Intrinsic Structural and Functional Determinants within the Amino Acid Sequence of Mature Pulmonary Surfactant Protein SP-B

Abstract

Pulmonary surfactant protein SP-B is absolutely required for proper function of surfactant in the alveoli, and is an important component of therapeutical surfactant preparations used to treat respiratory pathologies. To explore inherent structural and functional determinants within the amino acid sequence of mature SP-B, porcine SP-B has been subjected to extensive disulfide reduction under highly denaturing conditions and to cysteine carboxyamidomethylation, and the structure, lipid-protein interactions, and surface activity of this modified form have been characterized. Refolding of the reduced protein yielded a form (SP-Br) with secondary structure practically identical to that of the native disulfide-linked SP-B dimer. Reduced SP-Br exhibited higher structural flexibility than native SP-B, as indicated by a higher susceptibility of fluorescence emission to quenching by acrylamide and biphasic behavior during interaction of the protein with lipid bilayers and monolayers. SP-Br had, however, effects similar to those of native SP-B on the thermotropic properties of dipalmitoylphosphatidylcholine (DPPC) bilayers. SP-Br was more effective than native SP-B in promoting interfacial adsorption of phospholipid bilayers into interfacial films, presumably because of its higher structural flexibility, and retained the ability of native SP-B to stabilize DPPC interfacial films compressed to pressures near collapse against spontaneous relaxation. SP-Br also mimicked the behavior of native SP-B in lipid-protein films subjected to dynamic compression-expansion cycling in a captive bubble surfactometer, but only in the presence of phosphatidylglycerol (PG), the main anionic phospholipid in surfactant. The presence of PG appears to be required for SP-Br to acquire the appropriate tertiary folding to produce progressively more efficient lipid-protein films capable of reaching very high pressures upon limited compression with almost no hysteresis.

A model for the structure and mechanism of action of pulmonary surfactant protein B

“…This interpretation of the participation of the different segments of SP‐B in the protein‐lipid interactions is fully compatible with the functional activities assigned to SP‐B segments as a result of experiments that used partial synthetic peptides of this protein (8). The strict requirement of the N‐terminal segment to achieve an appropriate surface active behavior seems to rely on its function as a membrane anchor (41–43) in which residue W9 is particularly involved (44). N‐ and C‐terminal helices 1 and 5 mediate electrostatic interactions with the membranes, and they would also be important for the surface activity of SP‐B as well as for membrane fusion and lysis (8, 44) because, according to our model, these helices create the main surface that lays on the surface of the surfactant layers.…”

Close mimicry of lung surfactant protein B by “clicked” dimers of helical, cationic peptoids

“…Although chemically synthesized SP-B 1-78 has exhibited good in vitro surface activity, it was still inferior when compared to the naturally isolated protein29,56. Biophysical functioning of natural SP-B was inhibited if the protein was completely reduced57, or if homo-dimerization was prohibited transgenically or through the position-specific substitution of Cys48 → Ser in the sequence58,59. Although totally reduced forms of natural SP-B demonstrated surface activity, the protein behaved in a manner that is congruent with increased structural flexibility57.…”

Protein–lipid interactions and surface activity in the pulmonary surfactant system

“…Whatever the molecular mechanism, SP-B is the most active polypeptide, among surfactant proteins, in promoting phospholipid adsorption from the hypophase to the air-liquid interface (OosterlakenDijksterhuis et al, 1991a,b;Nag et al, 1999;Cruz et al, 2000), although additive and synergistic effects have also been reported for the simultaneous presence of SP-C (Oosterlaken-Dijksterhuis et al, 1991a,b;Wang et al, 1996) or SP-A (Venkitaraman et al, 1990;Schürch et al, 1992;Bi et al, 2001;Possmayer et al, 2001), respectively. On the other hand, SP-B has also a potential role in maintaining the stability of highly compressed films (Nag et al, 1999;Cruz et al, 2000;Krol et al, 2000;Serrano et al, 2005) as well as in facilitating the respreading of surfactant material during expansion, so that minimum surface tension can still be attained after successive compression-expansion cycles (OosterlakenDijksterhuis et al, 1991a,b;Taneva and Keough, 1994;Krol et al, 2000;Veldhuizen et al, 2000;Zaltash et al, 2000). It has been proposed that SP-B could act as a bridge between bilayers or bilayers and monolayers (Fig.…”