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Biophys J. 2010 Apr 21; 98(8): 1549–1557.
doi: 10.1016/j.bpj.2009.12.4302
PMCID: PMC2856140
PMID: 20409474

Hydrophobic Surfactant Proteins Induce a Phosphatidylethanolamine to Form Cubic Phases

Mariya Chavarha,†‡§ Hamed Khoojinian,†‡§▵ Leonard E. Schulwitz, Jr.,†‡§▵ Samares C. Biswas,†‡§ Shankar B. Rananavare, and Stephen B. Hall†‡§∗
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Associated Data

Supplementary Materials

Abstract

The hydrophobic surfactant proteins SP-B and SP-C promote rapid adsorption of pulmonary surfactant to an air/water interface. Previous evidence suggests that they achieve this effect by facilitating the formation of a rate-limiting negatively curved stalk between the vesicular bilayer and the interface. To determine whether the proteins can alter the curvature of lipid leaflets, we used x-ray diffraction to investigate how the physiological mixture of these proteins affects structures formed by 1-palmitoyl-2-oleoyl phosphatidylethanolamine, which by itself undergoes the lamellar-to-inverse hexagonal phase transition at 71°C. In amounts as low as 0.03% (w:w) and at temperatures as low as 57°C, the proteins induce formation of bicontinuous inverse cubic phases. The proteins produce a dose-related shift of diffracted intensity to the cubic phases, with minimal evidence of other structures above 0.1% and 62°C, but no change in the lattice-constants of the lamellar or cubic phases. The induction of the bicontinuous cubic phases, in which the individual lipid leaflets have the same saddle-shaped curvature as the hypothetical stalk-intermediate, supports the proposed model of how the surfactant proteins promote adsorption.

Introduction

Pulmonary surfactant contains small amounts of two very hydrophobic proteins, SP-B and SP-C, at least one of which is essential for normal function of the lungs. Ventilation in the absence of SP-B produces an injury to the alveolocapillary barrier (1–3) equivalent to the insult caused by ventilation when surface tension is elevated because the complete mixture of surfactant-constituents is missing (4,5). A deficiency of SP-C produces effects with a more gradual onset, but it too leads to altered lungs (6,7). The hydrophobic proteins in vitro greatly accelerate the adsorption of surfactant vesicles to an air/water interface (8–10). These results suggest that the essential physiological function of these proteins is to promote rapid formation of the alveolar film.

Currently available data specify features that must be present in any model of how the proteins facilitate adsorption (11). In contrast to classical molecular surfactants, which insert into the interface as individual monomers, components of pulmonary surfactant adsorb collectively (12–14), suggesting that the surfactant vesicles fuse with the surface to deliver their complete set of constituents. Factors that accelerate adsorption have similar effects whether they are confined to the interface or an adsorbing vesicle (9,15,16), suggesting that a rate-limiting structure must be equally accessible from both locations. Compounds not present in pulmonary surfactant generally promote or inhibit adsorption according to their tendency to produce negative or positive curvature (9,17–19), in which the hydrophilic face of a phospholipid leaflet is concave or convex, respectively. Together, these results suggest a model in which components adsorb through negatively curved leaflets that extend from the vesicular bilayer to the interface (Fig. 1 A), analogous to the stalk-intermediate proposed as a key step in the fusion of two bilayers (20). The hydrophobic proteins would accelerate adsorption by promoting the formation of this rate-limiting structure.

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Schematic representation of saddle-shaped structures. The radii R1 and R2 each define a corresponding principal curvature of the structure, c1 and c2, with c = 1/R. Scales for the two diagrams are different. (A) Hypothetical rate-limiting kinetic intermediate formed during adsorption of the vesicular bilayer to the air/water interface. The radii of the leaflets are defined at the pivotal surface, which lies approximately at the junction between the hydrophobic acyl tails and the hydrophilic headgroup. (B) Segment of the inverse bicontinuous cubic phase with space group Im3¯m. In contrast to the individual leaflets, the curvature of the bilayer is defined at its midpoint, which for the inverse bicontinuous cubic phases lies along an infinite periodic minimal surface at which the two radii of curvature are equal in magnitude and opposite in sign, resulting in no net curvature.

