Abstract
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.
Highlights
Lung surfactant is a complex mixture of lipids and proteins that is required for normal breathing, due to its ability to reduce alveolar surface tension to very low values
Final oxidized MB and Super Mini-B (S-MB) were repurified by reverse phase HPLC, verified in molecular mass via MALDI-TOF, and disulfide connectivity was confirmed by mass spectroscopy of enzyme-digested fragments
Representative Fourier transform infrared (FTIR) spectra of the amide I band for MB in these environments (Fig. 3A) were all similar, each indicating a major component centered at,1651–1657 cm21 with a small low-field shoulder at,1620 cm21
Summary
Lung surfactant is a complex mixture of lipids (mostly phospholipids) and proteins that is required for normal breathing, due to its ability to reduce alveolar surface tension to very low values. Surfactant therapy using bovine or porcine lung extracts surfactant extracts, which contain only polar lipids and native SP-B and SP-C, has greatly improved the therapeutic outcomes of neonates with respiratory distress (NRDS). 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; Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. 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 involved in surfactant activity
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