Hydrophobic Surfactant Proteins Induce a Phosphatidylethanolamine to Form Cubic Phases

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Biophysical Journal Volume 98 April 2010 1549–1557 1549

Mariya Chavarha,†‡§ Hamed Khoojinian,†‡§6 Leonard E. Schulwitz Jr.,†‡§6 Samares C. Biswas,†‡§ Shankar B. Rananavare,{ and Stephen B. Hall†‡§* †Department of Molecular Biology and Biochemistry, ‡Department of Medicine, and §Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon; and {Department of Chemistry, Portland State University, Portland, Oregon

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 71C. In amounts as low as 0.03% (w:w) and at temperatures as low as 57C, 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 62C, 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 in which the hydrophilic face of a phospholipid leaflet is hydrophobic proteins, SP-B and SP-C, at least one of which concave or convex, respectively. Together, these results is essential for normal function of the lungs. Ventilation in suggest a model in which components adsorb through nega- the absence of SP-B produces an injury to the alveolocapil- tively curved leaflets that extend from the vesicular bilayer lary barrier (1–3) equivalent to the insult caused by ventila- to the interface (Fig. 1 A), analogous to the stalk-intermediate tion when surface tension is elevated because the complete proposed as a key step in the fusion of two bilayers (20). The mixture of surfactant-constituents is missing (4,5). A defi- hydrophobic proteins would accelerate adsorption by ciency of SP-C produces effects with a more gradual onset, promoting the formation of this rate-limiting structure. but it too leads to altered lungs (6,7). The hydrophobic To the best of our knowledge, however, no direct evidence proteins in vitro greatly accelerate the adsorption of surfac- shows that the surfactant proteins (SPs) can affect the ten- tant vesicles to an air/water interface (8–10). These results dency of lipids to form curved structures. The absence of suggest that the essential physiological function of these such data may reflect the lipids with which the proteins proteins is to promote rapid formation of the alveolar film. have been studied. Like most biological mixtures of lipids, Currently available data specify features that must be pulmonary surfactant forms lamellar bilayers (9). Any spon- present in any model of how the proteins facilitate adsorption taneous curvature in the individual leaflets is cancelled by the (11). In contrast to classical molecular surfactants, which presence of the oppositely oriented, paired leaflet (21). Any insert into the interface as individual monomers, components effect of the proteins on the ability of the leaflets to curve is of pulmonary surfactant adsorb collectively (12–14), suggest- undetectable. The studies reported here tested how the ing that the surfactant vesicles fuse with the surface to deliver proteins affect a phospholipid that does form curved struc- their complete set of constituents. Factors that accelerate tures. Because 1-palmitoyl-2-oleoyl phosphatidylethanol- adsorption have similar effects whether they are confined to amine (POPE) has a sufficiently inverted conical shape, the interface or an adsorbing vesicle (9,15,16), suggesting high temperatures convert lamellar bilayers to the inverse that a rate-limiting structure must be equally accessible from hexagonal (HII) phase, in which unpaired leaflets approxi- both locations. Compounds not present in pulmonary surfac- mate their spontaneous curvature (21). Lipids that can tant generally promote or inhibit adsorption according to their form such structures should more readily express any effects tendency to produce negative or positive curvature (9,17–19), of the proteins on curvature.

Submitted October 5, 2009, and accepted for publication December 15, MATERIALS AND METHODS 2009. Materials 6Hamed Khoojinian and Leonard E. Schulwitz Jr., contributed equally to this work. POPE was obtained from Avanti Polar Lipids (Alabaster, AL) and used *Correspondence: [email protected] without further characterization or puriﬁcation. Extracts of pulmonary surfactant from calf lungs (ONY, Amherst, NY) were prepared as described Editor: Lukas K. Tamm. Ó 2010 by the Biophysical Society 0006-3495/10/04/1549/9 $2.00 doi: 10.1016/j.bpj.2009.12.4302 1550 Chavarha et al.

