Different modes of interaction of pulmonary surfactant protein SP-B in phosphatidylcholine bilayers

Abstract

Pulmonary surfactant-associated protein B (SP-B) has been incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (PC) by two different procedures to characterize the dependence of lipid–protein interactions on the method of reconstitution. In method A the protein was dissolved in a small volume of either methanol or 60% (v/v) acetonitrile and injected into an aqueous phase containing phospholipid vesicles. In method B the vesicles were prepared by injection of a mixture of phospholipid and SP-B dissolved in methanol or aqueous acetonitrile. Both methods of reconstitution led to the extensive interaction of SP-B with PC bilayers as demonstrated by co-migration during centrifugation, marked protection against proteolysis, change in the fluorescence emission intensity of SP-B, and protection of SP-B tryptophan fluorescence from quenching by acrylamide. SP-B promoted the rapid adsorption of DPPC on an air/liquid interface irrespective of the method of protein reconstitution. However, the interfacial adsorption activity of SP-B reconstituted by method B remained stable for hours, but that of SP-B prepared by method A decreased with time. Electron microscopy showed that the injection of SP-B into an aqueous phase containing PC or DPPC vesicles (method A) induced a rapid aggregation of vesicles. By contrast, a much longer time was required for detecting vesicle aggregation when the protein was reconstituted by co-injection of SP-B and phospholipids (method B). The presence of 5% (w/w) SP-B in DPPC bilayers prepared by method B broadened the differential scanning calorimetry thermogram and decreased the enthalpy of the transition. In contrast, the injection of SP-B into preformed DPPC vesicles (method A) did not influence the gel-to-liquid phase transition of DPPC bilayers. Taken together, these results indicate that the mode and extent of interaction of SP-B with surfactant phospholipids depends on the conditions of preparation of lipid/protein samples, and that care should be taken in the interpretation of findings from reconstituted systems on the role of these surfactant proteins in the alveolar space.