Hydrophobic Surfactant Proteins Induce a Phosphatidylethanolamine to Form Cubic Phases

Biophysical Journal Volume 98 April 2010 1549–1557 1549

Hydrophobic Surfactant Proteins Induce a Phosphatidylethanolamineto Form Cubic Phases

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 negativelycurved stalk between the vesicular bilayer and the interface. To determine whether the proteins can alter the curvature of lipidleaflets, we used x-ray diffraction to investigate how the physiological mixture of these proteins affects structures formed by1-palmitoyl-2-oleoyl phosphatidylethanolamine, which by itself undergoes the lamellar-to-inverse hexagonal phase transitionat 71�C. In amounts as low as 0.03% (w:w) and at temperatures as low as 57�C, the proteins induce formation of bicontinuousinverse cubic phases. The proteins produce a dose-related shift of diffracted intensity to the cubic phases, with minimal evidenceof other structures above 0.1% and 62�C, but no change in the lattice-constants of the lamellar or cubic phases. The induction ofthe bicontinuous cubic phases, in which the individual lipid leaflets have the same saddle-shaped curvature as the hypotheticalstalk-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 alveolocapil-

lary barrier (1–3) equivalent to the insult caused by ventila-

tion when surface tension is elevated because the complete

mixture of surfactant-constituents is missing (4,5). A defi-

ciency 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 surfac-

tant 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), suggest-

ing 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 surfac-

tant generally promote or inhibit adsorption according to their

tendency to produce negative or positive curvature (9,17–19),

Submitted October 5, 2009, and accepted for publication December 15,

2009.6Hamed Khoojinian and Leonard E. Schulwitz Jr., contributed equally to

this work.

*Correspondence: [email protected]

Editor: Lukas K. Tamm.

� 2010 by the Biophysical Society

0006-3495/10/04/1549/9 $2.00

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 nega-

tively 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.

To the best of our knowledge, however, no direct evidence

shows that the surfactant proteins (SPs) can affect the ten-

dency 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 spon-

taneous 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 struc-

tures. Because 1-palmitoyl-2-oleoyl phosphatidylethanol-

amine (POPE) has a sufficiently inverted conical shape,

high temperatures convert lamellar bilayers to the inverse

hexagonal (HII) phase, in which unpaired leaflets approxi-

mate 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 surfac-

tant from calf lungs (ONY, Amherst, NY) were prepared as described

doi: 10.1016/j.bpj.2009.12.4302

mailto:[email protected]

FIGURE 1 Schematic representation of saddle-shaped structures. The

radii R1 and R2 each define a corresponding principal curvature of the struc-

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

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 Im3m.

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.

1550 Chavarha et al.

previously (22). Hydrophobic SPs were obtained from the extracted surfac-

tant 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

Biophysical Journal 98(8) 1549–1557

(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

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 A 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 circu-

lating 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.

FIGURE 2 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 character-

istic of the Lb0 phase (33). (B) POPE alone, 30�C; lamellar spacing, consis-

tent with the La 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

FIGURE 3 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 Pn3m (Pn) or Im3m (Im) space groups based on the

fits of the observed spacings to allowed peaks (Fig. 4).

Hydrophobic Surfactant Proteins Induce Cubic Phases 1551

RESULTS

Samples containing POPE with 0–3% (w:w) SP at specific

temperatures of 11–90�C in all cases produced rings indi-

cating 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 Lb0 phase

(33) (Fig. 2 A). A second set of lamellar peaks appeared at

25�C with lower d-spacing (Fig. 2 B), consistent with forma-

tion of the La phase close to the previously reported main

transition temperature of 26�C (34). The La 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 rea-

sonably with the expected formation of the HII phase at

71�C (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 transi-

tion 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 phosphatidyletha-

nolamines. 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 struc-

tures with Pn3m and Im3m space groups (Fig. 4). At lower

temperatures, although the shift to lower q-values caused

with simultaneous diffraction from cubic structures with Pn3m and Im3m

space groups, indicating coexistence of inverse bicontinuous cubic (QII)

phases with the diamond and primitive minimal surfaces, respectively.


FIGURE 4 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 þ hk þ k2)1/2 for the hexagonal phase, and

(h2 þ k2 þ 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 exper-

imentally in other systems of phospholipids or suggested by simulation (54):

lamellar (L; space group pm, No.3 in the International Tables of Crystallog-

raphy (55)); hexagonal (H;p6m, No.17); Pn3m (P; No.224); Im3m (Im;

No.229); Fd3m (F; No.227); and Ia3d (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 Pn3m and Im3m 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 ¼ 2p/slope)

for the lamellar and cubic phases, and (ao¼ 4p/(31/2 $ slope)) for the hexag-

onal phase.


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 5 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

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

phases with diamond (QIID) and primitive (QII

P) 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

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 Pn3m diffraction.

For protein R 0.1%, hexagonal diffraction disappeared com-

pletely, 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 QII

P

(Fig. 5 and Fig. S1). More protein also induced a broadening

of the peaks. Consequently, although the integrated intensity

from the Im3m peaks continued to increase at 3% (Fig. 6),

the height of the peaks fell.

The temperature at which cubic diffraction first emerged

showed limited dependence on the amount of protein pres-

ent. 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 transi-

tion-temperature or simply a dose-dependent variation in

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.

A

B

FIGURE 6 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.

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).


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 essen-

tially 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.

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 La-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 experi-

ments, 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 temper-

ature of the La-Lb0 transition, below which they were absent.

Although the diffraction of any sample during heating from

the Lb0 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

La-QII transition temperature. The results confirmed,

however, that although the proteins greatly facilitated forma-

tion 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 temper-

atures at which the cubic phases first appeared and disap-

peared. Like the samples with POPE alone, the lattice

constant for the lamellar phases depended only on tempera-

ture, irrespective of how the samples had been treated


FIGURE 8 Hysteresis of POPE with 0.3% SP during the initial cycle of

heating and cooling. Diffraction was recorded during heating from 11�Cto 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.


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 Lb0 phase. Crys-

tallization of the acyl chains was unnecessary to promote

return from the QII phases.

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 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

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 La-HII tran-

sition. 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 inacces-

sible. 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 possibil-

ities. 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 struc-

tures. 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 num-

ber 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 spon-

taneous 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 phospho-

lipids. 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 quanti-

ties, 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 surfac-

tant 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 hydra-

tion (50). For preparations of most therapeutic surfactants,

and for the multiple mixtures with lipids in which the hydro-

phobic 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) phospha-

tidylethanolamine (23,51), and the phosphatidylcholines,

which constitute ~80% of the lipids, contain acyl con-

stituents 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 bila-

yers, 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 motiva-

tion 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 La 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.

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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Hydrophobic Surfactant Proteins Induce a Phosphatidylethanolamine to Form Cubic Phases

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