To the best of our knowledge, however, no direct evidence shows that the surfactant proteins (SPs) can affect the tendency of lipids to form curved structures. The absence of such data may reflect the lipids with which the proteins have been studied. Like most biological mixtures of lipids, pulmonary surfactant forms lamellar bilayers (9). Any spontaneous curvature in the individual leaflets is cancelled by the presence of the oppositely oriented, paired leaflet (21). Any effect of the proteins on the ability of the leaflets to curve is undetectable. The studies reported here tested how the proteins affect a phospholipid that does form curved structures. Because 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) has a sufficiently inverted conical shape, high temperatures convert lamellar bilayers to the inverse hexagonal (HII) phase, in which unpaired leaflets approximate their spontaneous curvature (21). Lipids that can form such structures should more readily express any effects of the proteins on curvature.

Materials and Methods

Materials

POPE was obtained from Avanti Polar Lipids (Alabaster, AL) and used without further characterization or purification. Extracts of pulmonary surfactant from calf lungs (ONY, Amherst, NY) were prepared as described previously (22). Hydrophobic SPs were obtained from the extracted surfactant by minor modifications of a previously described protocol based on gel permeation chromatography (23). Samples were eluted from a 150 × 2.5 cm column (Spectrum Chromatography, Houston, TX) packed with LH-20 matrix (24) using a solvent of chloroform/methanol (1:1, v:v) at constant flow (1 mL/min) driven by gravity. The content of phospholipid and protein was monitored qualitatively by measuring the optical density of the eluted fractions at 240 and 280 nm (25). Fractions containing SPs were pooled, concentrated initially by rotary evaporation and then with a stream of nitrogen, and stored at 4°C before mixing in appropriate ratios with POPE.

The following reagents were purchased commercially and used without further purification: chloroform and methanol (Fisher Scientific, Pittsburgh, PA); Na2EDTA-2H2O (Gibco, Grand Island, NY); and NaN3 (Sigma, St. Louis, MO). Water was processed and photooxidized with ultraviolet light using a NANOpure Diamond TOC-UV water-purification apparatus (Barnstead/Thermolyne, Dubuque, IA). All chemicals and solvents were ACS grade.

Methods

Biochemical determinations

Protein content was determined quantitatively by amido black assay on material precipitated with trichloroacetic acid using a standard of bovine serum albumin (26). Contamination of protein by residual phospholipid, as determined by measuring the content of phosphate (27), was assessed at 85 ng of lipid for each microgram of protein. This level represented a purification of the proteins from the initial extracted surfactant by approximately 3 orders of magnitude.

Samples for x-ray diffraction

Samples of protein and POPE were mixed in chloroform, concentrated under a stream of N2 until they reached a homogeneous viscous consistency, deposited as a thin uniform film at the bottom of a test tube, and then held overnight under vacuum at room temperature to remove any residual solvent. Samples were resuspended in 2 mM EDTA with 0.002% (w/w) NaN3 for a final phospholipid concentration of 50 mM, flushed with N2 and sealed with Teflon tape, agitated briefly, and then left to hydrate at 4°C overnight. Cyclic freezing and thawing along with vigorous vortexing achieved homogeneous dispersions. The hydrated samples were transferred to special glass capillaries (1.0 mm diameter, 0.01 mm wall thickness; Charles Supper, Natick, MA), both ends of which were sealed first by flame and then with epoxy. The capillaries were centrifuged at 640 × gmax for 10 min to concentrate the aggregated samples at one end of the tube, and then stored at 4°C until needed. Heating and cooling of the samples through the lamellar-HII transition temperature, which is commonly used (28) to induce formation of inverse cubic phases (QII), was avoided before obtaining the initial measurements.

The samples contained 0–3% (w:w) protein/phospholipid. Other studies of similar phenomena with different proteins have expressed compositions as % (mol:mol) (29). Results from amino acid analyses (30) suggest that the physiological mixture of SP-B and SP-C obtained by gel permeation chromatography is roughly equimolar. Given the known molecular weights of the different constituents, our samples with a protein/phospholipid content of 1% (w:w) were roughly 0.1% (mol:mol).

