External reflection absorption infrared spectroscopy study of lung surfactant proteins SP-B and SP-C in phospholipid monolayers at the air/water interface

Abstract

The interactions of the hydrophobic pulmonary surfactant proteins SP-B and SP-C with 1,2-dipalmitoylphosphatidylcholine in mixed, spread monolayer films have been studied in situ at the air/water interface with the technique of external reflection absorption infrared spectroscopy (IRRAS). SP-C has a mostly alpha-helical secondary structure both in the pure state and in the presence of lipids, whereas SP-B secondary structure is a mixture of alpha-helical and disordered forms. When films of SP-B/1,2-dipalmitoylphosphatidylcholine are compressed to surface pressures (pi) greater than approximately 40-43 mN/m, the protein is partially (15-35%) excluded from the surface, as measured by intensity ratios of the peptide bond amide l/lipid C==O stretching vibrations. The extent of exclusion increases as the protein/lipid ratio in the film increases. In contrast, SP-C either remains at the surface at high pressures or leaves accompanied by lipids. The amide l peak of SP-C becomes asymmetric as a result of the formation of intermolecular sheet structures (1615-1630 cm-1) suggestive of peptide aggregation. The power of the IRRAS experiment for determination of film composition and molecular structure, i.e., as a direct test of the squeeze-out hypothesis of pulmonary surfactant function, is evident from this work.

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

“…The exact three-dimensional structure of SP-B is still unknown. There are abundant experimental data obtained from indirect techniques such us circular dichroism (Andersson et al, 1995), infrared spectroscopy (Vandenbussche et al, 1992;Pastrana-Rios et al, 1995), spin resonance (Perez-Gil et al, 1995) or fluorescence anisotropy (Baatz et al, 1990), that provide a good picture of the conformation and disposition of the polypeptide, either in solvents or in membrane environments. Also, sequence alignments reveal that the location of the Cys residues in SP-B is a common feature among a group of proteins referred to as the saposin-like proteins (SAPLIP) (Munford et al, 1995).…”

Synthetic Pulmonary Surfactant Preparations: New Developments and Future Trends

“…However, a molecular structure of SP-B, the only member of the saposin family that is permanently associated with membranes, is still not available despite the fact that some molecular modelling efforts have been reported [69,70]. Numerous studies have approached the analysis of structural determinants in SP-B by means of indirect techniques such as circular dichroism [71,72] [73], fluorescence [74] or infrared spectroscopies [75,76], or electron spin resonance (ESR) [77]. The protein structure is dominated by a 40-50% "-helix, predicted to be in the form of amphipathic helical segments with well-defined polar and non-polar faces.…”

Reflection–absorption FT-IR spectroscopy of pentadecanoic acid at the air/water interface

“…Increases in the integrated peak area of w a (CH 2 ) and w s (CH 2 ) as the monolayer is compressed result not only from an increase in the concentration of film molecules on the surface but may also arise from a change in the direction of the dipole moment for the stretching vibration indicating a decrease in the average tilt angle of the ordered hydrocarbon chains [7,43,44]. Gericke et al [45,46] have made extensive use of relative peak intensities and integrated peak areas for w a (CH 2 ) to gain qualitative information on changes in the tilt angle.…”