Synthesis, secondary structure and folding of the bend region of lung surfactant protein B

Previous theoretical analysis of the primary structure of lung surfactant protein SP-B indicates a disulfide-linked, hydrophobic midsequence that forms a hairpin-like motif. Here, we experimentally investigate the secondary structure of the disulfide-stabilized bend region by synthesizing two 12-residue analogs of the SP-B midsequence. The native peptide has the same sequence for residues 35 to 46 as native human SP-B, while, in the second mimic peptide, Leu40 and Val41 were replaced with D-Ser and L-His. Both peptides contain cysteine residues at the N- and C-terminus (Cys35 and Cys46, respectively). Oxidation/reduction experiments with fast atom bombardment mass spectrometry showed mass shifts of approximately 2 daltons, consistent with the oxidized peptides existing in solution as monomers, each with one internal disulfide bond (Cys35-Cys46). Since circular dichroism and Fourier-transform infrared measurements show that both peptides assume turn conformations in structure-promoting solvents such as trifluoroethanol (TFE), a structural model is proposed in which Cys35 and Cys46 are brought in close apposition through an internal bend in the peptide. Consistent with this model are electron spin resonance (ESR) results of the mimic peptide nitroxide spin-labeled at Cys35 and Cys46. For the double spin-labeled mimic peptide in TFE. ESR spectra indicated broadening characteristic of either radical interactions or decreased mobility, or both. Increases in radical interactions for the double spin-labeled mimic peptide would be expected for Cys35 and Cys46 approaching within 14 A in structure-promoting solvents, while decreases in spin-label mobility could be due to the formation of a loop. Based on these observations with peptide analogs, residues 35 to 46 probably form a similar bend in the full-length protein.