Thermal stability and DPPC/Ca2+ interactions of pulmonary surfactant SP-A from bulk-phase and monolayer IR spectroscopy

Surfactant protein A (SP-A), the most abundant pulmonary surfactant protein , is implicated in multiple biological functions including surfactant homeo stasis, biophysical activity, and host defense. SP-A forms ternary complexe s with lipids and Ca2+ which are important for protein function. The curren t study uses infrared (IR) transmission spectroscopy to investigate the bul k-phase interaction between SPA, 1,2-dipalmitoylphosphatidylcholine (DPPC), and Ca2+ ions along with IR reflection-absorption spectroscopy (IRRAS) to examine protein secondary structure and lipid orientational order in monola yer films in situ at the air/water interface. The amide I contour of SP-A r eveals two features at 1653 and 1636 cm(-1) arising from the collagen-like domain and a broad feature at 1645 cm(-1) suggested to arise from the carbo hydrate recognition domain (CRD). SP-A secondary structure is unchanged in lipid monolayers. Thermal denaturation of SP-A in the presence of either DP PC or Ca2+ ion reveals a sequence of events involving the initial melting o f the collagen-like region, followed by formation of intermolecular extende d forms. Interestingly, these spectral changes were inhibited in the ternar y system, showing that the combined presence of both DPPC and Ca2+ confers a remarkable thermal stability upon SP-A. The ternary interaction was revea led by the enhanced intensity of the asymmetric carboxylate stretching vibr ation. The IRRAS measurements indicated that incorporation of SP-A into pre formed DPPC monolayers at a surface pressure of 10 mN/m induced a decrease in the average acyl chain tilt angle from 35 to 28. In contrast, little cha nge in chain tilt was observed at surface pressures of 25 or 40 mN/m. These results are consistent with and extend the fluorescence microscopy studies of Keough and co-workers [guano, M. L. F., et al. (1998) Biophys. J. 74, 1 101-1109] in which SP-A was suggested to accumulate at the liquid-expanded/ liquid-condensed boundary. Overall these experiments reveal the remarkable stability of SP-A in diverse, biologically relevant environments.