An anionic phospholipid enables the hydrophobic surfactant proteins to alter spontaneous curvature

Abstract

The hydrophobic surfactant proteins, SP-B and SP-C, greatly accelerate the adsorption of the surfactant lipids to an air/water interface. Previous studies of factors that affect curvature suggest that vesicles may adsorb via a rate-limiting structure with prominent negative curvature, in which the hydrophilic face of the lipid leaflets is concave. To determine if SP-B and SP-C might promote adsorption by inducing negative curvature, we used small-angle x-ray scattering to test whether the physiological mixture of the two proteins affects the radius of cylindrical monolayers in the inverse hexagonal phase. With dioleoyl phosphatidylethanolamine alone, the proteins had no effect on the hexagonal lattice constant, suggesting that the proteins fail to insert into the cylindrical monolayers. The surfactant lipids also contain ∼10% anionic phospholipids, which might allow incorporation of the cationic proteins. With 10% of the anionic dioleoyl phosphatidylglycerol added to dioleoyl phosphatidylethanolamine, the proteins induced a dose-related decrease in the hexagonal lattice constant. At 30°C, the reduction reached a maximum of 8% relative to the lipids alone at ∼1% (w/w) protein. Variation of NaCl concentration tested whether the effect of the protein represented a strictly electrostatic effect that screening by electrolyte would eliminate. With concentrations up to 3 M NaCl, the dose-related change in the hexagonal lattice constant decreased but persisted. Measurements at different hydrations determined the location of the pivotal plane and proved that the change in the lattice constant produced by the proteins resulted from a shift in spontaneous curvature. These results provide the most direct evidence yet that the surfactant proteins can induce negative curvature in lipid leaflets. This finding supports the model in which the proteins promote adsorption by facilitating the formation of a negatively curved, rate-limiting structure.

Figure 1

Diffraction from phospholipid-protein mixtures. Traces depict radially integrated intensity plotted on a logarithmic scale for a limited range of q. The different sets of data are offset vertically without change in scale to allow inspection of the separated traces. The results show how DOPG, different levels of SP (%, w/w), and temperature affect diffraction from DOPE. Data at additional temperatures and concentrations of protein were omitted for clarity of presentation. Samples contain 2 μM EDTA and 10 mM HEPES, pH 7.0. (A) DOPE alone. (B) DOPE/DOPG (9:1, mol/mol).

Figure 2

Phase diagrams for DOPE (A–D) and DOPE/DOPG (9:1, mol/mol) (E–H) showing the effects of the hydrophobic proteins and temperature on the phases formed with different concentrations of NaCl. Labels on the graphs indicate regions with the following phases: inverse hexagonal phase (H_II); inverse bicontinuous cubic phase without distinction between the $\text{P}\text{n}\overline{3}\text{m}$ and $\text{I}\text{m}\overline{3}\text{m}$ space groups (Q_II); coexisting H_II and Q_II phases (Co); coexisting H_II and $\text{F}\text{d}\overline{3}\text{m}$ phases (H_II-Fd); and samples that scattered without diffraction (S). Diagrams for 100 mM NaCl and 1 M NaCl are omitted for clarity of presentation.

Figure 3

Effect of temperature on the lattice constant of structures formed by DOPE and DOPE/DOPG with different amounts of protein. The diffracting structures were identified according to the spacing of the diffraction peaks (Fig. 1) as H_II or as Q_II with space group either $\text{P}\text{n}\overline{3}\text{m}$ or $\text{I}\text{m}\overline{3}\text{m}$, corresponding to a bicontinuous structure along the diamond (Q_II^D) or primitive (Q_II^P) infinitely periodic minimal surface, respectively. The y axis is split to emphasize the shift in a_o(H_II) with different amounts of SP. Samples contained 10 mM HEPES, pH 7.0, and 2 mM EDTA. (A) DOPE alone. (B) DOPE/DOPG (9:1, mol/mol). The dashed black line indicates the values of a_o(H_II) for DOPE without DOPG or protein.

Figure 4

Variation of a_o(H_II) with different concentrations of protein. Traces compare values obtained from Fig. 3 at the same temperature. To correct for the variation of a_o(H_II) with temperature, values are expressed relative to the value without protein, a_o/a_o(SP = 0). Closed symbols indicate samples with minimal evidence of a coexisting phase; open symbols indicate samples with coexistence. Samples contained 10 mM HEPES, pH 7.0, and 2 mM EDTA. (A) DOPE. (B) DOPE/DOPG (9:1, mol/mol).

Figure 5

Response of a_o(H_II) to variable concentrations of electrolyte. Samples contained lipids combined with different amounts (%, w/w) of proteins and suspended in different concentrations of NaCl buffered with 10 mM HEPES, pH 7.0, at 60°C. HSC samples contained 150 mM NaCl, 1.5 mM CaCl₂, and 10 mM HEPES, pH 7.0. Closed symbols indicate measurements that detected only the hexagonal phase; open symbols indicate the presence of coexisting Q_II phases. (A) DOPE. (B) DOPE/DOPG (9:1, mol/mol).

Figure 6

Schematic diagram indicating key features of the H_II phase. The gray rings with the sketch of an individual phospholipid represent the cylindrical monolayers surrounding the white aqueous core. The arrows indicate: the lattice constant obtained directly from measurements of diffraction (a_o); the radius at the Luzzati surface at the outer surface of the aqueous core (R_w); the radius at the outer surface of the monolayer (R_o); the radius at the pivotal plane (dashed ring), where the molecular cross-sectional area remains constant during bending (R_p); and the molecular volume that separates the pivotal plane from the Luzzati surface $(\Delta {\overline{\text{V}}}_{\text{p}})$. The crosshatched areas represent regions that would be unfilled if the monolayers were circular with a uniform thickness.

Figure 7

Swelling of DOPE/DOPG (9:1, mol/mol) with different amounts of the combined surfactant proteins (%, w/w). Measurements were made at 30°C on samples containing HE added to achieve different levels of hydration. The data were restricted to samples that produced only hexagonal diffraction without evidence for a coexisting phase. The solid symbols indicate results obtained with HE added to excess.

Figure 8

Diagnostic plots for samples of DOPE/DOPG with different amounts of the SPs. The data from the ascending limb (0 < ϕ_w < 0.26) in Fig. 7 were used to calculate values of ${\overline{\text{A}}}_{\text{w}}^2$ and ${\overline{\text{A}}}_{\text{w}}/{\text{R}}_{\text{w}}$. The straight lines represent the least-squares linear fit to the data for each sample.