Small-angle x-ray diffraction

Measurements of diffraction were conducted on beamline 1-4 at the Stanford Synchrotron Radiation Lightsource. An x-ray beam with a wavelength of 1.488 Å was focused to an elliptical spot with approximate dimensions of 0.3 (horizontal) × 0.1 (vertical) mm. A beryllium window inserted into the Kapton film used to seal the helium-filled drift-tube between the sample and detector minimized background scattering from Kapton. Diffraction images were acquired at a sample-to-detector distance of 0.26 m, producing a range of accessible q-values from 0.29 to 5.40 nm−1. Spatial calibration was performed using both cholesterol myristate and silver behenate (31). Temperature was controlled with water circulated through the sample-holder, and measurements with a thermocouple established the relationship between temperatures in the bath and the capillary. With the use of two circulating baths, the temperatures changed rapidly—within 20 s for an increase of 10°C. The samples were then equilibrated for 10 min before diffraction was measured. Incubations longer than 10 min produced no further changes in relative intensities. Measurements of diffraction routinely exposed samples to the beam for 120 s. Continuous exposure of other samples for periods as long as 2.5 h to test for evidence of radiation-induced damage produced no changes in diffraction. The diffracted intensities were radially integrated using the program FIT2D (32). The results presented here, obtained with a single set of samples during a single visit to the Stanford Synchrotron Radiation Lightsource, were confirmed with two other sets of samples during separate visits that included measurements on beamline 4-2.

Results

Samples containing POPE with 0–3% (w:w) SP at specific temperatures of 11–90°C in all cases produced rings indicating powder diffraction. Results obtained with POPE alone agreed well with previously published results. Below 25°C, the samples produced diffraction with lamellar spacing and a prominent fourth-order peak characteristic of the Lβ′ phase (33) (Fig. 2 A). A second set of lamellar peaks appeared at 25°C with lower d-spacing (Fig. 2 B), consistent with formation of the Lα phase close to the previously reported main transition temperature of 26°C (34). The Lα peaks persisted to 72°C, above which diffraction converted to the spacing of a hexagonal phase, which remained present at the highest temperature of 90°C (Fig. 2 C). These results agreed reasonably with the expected formation of the HII phase at 71°C (35).

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Diffraction patterns. Negative images, obtained by exposing samples to the x-ray beam for 120 s, record one quadrant of the circularly symmetric diffraction rings, with the beam-stop located at the upper-right corner. The brightness and contrast of each entire image are adjusted to show higher-order rings. (A) POPE alone, 11°C; rings have lamellar spacing and the increased relative intensity of the fourth-order ring that is characteristic of the Lβ′ phase (33). (B) POPE alone, 30°C; lamellar spacing, consistent with the Lα phase. (C) POPE alone, 90°C; hexagonal spacing, consistent with the HII phase. (D) POPE with 0.3% (w:w) SP, 81°C; spacing consistent with simultaneous diffraction from cubic structures with Pn3¯m and Im3¯m space groups, indicating coexistence of inverse bicontinuous cubic (QII) phases with the diamond and primitive minimal surfaces, respectively.

The addition of the proteins had essentially no effect on diffraction at lower temperatures. The lattice constant of the lamellar phases remained unchanged, and the Lβ′-Lα transition occurred at approximately the same temperature. Above 53°C, a new set of rings appeared at q-values below the first-order lamellar peak (Fig. 2 D), suggesting the presence of the cubic phases found previously with other phosphatidylethanolamines. At the highest temperatures, with the diffraction peaks spread the farthest (Fig. 3), these peaks had the relative spacing expected for individual or coexisting cubic structures with Pn3¯m and Im3¯m space groups (Fig. 4). At lower temperatures, although the shift to lower q-values caused the overlap of some peaks, these patterns remained evident. When the two cubic structures coexisted, their lattice constants maintained a constant ratio of 1.28 ± 0.01. This value agreed with the ratio of 1.279 expected for the inverse bicontinuous cubic phases (QII) with those space groups interconverted by Bonnet transformation (28). Because six other space groups generate the same absent reflections as Im3¯m over the range detected here, and one other for Pn3¯m, the spacing of the diffraction rings alone was insufficient to make definitive assignments to those space groups. In combination with the ratio of lattice constants, however, the low values of q at which the peaks occurred, and the previous observations made with similar lipids, the pattern of diffraction strongly supported the presence of structures with space groups Pn3¯m and Im3¯m, corresponding to QII phases with diamond (QIID) and primitive (QIIP) infinitely periodic minimal surfaces, respectively.

