Combinations of fluorescently labeled pulmonary surfactant proteins SP-B and SP-C in phospholipid films

Abstract

Hydrophobic pulmonary surfactant (PS) proteins B (SP-B) and C (SP-C) modulate the surface properties of PS lipids. Epifluorescence microscopy was performed on solvent-spread monolayers of fluorescently labeled porcine SP-B (R-SP-B, labeled with Texas Red) and SP-C (F-SP-C, labeled with fluorescein) in dipalmitoylphosphatidylcholine (DPPC) (at protein concentrations of 10 and 20 wt%, and 10 wt% of both) under conditions of cyclic compression and expansion. Matrix-assisted laser desorption/ionization (MALDI) spectroscopy of R-SP-B and F-SP-C indicated that the proteins were intact and labeled with the appropriate fluorescent probe. The monolayers were compressed and expanded for four cycles at an initial rate of 0.64 A2 x mol(-1) x s(-1) (333 mm2 x s x [-1]) up to a surface pressure pi approximately 65 mN/m, and pi-area per residue (pi-A) isotherms at 22 +/- 1 degrees C were obtained. The monolayers were microscopically observed for the fluorescence emission of the individual proteins present in the film lipid matrix, and their visual features were video recorded for image analysis. The pi-A isotherms of the DPPC/protein monolayers showed characteristic "squeeze out" effects at pi approximately 43 mN/m for R-SP-B and 55 mN/m for F-SP-C, as had previously been observed for monolayers of the native proteins in DPPC. Both proteins associated with the expanded (fluid) phase of DPPC monolayers remained in or associated with the monolayers at high pi (approximately 65 mN/m) and redispersed in the monolayer upon its reexpansion. At comparable pi and area/molecule of the lipid, the proteins reduced the amounts of condensed (gel-like) phase of DPPC monolayers, with F-SP-C having a greater effect on a weight basis than did R-SP-B. In any one of the lipid/protein monolayers the amounts of the DPPC in condensed phase were the same at equivalent pi during compression and expansion and from cycle to cycle. This indicated that only minor loss of components from these systems occurred between compression-expansion cycles. This study indicates that hydrophobic PS proteins associate with the fluid phase of DPPC in films, some proteins remain at high surface pressures in the films, and such lipid-protein films can still attain high pi during compression.

Cholesterol Rules

“…2). The partitioning of SP-B and SP-C in NPSM and in bilayers made of surfactant organic extract resemble the distribution of the proteins observed in monolayer experiments, where both SP-B and SP-C preferentially situate in liquid-expanded areas (33). This distribution is particularly remarkable in the case of SP-C, because this protein should, in principle, have a different local environment in bilayers with respect to monolayers simply because of the different membrane thickness (SP-C in bilayers accommodate in a transmembrane orientation (1)).…”

Selective Labeling of Pulmonary Surfactant Protein SP-C in Organic Solution

“…In the past we used amine-reactive reagents to introduce different groups in SP-C, such as visible-emission fluorescent (22,23) or spin (24) probes. Derivatization of amines is critically dependent on pH as they are good nucleophiles only in their nonprotonated state.…”

“…This is a common procedure for derivatizing proteins and peptides in aqueous solutions but it is not trivial when labeling proteins in organic solvents. We have labeled amines in SP-B and SP-C by adding traces of buffer to the protein solution in chloroform/methanol, so allowing a certain control of apparent pH (22)(23)(24). However, alkaline pH can produce cleavage of the labile thioesther bonds which maintain the two cysteines of SP-C palmitoylated (2,22) and, in fact, we could not avoid total or partial depalmitoylation of the protein in previous studies.…”

“…An alternative is the site-selective introduction of an extrinsic fluorescent probe in SP-C, which may allow identification of structural features that could be directly assigned to local regions in the protein (21). Characterization of lipid-protein interactions of SP-C by other techniques such as epifluorescence microscopy (22,23) or electron spin resonance (24) has also required introduction of extrinsic probes in proper locations of the protein, without producing significant alterations of the native conformation. However, the procedure to derivatize SP-C in organic solutions has not been optimized, specially to ensure preservation of the palmitic chains bound to the structure of SP-C via thioesther linkages, and also to produce incorporation of single groups in selective sites.…”

Molecular Interactions in Pulmonary Surfactant Films

“…Surfactant proteins are differently sorted between condensed and expanded areas segregated during compression. SP-B and SP-C are excluded from DPPC-enriched condensed regions [62]. Compression of surfactant films up to pressures in the range of 30-40 mN/m produces regionalization in two types of areas.…”