(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 puri- ﬁcation 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 4C 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 4C 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 FIGURE 1 Schematic representation of saddle-shaped structures. The of the different constituents, our samples with a protein/phospholipid radii R and R each define a corresponding principal curvature of the struc- 1 2 content of 1% (w:w) were roughly 0.1% (mol:mol). ture, c1 and c2, with c ¼ 1/R. Scales for the two diagrams are different. (A) Hypothetical rate-limiting kinetic intermediate formed during adsorption of Small-angle x-ray diffraction 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 Measurements of diffraction were conducted on beamline 1-4 at the Stanford between the hydrophobic acyl tails and the hydrophilic headgroup. (B) Synchrotron Radiation Lightsource. An x-ray beam with a wavelength of Segment of the inverse bicontinuous cubic phase with space group Im3m. 1.488 A˚ was focused to an elliptical spot with approximate dimensions of In contrast to the individual leaflets, the curvature of the bilayer is defined 0.3 (horizontal) 0.1 (vertical) mm. A beryllium window inserted into the at its midpoint, which for the inverse bicontinuous cubic phases lies along Kapton film used to seal the helium-filled drift-tube between the sample an infinite periodic minimal surface at which the two radii of curvature and detector minimized background scattering from Kapton. Diffraction are equal in magnitude and opposite in sign, resulting in no net curvature. 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 nm1. Spatial calibration previously (22). Hydrophobic SPs were obtained from the extracted surfac- was performed using both cholesterol myristate and silver behenate (31). tant by minor modifications of a previously described protocol based on gel Temperature was controlled with water circulated through the sample- permeation chromatography (23). Samples were eluted from a 150 2.5 holder, and measurements with a thermocouple established the relationship cm column (Spectrum Chromatography, Houston, TX) packed with LH-20 between temperatures in the bath and the capillary. With the use of two circu- matrix (24) using a solvent of chloroform/methanol (1:1, v:v) at constant lating baths, the temperatures changed rapidly—within 20 s for an increase of flow (1 mL/min) driven by gravity. The content of phospholipid and protein 10C. The samples were then equilibrated for 10 min before diffraction was was monitored qualitatively by measuring the optical density of the eluted measured. Incubations longer than 10 min produced no further changes in fractions at 240 and 280 nm (25). Fractions containing SPs were pooled, relative intensities. Measurements of diffraction routinely exposed samples concentrated initially by rotary evaporation and then with a stream of to the beam for 120 s. Continuous exposure of other samples for periods as nitrogen, and stored at 4C before mixing in appropriate ratios with POPE. long as 2.5 h to test for evidence of radiation-induced damage produced no The following reagents were purchased commercially and used without changes in diffraction. The diffracted intensities were radially integrated further purification: chloroform and methanol (Fisher Scientific, Pittsburgh, using the program FIT2D (32). The results presented here, obtained with

PA); Na2EDTA-2H2O (Gibco, Grand Island, NY); and NaN3 (Sigma, a single set of samples during a single visit to the Stanford Synchrotron St. Louis, MO). Water was processed and photooxidized with ultraviolet Radiation Lightsource, were conﬁrmed with two other sets of samples during light using a NANOpure Diamond TOC-UV water-puriﬁcation apparatus separate visits that included measurements on beamline 4-2.

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FIGURE 3 Diffraction pattern for POPE with 1% SP at 90C. 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 Pn3m (Pn) or Im3m (Im) space groups based on the ﬁts of the observed spacings to allowed peaks (Fig. 4).

RESULTS Samples containing POPE with 0–3% (w:w) SP at speciﬁc temperatures of 11–90C in all cases produced rings indicating powder diffraction. Results obtained with POPE alone agreed well with previously published results. Below 25C, the samples produced diffraction with lamellar spacing and a prominent fourth-order peak characteristic of the Lb0 phase (33)(Fig. 2 A). A second set of lamellar peaks appeared at 25C with lower d-spacing (Fig. 2 B), consistent with formation of the La phase close to the previously reported main transition temperature of 26 C(34). The La peaks persisted to 72C, above which diffraction converted to the spacing of a hexagonal phase, which remained present at the highest temperature of 90C(Fig. 2 C). These results agreed rea- sonably with the expected formation of the HII phase at 71C(35). 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 Lb0-La transition occurred at approximately the same temperature. Above 53C, 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 phosphatidyletha- nolamines. At the highest temperatures, with the diffraction FIGURE 2 Diffraction patterns. Negative images, obtained by exposing peaks spread the farthest (Fig. 3), these peaks had the relative samples to the x-ray beam for 120 s, record one quadrant of the circularly spacing expected for individual or coexisting cubic struc- symmetric diffraction rings, with the beam-stop located at the upper-right 3m and Im3m space groups (Fig. 4). At lower corner. The brightness and contrast of each entire image are adjusted to tures with Pn show higher-order rings. (A) POPE alone, 11C; rings have lamellar spacing temperatures, although the shift to lower q-values caused and the increased relative intensity of the fourth-order ring that is characteristic of the Lb0 phase (33). (B) POPE alone, 30 C; lamellar spacing, consis- with simultaneous diffraction from cubic structures with Pn3m and Im3m tent with the La phase. (C) POPE alone, 90 C; hexagonal spacing, consistent space groups, indicating coexistence of inverse bicontinuous cubic (QII) with the HII phase. (D) POPE with 0.3% (w:w) SP, 81 C; spacing consistent phases with the diamond and primitive minimal surfaces, respectively.