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Diffraction pattern for POPE with 1% SP at 90°C. The radially integrated intensity is plotted as a function of the measured q. Labeled vertical lines assign peaks to diffraction with the indicated Miller indices from structures with Pn3¯m (Pn) or Im3¯m (Im) space groups based on the fits of the observed spacings to allowed peaks (Fig. 4).

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Analysis of the diffraction pattern for POPE with 1% SP at 90°C (Fig. 3) (53). The values of q measured for the diffraction peaks are plotted as horizontal lines. Vertical lines indicate all possible values of f(h,k,l), where h, k, and l are the Miller indices, and f(h,k,l) is given by h for the lamellar phases, (h2 + hk + k2)1/2 for the hexagonal phase, and (h2 + k2 + l2)1/2 for the cubic phases. Labels on the lower portion of the vertical lines indicate values that are allowed for diffraction from structures with the following symmetries, which have been either documented experimentally in other systems of phospholipids or suggested by simulation (54): lamellar (L; space group pm, No.3 in the International Tables of Crystallography (55)); hexagonal (H;p6m, No.17); Pn3¯m (P; No.224); Im3¯m (Im; No.229); Fd3¯m (F; No.227); and Ia3¯d (Ia; No.230). Symbols indicate assignment of peaks to (h,k,l) based on optimizing the linear fit of measured q-values at allowed values of f(h,k,l). Solid and open symbols fit well the diffraction predicted for Pn3¯m and Im3¯m space groups, respectively. Lines through the symbols, obtained by least-squares fit, provide the slope, which yields the lattice-constant (ao) of the unit cell according to (ao = 2π/slope) for the lamellar and cubic phases, and (ao = 4π/(31/2 · slope)) for the hexagonal phase.

The major response to increasing amounts of protein was an increase in the intensities of the cubic diffraction and the corresponding loss of signal from coexisting structures (Fig. 5 and Fig. S1 in the Supporting Material)). At 0.01% protein, only questionable peaks were present at the highest temperatures for values of q below the first-order hexagonal peak to suggest the possible presence of some QII structures. With 0.03% protein at temperatures above 67°C, definite rings occurred at low q-values (Fig. 5), with seven peaks at 76°C and 81°C fitting the spacing for Pn3¯m diffraction. For protein ≥ 0.1%, hexagonal diffraction disappeared completely, and the cubic phases became the only structures present at high temperatures (Fig. 5). Increasing amounts of protein shifted intensity from the QIID phase to the larger QIIP structures. Samples with 0.03% SP produced peaks only for the QIID phase; diffraction at 3% SP showed only QIIP (Fig. 5 and Fig. S1). More protein also induced a broadening of the peaks. Consequently, although the integrated intensity from the Im3¯m peaks continued to increase at 3% (Fig. 6), the height of the peaks fell.

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Diffraction patterns at different temperatures and contents of protein. Curves give the radially integrated intensity in arbitrary units, plotted on a logarithmic scale, over a limited range of q-values for each sample at each temperature. Traces are offset by a fixed amount for each temperature.

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Intensities of the cubic phases with different quantities of SP. Intensity is expressed for each phase as the integrated area above the baseline of the peak with Miller indices 110 (I), normalized relative to its value (I1) at the temperature and % SP at which I is greatest.

The temperature at which cubic diffraction first emerged showed limited dependence on the amount of protein present. Increasing the protein concentration from 0.03% to 0.10% lowered the temperature at which the peaks first appeared at low q-values from 67°C to 57°C. At 3% SP, however, that temperature increased from 57°C to 62°C. Whether these changes reflected true shifts in the transition-temperature or simply a dose-dependent variation in peak-intensity remained unresolved.

