Calcium triggered L alpha-H2 phase transition monitored by combined rapid mixing and time-resolved synchrotron SAXS

Abstract

Background: Awad et al. reported on the Ca(2+)-induced transitions of dioleoyl-phosphatidylglycerol (DOPG)/monoolein (MO) vesicles to bicontinuous cubic phases at equilibrium conditions. In the present study, the combination of rapid mixing and time-resolved synchrotron small-angle X-ray scattering (SAXS) was applied for the in-situ investigations of fast structural transitions of diluted DOPG/MO vesicles into well-ordered nanostructures by the addition of low concentrated Ca(2+) solutions.

Methodology/principal findings: Under static conditions and the in absence of the divalent cations, the DOPG/MO system forms large vesicles composed of weakly correlated bilayers with a d-spacing of approximately 140 A (L(alpha)-phase). The utilization of a stopped-flow apparatus allowed mixing these DOPG/MO vesicles with a solution of Ca(2+) ions within 10 milliseconds (ms). In such a way the dynamics of negatively charged PG to divalent cation interactions, and the kinetics of the induced structural transitions were studied. Ca(2+) ions have a very strong impact on the lipidic nanostructures. Intriguingly, already at low salt concentrations (DOPG/Ca(2+)>2), Ca(2+) ions trigger the transformation from bilayers to monolayer nanotubes (inverted hexagonal phase, H(2)). Our results reveal that a binding ratio of 1 Ca(2+) per 8 DOPG is sufficient for the formation of the H(2) phase. At 50 degrees C a direct transition from the vesicles to the H(2) phase was observed, whereas at ambient temperature (20 degrees C) a short lived intermediate phase (possibly the cubic Pn3m phase) coexisting with the H(2) phase was detected.

Conclusions/significance: The strong binding of the divalent cations to the negatively charged DOPG molecules enhances the negative spontaneous curvature of the monolayers and causes a rapid collapsing of the vesicles. The rapid loss of the bilayer stability and the reorganization of the lipid molecules within ms support the argument that the transition mechanism is based on a leaky fusion of the vesicles.

Figure 1. Schematic of the combined stopped-flow and synchrotron SAXS set-up.

In the stopped-flow apparatus, one syringe contained a buffer with Ca²⁺ ions, whereas the other contained DOPG/MO-based vesicles. The rapid mixing was conducted within 10 ms and the formation of the inverse hexagonal phase (H₂) was followed by millisecond time-resolved SAXS.

Figure 2. Background subtracted SAXS pattern of DOPG/MO-based vesicles at 25°C in the absence of Ca²⁺ ions.

The investigated vesicles are composed of DOPG and MO at a molar ratio of 30∶70 with a total lipid content of 7 wt%. The best global fit to the experimental data is given by a solid red line (cp. data analysis). The inset shows the quality of the fit at higher q-values. The structural bilayer parameters are summarized in Table 1.

Figure 3. Electron density bilayer model of the DOPG/MO vesicles at 25°C in the absence of Ca²⁺ ions.

This model is composed of one Gaussian representing the electron density distribution of the polar headgroups at ±z_H and a second for the hydrophobic core with its centre at the bilayer mid-plane. The corresponding widths of the Gaussians are given by the standard deviations σ_H and σ_C, respectively. d defines the lattice spacing of the L_α phase. The obtained results refer to the data of Figure 2.

Figure 4. Time-resolved X-ray pattern of the rapid calcium-triggered H₂ phase formation at 50°C.

The vesicle dispersion contained DOPG/MO with a molar ratio 30∶70 (7 wt% lipid), and the final salt concentration was 20 mM. The contour plot clearly displays the first three reflections of the H₂ phase; no indication for the formation of an intermediate phase is spotted.

Figure 5. Time dependence of the intensity (A), and the d-spacing (B) of the first order reflection of the rapidly formed H₂ phase referring to the experiment given in Figure 4.

The solid red line shows the best single exponential fit to the data. The time constant, k, was determined to be 0.10±0.03 and 0.12±0.02 sec⁻¹ for panels (A) and (B), respectively.

Figure 6. Comparison of the calcium-induced H₂ phase in dependence of the final salt concentration.

All rapid-mixing experiments were carried out at 50°C. (A) The SAXS patterns of the DOPG/MO-based aqueous dispersions are displayed 71 s after the rapid mixing, i.e. approximately one minute after the turnovers were completed (Figure 5). It should be pointed out that for the two lowest salt concentrations the H₂ phase is coexisting with weakly correlated bilayers (the positions of the very weak first two diffraction orders are marked by arrows). (B) The d₁₀-spacing of the H₂ phase is displayed as a function of the final Ca²⁺ concentration. The inset shows the d-spacing in dependence of the DOPG/Ca²⁺ ratio. The electroneutral regime is highlighted in light grey.

Figure 7. Time-resolved X-ray pattern of the calcium induced H₂ phase formation at 20°C.

The vesicle dispersion contained DOPG/MO with a molar ratio 30∶70 (7 wt% lipid), and the final salt concentration was 34 mM. The contour plot displays the first three reflections of the H₂ phase, but its formation is not immediate. In the first 400 ms, an intermediate phase is apparent. Two strong reflections are indicated by arrows and one weaker peak is circled by a dashed line.

Figure 8. Intermediate formation referring to the rapid-mixing experiment of Figure 7.

(A) The SAXS pattern is averaged from the data taken in the range of 100–400 ms after the rapid mixing. It indicates a possible formation of bicontinuous cubic phase of the symmetry Pn3m. The observed Bragg peaks and their presented q-values, suggest a coexistence with the H₂ phase (for details see text). (B) Temporal evolution of the observed lattice spacings.

Figure 9. Two schematics of the proposed pathways from the bilayer to the inverted monolayer tube transition.

On the left hand side, the classical vesicle fusion route is depicted. The formation of pores is widely believed to be the prerequisite for the formation of bicontinuous cubic (Q₂) nanostructures , which upon further curvature frustration may transform into self-assembled monolayer tubes (H₂ phase). On the right hand side, the direct formation of an inverse lipid nanotube between two opposed bilayers is illustrated. For better understanding of the structural conversions, the headgroups of opposed monolayers are shown in light blue whereas the rest are depicted in blue (for further details see text).