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interconverted by Bonnet transformation (28). Because six other space groups generate the same absent reflections as Im3m over the range detected here, and one other for Pn3m, the spacing of the diffraction rings alone was insuffi- cient 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 Pn3m and Im3m, corresponding to QII D P phases with diamond (QII ) and primitive (QII ) infinitely periodic minimal surfaces, respectively. 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 FIGURE 4 Analysis of the diffraction pattern for POPE with 1% SP at peak to suggest the possible presence of some QII structures. 90C(Fig. 3)(53). The values of q measured for the diffraction peaks are With 0.03% protein at temperatures above 67C, definite plotted as horizontal lines. Vertical lines indicate all possible values of rings occurred at low q-values (Fig. 5), with seven peaks f(h,k,l), where h, k, and l are the Miller indices, and f(h,k,l) is given by h at 76C and 81C fitting the spacing for Pn3m diffraction. for the lamellar phases, (h2 þ hk þ k2)1/2 for the hexagonal phase, and 2 2 2 1/2 For protein R 0.1%, hexagonal diffraction disappeared com- (h þ k þ l ) for the cubic phases. Labels on the lower portion of the vertical lines indicate values that are allowed for diffraction from structures pletely, and the cubic phases became the only structures with the following symmetries, which have been either documented exper- present at high temperatures (Fig. 5). Increasing amounts D imentally in other systems of phospholipids or suggested by simulation (54): of protein shifted intensity from the QII phase to the larger lamellar (L; space group pm, No.3 in the International Tables of Crystallog- P QII structures. Samples with 0.03% SP produced peaks only raphy (55)); hexagonal (H;p6m, No.17); Pn3m (P; No.224); Im3m (Im; D P No.229); Fd3m (F; No.227); and Ia3d (Ia; No.230). Symbols indicate for the QII phase; diffraction at 3% SP showed only QII assignment of peaks to (h,k,l) based on optimizing the linear fit of measured (Fig. 5 and Fig. S1). More protein also induced a broadening q-values at allowed values of f(h,k,l). Solid and open symbols fit well the of the peaks. Consequently, although the integrated intensity diffraction predicted for Pn3m and Im3m space groups, respectively. Lines from the Im3m peaks continued to increase at 3% (Fig. 6), through the symbols, obtained by least-squares fit, provide the slope, which the height of the peaks fell. yields the lattice-constant (ao) of the unit cell according to (ao ¼ 2p/slope) 1/2 The temperature at which cubic diffraction first emerged for the lamellar and cubic phases, and (ao ¼ 4p/(3 $ slope)) for the hexagonal phase. 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 the overlap of some peaks, these patterns remained evident. appeared at low q-values from 67Cto57C. At 3% SP, When the two cubic structures coexisted, their lattice however, that temperature increased from 57Cto62C. constants maintained a constant ratio of 1.28 5 0.01. This Whether these changes reflected true shifts in the transi- value agreed with the ratio of 1.279 expected for the inverse tion-temperature or simply a dose-dependent variation in bicontinuous cubic phases (QII) with those space groups peak-intensity remained unresolved.

FIGURE 5 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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FIGURE 7 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).

as cycling between temperatures above and below the La-HII FIGURE 6 Intensities of the cubic phases with different quantities of SP. transition, can induce cubic phases in lipids, including POPE Intensity is expressed for each phase as the integrated area above the baseline (29,37), that form only lamellar and hexagonal structures of the peak with Miller indices 110 (I), normalized relative to its value (I1)at the temperature and % SP at which I is greatest. during initial heating. Under the conditions of our experiments, when POPE was cooled from 90C without protein, it produced low-intensity rings at q-values consistent with P The added protein had little effect on the dimensions of the QII phase (Fig. S3). These peaks persisted to the temper- the unit cell for the different diffracting structures (Fig. 7). ature of the La-Lb0 transition, below which they were absent. At any particular temperature, the lattice constants (ao) for Although the diffraction of any sample during heating from both the lamellar and the hexagonal structures were essen- the Lb0 phase was indistinguishable, regardless of its history, P tially invariant with different amounts of protein (Fig. 7 during sequential cycles of cooling, the intensity of the QII and Fig. S2, A–C). Hexagonal diffraction, however, was peaks increased (Fig. S3). The marked hysteresis of phase limited to samples containing 0–0.03% SP (Fig. 7 and behavior, with the cubic phases absent during heating but Fig. S2 C), which prevented determination of how the present during cooling, prevented any estimation of the proteins affected the size of the hexagonal lattice. The cubic La-QII transition temperature. The results confirmed, lattices showed more dependence on temperature, but however, that although the proteins greatly facilitated forma- no consistent response to increasing amounts of protein tion of the QII phases, they were not essential components of (Fig. 7 and Fig. S2, D and E). The proteins induced a dose- those structures. related shift in the extent of the two cubic phases, but the The cubic phases also showed significant hysteresis in structures formed were roughly constant, with dimensions samples with protein (Fig. 8). In contrast to the samples at that showed no clear change in response to larger amounts high temperatures that contained only lipid, with the protein of protein. present, the QII phases represented the dominant structures. The results of these measurements suggested that the SPs Experiments could therefore compare the cubic lattice induced POPE to form structures that would not occur for the constants during heating and cooling as well as the temper- lipid alone. Previous theoretical and experimental studies, atures at which the cubic phases first appeared and disap- however, have suggested that at increasing temperatures, peared. Like the samples with POPE alone, the lattice the QII phases should exist between lamellar and hexagonal constant for the lamellar phases depended only on tempera- structures (21,36). Cooling from high temperatures, as well ture, irrespective of how the samples had been treated