The added protein had little effect on the dimensions of the unit cell for the different diffracting structures (Fig. 7). At any particular temperature, the lattice constants (ao) for both the lamellar and the hexagonal structures were essentially invariant with different amounts of protein (Fig. 7 and Fig. S2, A–C). Hexagonal diffraction, however, was limited to samples containing 0–0.03% SP (Fig. 7 and Fig. S2 C), which prevented determination of how the proteins affected the size of the hexagonal lattice. The cubic lattices showed more dependence on temperature, but no consistent response to increasing amounts of protein (Fig. 7 and Fig. S2, D and E). The proteins induced a dose-related shift in the extent of the two cubic phases, but the structures formed were roughly constant, with dimensions that showed no clear change in response to larger amounts of protein.

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Effect of temperature on the lattice constants for POPE with different amounts of protein. Structural symmetries were assigned based on the spacing of the diffraction rings (Fig. 4). The slope of q versus f(h,k,l) provided the lattice constant (ao) (Fig. 4).

The results of these measurements suggested that the SPs induced POPE to form structures that would not occur for the lipid alone. Previous theoretical and experimental studies, however, have suggested that at increasing temperatures, the QII phases should exist between lamellar and hexagonal structures (21,36). Cooling from high temperatures, as well as cycling between temperatures above and below the Lα-HII transition, can induce cubic phases in lipids, including POPE (29,37), that form only lamellar and hexagonal structures during initial heating. Under the conditions of our experiments, when POPE was cooled from 90°C without protein, it produced low-intensity rings at q-values consistent with the QIIP phase (Fig. S3). These peaks persisted to the temperature of the Lα-Lβ′ transition, below which they were absent. Although the diffraction of any sample during heating from the Lβ′ phase was indistinguishable, regardless of its history, during sequential cycles of cooling, the intensity of the QIIP peaks increased (Fig. S3). The marked hysteresis of phase behavior, with the cubic phases absent during heating but present during cooling, prevented any estimation of the Lα-QII transition temperature. The results confirmed, however, that although the proteins greatly facilitated formation of the QII phases, they were not essential components of those structures.

The cubic phases also showed significant hysteresis in samples with protein (Fig. 8). In contrast to the samples at high temperatures that contained only lipid, with the protein present, the QII phases represented the dominant structures. Experiments could therefore compare the cubic lattice constants during heating and cooling as well as the temperatures at which the cubic phases first appeared and disappeared. Like the samples with POPE alone, the lattice constant for the lamellar phases depended only on temperature, irrespective of how the samples had been treated previously. With the proteins present, the lattice constants of the QII phases during cooling were significantly smaller than during heating, suggesting that the smaller unit cell observed at higher temperatures grew during cooling with difficulty (Fig. 8). As with the samples of POPE alone, the QII phases persisted during cooling to temperatures well below the levels at which they formed during heating. With the proteins present, however, the cubic phases reverted during cooling to lamellar structures well before reaching the Lβ′ phase. Crystallization of the acyl chains was unnecessary to promote return from the QII phases.

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Hysteresis of POPE with 0.3% SP during the initial cycle of heating and cooling. Diffraction was recorded during heating from 11°C to 90°C, and then during subsequent cooling to 6°C. Open symbols with solid lines indicate the lattice constants (ao) during the initial heating; solid symbols with dotted lines provide the values during the subsequent cooling.

Discussion

Our experiments show that the hydrophobic SPs induce POPE to form QII phases in a dose-dependent fashion. The bicontinuous structures share important features of the key kinetic intermediate in the most widely considered model of how those proteins promote adsorption at the alveolar air/water interface (Fig. 1). The available evidence suggests that, at least initially, the lipids flow from the adsorbing vesicle to an air/water interface through a stalk that connects the two locations (38,39). The common two-dimensional representation of this structure emphasizes that the curvature in the plane perpendicular to the interface (c1) would be negative (Fig. 1 A). However, the second principal curvature (c2) in the plane parallel to the interface would be positive (Fig. 1 A). The stalk would have a Gaussian curvature (c1 · c2) that would be negative. The correlation between the molecular shape of added constituents and their effect on adsorption (11) suggests that the net curvature (c1 + c2) of the stalk would be negative. The proteins would promote adsorption by facilitating formation of this rate-limiting structure.

