The detailed topographical mapping of SP-B(1-25) and its dimer in PB provides new insights into the conformational organization of the lung pulmonary surfactant proteins in the environment that mimics the native state. The environment-specific conformational flexibility of the hydrophobic domain created by SP-B folding may explain the key functional properties of SP-B including their impact on phospholipid transport between the lipid phases and in modulating the cell inflammatory response during respiratory distress syndrome.
Health Effects Laboratory Division, National Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown, WV 26505-2888