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(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 þ 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 FIGURE 8 Hysteresis of POPE with 0.3% SP during the initial cycle of roughly at the junction between the headgroup and the heating and cooling. Diffraction was recorded during heating from 11C to 90C, and then during subsequent cooling to 6C. Open symbols with acyl residues (21)) from the minimal surface at the midpoint solid lines indicate the lattice constants (ao) during the initial heating; solid of the bilayer has opposite effects on the magnitude of the symbols with dotted lines provide the values during the subsequent cooling. two principal curvatures. The leaflet curvature with negative sign becomes more negative, and the positive curvature becomes smaller, resulting in a negative net curvature. previously. With the proteins present, the lattice constants of Although the leaflets are paired in the cubic phase and the QII phases during cooling were significantly smaller than unpaired in the stalk, the net and Gaussian curvatures in during heating, suggesting that the smaller unit cell observed the two cases are at least qualitatively similar. at higher temperatures grew during cooling with difficulty The proteins may induce formation of the QII phases by (Fig. 8). As with the samples of POPE alone, the QII phases either thermodynamic or kinetic effects. The distinction persisted during cooling to temperatures well below the depends on the ultimate stability of the different phases for levels at which they formed during heating. With the proteins the lipids alone. Without the proteins, the QII phases only present, however, the cubic phases reverted during cooling to appear after exposure to temperatures above the La-HII tran- lamellar structures well before reaching the Lb0 phase. Crys- sition. If this behavior represents true equilibrium, then the tallization of the acyl chains was unnecessary to promote observed formation of the QII structures at much lower return from the QII phases. 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 DISCUSSION that under these conditions, the curved structures might be Our experiments show that the hydrophobic SPs induce more stable than the lamellar phases, but kinetically inacces- POPE to form QII phases in a dose-dependent fashion. The sible. The proteins would then induce the QII phases by bicontinuous structures share important features of the key promoting their faster formation. The behavior of POPE by kinetic intermediate in the most widely considered model itself prevents a clear distinction between these two possibil- of how those proteins promote adsorption at the alveolar ities. The marked hysteresis during heating and cooling air/water interface (Fig. 1). The available evidence suggests prevents determination of the noncubic/cubic transition that, at least initially, the lipids flow from the adsorbing temperature that would allow assessment of thermodynamic vesicle to an air/water interface through a stalk that connects stability. The proteins narrow the hysteresis of the QII phases the two locations (38,39). The common two-dimensional (Fig. 8), suggesting that their effect may be kinetic, and that representation of this structure emphasizes that the curvature they accelerate transitions both to and from the cubic struc- in the plane perpendicular to the interface (c1) would be tures. Both mechanisms would be physiologically relevant. negative (Fig. 1 A). However, the second principal curvature Whether the proteins stabilize the bridging stalk or simply (c2) in the plane parallel to the interface would be positive facilitate its formation, the result would be faster adsorption.