In the QII phases, the lipids form bilayers, in contrast to the unpaired monolayers of the hypothetical stalk (Fig. 1). Each individual leaflet in the cubic phases, however, shares the basic structural features of the monolayers in the stalk. In both cases, the Gaussian curvature is negative (21,28,36). The net curvature of the individual leaflets in both structures is also negative. In the QII phases, the midpoint of the bilayer lies along a minimal surface, at which the two principal curvatures are equal in magnitude and opposite in sign, resulting in no net curvature. The individual leaflets are shifted from that surface. The curvature of the monolayers that constitute the bilayer is defined empirically at the pivotal surface, at which the cross-sectional area of constituents neither expands nor contracts during bending (40). For the leaflets, the displacement of the pivotal surface (located roughly at the junction between the headgroup and the acyl residues (21)) from the minimal surface at the midpoint of the bilayer has opposite effects on the magnitude of the two principal curvatures. The leaflet curvature with negative sign becomes more negative, and the positive curvature becomes smaller, resulting in a negative net curvature. Although the leaflets are paired in the cubic phase and unpaired in the stalk, the net and Gaussian curvatures in the two cases are at least qualitatively similar.

The proteins may induce formation of the QII phases by either thermodynamic or kinetic effects. The distinction depends on the ultimate stability of the different phases for the lipids alone. Without the proteins, the QII phases only appear after exposure to temperatures above the Lα-HII transition. If this behavior represents true equilibrium, then the observed formation of the QII structures at much lower temperatures with the proteins would reflect stabilization. With the lipid alone, however, the persistence of the QII phases to these lower temperatures during cooling suggests that under these conditions, the curved structures might be more stable than the lamellar phases, but kinetically inaccessible. The proteins would then induce the QII phases by promoting their faster formation. The behavior of POPE by itself prevents a clear distinction between these two possibilities. The marked hysteresis during heating and cooling prevents determination of the noncubic/cubic transition temperature that would allow assessment of thermodynamic stability. The proteins narrow the hysteresis of the QII phases (Fig. 8), suggesting that their effect may be kinetic, and that they accelerate transitions both to and from the cubic structures. Both mechanisms would be physiologically relevant. Whether the proteins stabilize the bridging stalk or simply facilitate its formation, the result would be faster adsorption.

The proteins could induce the QII phases, whether by stabilization or improving kinetic access, by a limited number of mechanisms. The kinetics of forming the QII phases, which have received less attention than their stability, would presumably depend on the rigidity of the leaflets, expressed as their moduli of bending. The stability of the QII phases has generally been considered in terms of the energy of elastic bending for the lipid leaflets. That energy depends on the spontaneous curvature of the leaflets, and their moduli of simple-splay and saddle-splay bending (41,42). A recent report, however, suggested that the energy of unbinding adjacent bilayers might also affect the stability of the QII phases (43). The transition of lamellar to QII phases requires a significant separation of adjacent bilayers. The contribution of the unbinding energy might explain, in particular, the order in which phases generally appear during heating (43), with QII structures only forming after the HII structures, opposite to the sequence predicted strictly from their bending energies. The proteins might then induce the QII phases or facilitate formation of the hypothetical stalk-intermediate by shifting either the modulus of bending, by altering the spontaneous curvature, or by reducing the energy of unbinding.

Our studies included no experiments, such as with osmotic stress, designed to probe these different possibilities. The proteins nonetheless might well generate spontaneous changes in the lattice constants for the different phases that might distinguish the different mechanisms. A decrease in the modulus of splay-bending or the unbinding energy could result in greater undulations of stacked bilayers and an increase in spacing of the lamellar phases. A change in spontaneous curvature would shift the lattice constant of the HII phase. A lower modulus of saddle-splay bending could increase the size of the cubic unit cells. Unfortunately, our results provide no insights concerning which mechanism is more likely. The limited range of 0.00–0.03% SP over which the HII phase is present prevents any assessment of how the proteins affect spontaneous curvature. The lattice constants for the lamellar and cubic phases are unaffected by the content of protein. The mechanism by which the proteins induce the QII phases therefore remains undetermined.

