Secondary Structure and Lipid Interactions of the N-Terminal Segment of Pulmonary Surfactant SP-C in Langmuir Films:  IR Reflection−Absorption Spectroscopy and Surface Pressure Studies

Abstract

Pulmonary surfactant, a thin lipid/protein film lining mammalian lungs, functions in vivo to reduce the work of breathing and to prevent alveolar collapse. Analogues of two hydrophobic surfactant proteins, SP-B and SP-C, have been incorporated into therapeutic agents for respiratory distress syndrome, a pathological condition resulting from deficiency in surfactant. To facilitate rational design of therapeutic agents, a molecular level understanding of lipid interaction with surfactant proteins or their analogues in aqueous monolayer films is necessary. The current work uses infrared reflection-absorption spectroscopy (IRRAS) to determine peptide conformation and the effects of S-palmitoylation on the lipid interactions of a synthetic 13 residue N-terminal peptide [SP-C13(palm)(2)] of SP-C, in mixtures with 1,2-dipalmitoylphosphatidylcholine (DPPC) or 1,2-dipalmitoylphosphatidylglycerol (DPPG). Two Amide I' features, at approximately 1655 and approximately 1639 cm(-1) in the peptide IRRAS spectra, are assigned to alpha-helical peptide bonds in hydrophobic and aqueous environments, respectively. In binary DPPC/SP-C13(palm)(2) films, the proportion of hydrated/hydrophobic helix increases reversibly with surface pressure (pi), suggestive of the peptide being squeezed out from hydrophobic regions of the monolayer. No such effect was observed for DPPG/peptide monolayers, indicative of stronger, probably electrostatic, interactions. Depalmitoylation produced a weakened interaction with either phospholipid as deduced from IRRAS spectra and from pi-area isotherms. S-Palmitoylation may modulate peptide hydration and conformation in the N-terminal region of SP-C and may thus permit the peptide to remain in the film at the high surface pressures present during lung compression. The unique capability of IRRAS to detect the surface pressure dependence of protein or peptide structure/interactions in a physiologically relevant model for surfactant is clearly demonstrated.

Comparison between the behavior of different hydrophobic peptides allowing membrane anchoring of proteins

“…The amide I band of the spread peptide is much broader than that of the injected peptide, which strongly suggests that the spreading procedure leads to the formation of disordered structures. Previous reports have shown a slight or no effect of monolayer compression with some lipid-peptide mixtures [295,311] whereas a large modification of the peptide structure was found to take place for other lipid-peptide mixtures [177,313,325]. Although measurements with additional peptides should be performed to definitively clarify this issue, the present data suggest that it is preferable to perform measurements by injecting peptides into the lipid monolayer subphase.…”

Pulmonary Surfactant Pathophysiology: Current Models and Open Questions

“…SP-B seems to provide mechanical stability to compressed films (23), possibly through increasing cohesivity between surfactant layers during maximal compression of the surface film. SP-C could promote association of excluded surfactant structures with the maximally compressed interface, likely through its NH 2 -terminal segment and/or insertion of palmitoylated cysteines of the protein into tightly packed interfacial films (4) (11,102). Finally, both SP-B (23, 90,106) and SP-C (73,74,81) seem to promote insertion (5) and re-spreading (6) of phospholipids from subsurface compartments into the interface during expansion.…”

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

“…The three-dimensional structure of SP-C was determined by NMR in apolar solvents (Johansson et al, 1994), but at that time the N-terminus tail appeared as a "flexible disordered" segment. More recently the study of the N-terminal segment of the protein has been addressed using synthetic peptides based in the sequence of this region of the lipopeptide (Plasencia et al, 2001(Plasencia et al, , 2004(Plasencia et al, , 2005Bi et al, 2002). These peptides from the N-terminal tail of SP-C seem to adopt an amphipathic conformation, with strong tendency to partition into the interface of phospholipid membranes and monolayers, even in the absence of palmitoylated cysteines (Plasencia et al, 2004(Plasencia et al, , 2005.…”

“…In this regard, SP-C seems to have an activity which is partly redundant, although with lower efficiency, with that of SP-B. The role of palmitoylation for the activity of SP-C has been outlined by studying the behaviour of palmitoylated and non-palmitoylated versions of peptides from the N-terminal segment of SP-C in lipid/protein monolayers subjected to compression/expansion dynamics, by infrared spectroscopy (Bi et al, 2002). It was determined that palmitoylation was required for the peptides to maintain association with phospholipid films compressed to the highest pressures (the lowest surface tensions).…”