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The proteins could induce the QII phases, whether by The surfactant lipids differ in several respects from the stabilization or improving kinetic access, by a limited num- compound used in our studies. The phospholipids in surfac- ber of mechanisms. The kinetics of forming the QII phases, tant from common mammals, for instance, contain ~10% which have received less attention than their stability, would anionic compounds, which may contribute to specific aspects presumably depend on the rigidity of the leaflets, expressed of adsorption (16,39), and which interact with the cationic as their moduli of bending. The stability of the QII phases has hydrophobic proteins. Because POPE should be zwitterionic generally been considered in terms of the energy of elastic in our samples, those interactions are absent. bending for the lipid leaflets. That energy depends on the The spontaneous curvature of POPE and the surfactant spontaneous curvature of the leaflets, and their moduli of lipids should also be different. POPE has a spontaneous simple-splay and saddle-splay bending (41,42). A recent curvature sufficient to form the HII phase. In excess water, report, however, suggested that the energy of unbinding the surfactant lipids form only lamellar bilayers. Like other adjacent bilayers might also affect the stability of the QII biological phospholipids, pulmonary surfactant can form phases (43). The transition of lamellar to QII phases requires HII structures if water is sufficiently restricted (50). That a significant separation of adjacent bilayers. The contribution effect, however, apparently occurs only at very low hydra- of the unbinding energy might explain, in particular, the tion (50). For preparations of most therapeutic surfactants, order in which phases generally appear during heating and for the multiple mixtures with lipids in which the hydro- (43), with QII structures only forming after the HII structures, phobic proteins promote adsorption in vitro, water is present opposite to the sequence predicted strictly from their bending in excess. Spontaneous curvature should be significantly energies. The proteins might then induce the QII phases or more negative for POPE than for fully hydrated surfactant facilitate formation of the hypothetical stalk-intermediate lipids. Calf surfactant contains only 3% (mol:mol) phospha- by shifting either the modulus of bending, by altering the tidylethanolamine (23,51), and the phosphatidylcholines, spontaneous curvature, or by reducing the energy of which constitute ~80% of the lipids, contain acyl con- unbinding. stituents that are remarkably low in the double bonds that Our studies included no experiments, such as with osmotic would contribute to negative curvature (51). Cholesterol stress, designed to probe these different possibilities. The represents the only major component that should favor the proteins nonetheless might well generate spontaneous HII phase. changes in the lattice constants for the different phases that The difference between the surfactant lipids and POPE is might distinguish the different mechanisms. A decrease in essential for our studies. Lipids adopt structures according to the modulus of splay-bending or the unbinding energy could the competing energies of bending and chain-packing (21). result in greater undulations of stacked bilayers and an When chain-packing dominates and lipids form planar bila- increase in spacing of the lamellar phases. A change in spon- yers, the spontaneous curvature of the individual leaflets is taneous curvature would shift the lattice constant of the HII unknown. To determine how the proteins affect the tendency phase. A lower modulus of saddle-splay bending could of the lipids to curve, the lipids must form structures that can increase the size of the cubic unit cells. Unfortunately, our express their curvature. results provide no insights concerning which mechanism is Our studies therefore substitute a nonphysiological motiva- more likely. The limited range of 0.00–0.03% SP over which tion for the formation of saddle-shaped structures. In the the HII phase is present prevents any assessment of how the lungs, a reduction in interfacial energy caused by the lower proteins affect spontaneous curvature. The lattice constants surface tension would drive adsorption and the transient for the lamellar and cubic phases are unaffected by the formation of the hypothetical stalk. In our studies with content of protein. The mechanism by which the proteins POPE, away from the interface, the lower energy of bending induce the QII phases therefore remains undetermined. present in the QII phases drives conversion from the planar Remarkably low levels of the proteins induce formation of bilayers. For both the stalk and the QII phases, the proteins the QII phases. With 0.1% (w:w) SP, for which diffraction at may promote formation of the saddle-shaped structure by high temperatures detects only the cubic structures, the modulating the energy of bending. In each circumstance, molecular ratio is roughly one protein per 10,000 phospho- the proteins should have similar effects on the factors that lipids. In this respect, the SPs extend findings with other determine the energy of bending for the two sets of lipids. peptides that induce QII phases in HII-forming phospholipids The spontaneous curvature of a lipid leaflet generally reflects (29,44–48). SP-B, like those peptides, consists largely of the additive effects of its constituents (52). Effects on the amphipathic helices (49). Like the SPs, these other peptides moduli of simple-splay and saddle-splay bending should be produce their structural effects on the lipids in small quanti- similar for different lipids. Despite the different driving ties, although none has demonstrated the effectiveness of the forces, the processes by which the proteins induce POPE to SPs. In native pulmonary surfactant, hydrophobic proteins form the QII phases should also facilitate formation by the account for ~1.5% (w:w, protein/phospholipid) of the surfactant lipids of the hypothetical stalk during adsorption. mixture. The effects demonstrated here at levels as low as In conclusion, the physiological mixture of SP-B and 0.03% therefore occur well within the physiological range. SP-C, in amounts as low as 0.03%, induces POPE to form

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