Remarkably low levels of the proteins induce formation of the QII phases. With 0.1% (w:w) SP, for which diffraction at high temperatures detects only the cubic structures, the molecular ratio is roughly one protein per 10,000 phospholipids. In this respect, the SPs extend findings with other peptides that induce QII phases in HII-forming phospholipids (29,44–48). SP-B, like those peptides, consists largely of amphipathic helices (49). Like the SPs, these other peptides produce their structural effects on the lipids in small quantities, although none has demonstrated the effectiveness of the SPs. In native pulmonary surfactant, hydrophobic proteins account for ∼1.5% (w:w, protein/phospholipid) of the mixture. The effects demonstrated here at levels as low as 0.03% therefore occur well within the physiological range.

The surfactant lipids differ in several respects from the compound used in our studies. The phospholipids in surfactant from common mammals, for instance, contain ∼10% anionic compounds, which may contribute to specific aspects of adsorption (16,39), and which interact with the cationic hydrophobic proteins. Because POPE should be zwitterionic in our samples, those interactions are absent.

The spontaneous curvature of POPE and the surfactant lipids should also be different. POPE has a spontaneous curvature sufficient to form the HII phase. In excess water, the surfactant lipids form only lamellar bilayers. Like other biological phospholipids, pulmonary surfactant can form HII structures if water is sufficiently restricted (50). That effect, however, apparently occurs only at very low hydration (50). For preparations of most therapeutic surfactants, and for the multiple mixtures with lipids in which the hydrophobic proteins promote adsorption in vitro, water is present in excess. Spontaneous curvature should be significantly more negative for POPE than for fully hydrated surfactant lipids. Calf surfactant contains only 3% (mol:mol) phosphatidylethanolamine (23,51), and the phosphatidylcholines, which constitute ∼80% of the lipids, contain acyl constituents that are remarkably low in the double bonds that would contribute to negative curvature (51). Cholesterol represents the only major component that should favor the HII phase.

The difference between the surfactant lipids and POPE is essential for our studies. Lipids adopt structures according to the competing energies of bending and chain-packing (21). When chain-packing dominates and lipids form planar bilayers, the spontaneous curvature of the individual leaflets is unknown. To determine how the proteins affect the tendency of the lipids to curve, the lipids must form structures that can express their curvature.

Our studies therefore substitute a nonphysiological motivation for the formation of saddle-shaped structures. In the lungs, a reduction in interfacial energy caused by the lower surface tension would drive adsorption and the transient formation of the hypothetical stalk. In our studies with POPE, away from the interface, the lower energy of bending present in the QII phases drives conversion from the planar bilayers. For both the stalk and the QII phases, the proteins may promote formation of the saddle-shaped structure by modulating the energy of bending. In each circumstance, the proteins should have similar effects on the factors that determine the energy of bending for the two sets of lipids. The spontaneous curvature of a lipid leaflet generally reflects the additive effects of its constituents (52). Effects on the moduli of simple-splay and saddle-splay bending should be similar for different lipids. Despite the different driving forces, the processes by which the proteins induce POPE to form the QII phases should also facilitate formation by the surfactant lipids of the hypothetical stalk during adsorption.

In conclusion, the physiological mixture of SP-B and SP-C, in amounts as low as 0.03%, induces POPE to form QII phases at temperatures 15°C below the point at which the lipid alone converts during heating from the Lα to the HII phase. These results fit with a model in which the proteins facilitate adsorption by promoting the formation of a rate-limiting structure with negative Gaussian and net curvatures that bridges the gap between the vesicular bilayer and the air/water interface.

Supporting Material

One table and three figures are available at http://www.biophysj.org/biophysj/supplemental/S0006-3495(10)00003-2.

Supporting Material

Document S1. Figures and table:

Acknowledgments

The extracted calf surfactant from which the SPs were isolated was provided by Dr. Edmund Egan (ONY Inc.). Diffraction was measured at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the Office of Basic Energy Sciences, U.S. Department of Energy.

This study was funded by the National Institutes of Health (HL 54209).

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