BackgroundSurfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., ∼residues 8–25 and 63–78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1–7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity.Methodology/ResultsFTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary α-helix and secondary β-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a “saposin-like” fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B.ConclusionSuper Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.

Surfactant protein B (SP-B) is essential for normal lung surfactant function. Theoretical models predict that the disulfide cross-linked, N- and C-terminal domains of SP-B fold as charged amphipathic helices, and suggest that these adjacent helices participate in critical surfactant activities. This hypothesis is tested using a disulfide-linked construct (Mini-B) based on the primary sequences of the N- and C-terminal domains. Consistent with theoretical predictions of the full-length protein, both isotope-enhanced Fourier transform infrared (FTIR) spectroscopy and molecular modeling confirm the presence of charged amphipathic alpha-helices in Mini-B. Similar to that observed with native SP-B, Mini-B in model surfactant lipid mixtures exhibits marked in vitro activity, with spread films showing near-zero minimum surface tensions during cycling using captive bubble surfactometry. In vivo, Mini-B shows oxygenation and dynamic compliance that compare favorably with that of full-length SP-B. Mini-B variants (i.e. reduced disulfides or cationic residues replaced by uncharged residues) or Mini-B fragments (i.e. unlinked N- and C-terminal domains) produced greatly attenuated in vivo and in vitro surfactant properties. Hence, the combination of structure and charge for the amphipathic alpha-helical N- and C-terminal domains are key to SP-B function.