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

<p>(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 <i>q</i>-values, suggest a coexistence with the H₂ phase (for details see text). (B) Temporal evolution of the observed lattice spacings.</p

Unit cell parameter, <i>a</i>, of the ternary MO/A₆D/water and MO/KA₆/water systems.

<p>The experiments were carried out at four different temperatures in the range of 25–70°C. The samples were formed at pH = 7.4 and contain 18 wt% lipid mixture (MO & peptide surfactant).</p

Phase diagrams of MO-based systems with differing A₆D (A) and KA₆ content (B), respectively.

<p>The structural investigations were carried out for <i>R</i> values in the range of 0–0.2. The Pn3m phase regions in these diagrams have been highlighted in light grey and approximate phase boundaries are depicted. The lattice parameter values, <i>a</i>, for some of the Pn3m (•) and H₂ (O) phases are given in Ångstrøms.</p

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

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

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

<p>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.</p

Contour X-ray diffraction plots covering the phase regime of the L_α, Im3m and Pn3m phases and displaying all the strong peaks as a function of <i>q</i> and temperature.

<p>(A) In a temperature scan from 25 to 80°C with a rate of 1°C/min, the ME aqueous dispersion underwent the internal L_α<i>via</i> Im3m to Pn3m structural transition. Every minute 3 X-ray patterns were recorded, i.e. the temperature resolution was 0.33°C. The exposure time was 5 sec (wait interval 15 sec). (B) The fully hydrated ME/water system displays the same three phases in a narrower temperature range, i.e. within 10°C only. Hence, a lower scan rate of 0.5°C/min was applied. It should be noted that at 1°C/min the formation of the Im3m phase is only scarcely visible (data not shown). X-ray patterns were recorded every half a minute, i.e. the temperature resolution was 0.25°C. The exposure time for each X-ray scattering curve was 10 sec (wait interval 20 sec). The color scale of the diffraction intensity is mentioned in the insets.</p

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

<p>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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002072#pone-0002072-g005" target="_blank">Figure 5</a>). 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 <i>d₁₀</i>-spacing of the H₂ phase is displayed as a function of the final Ca²⁺ concentration. The inset shows the <i>d</i>-spacing in dependence of the DOPG/Ca²⁺ ratio. The electroneutral regime is highlighted in light grey.</p

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

<p>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.</p

The molar ratios of DOPG/[Ca²⁺] and (DOPG+MO)/[Ca²⁺] after rapidly mixing the DOPG/MO-based vesicles with PIPES buffer (pH 7.0) containing 68 mM Ca²⁺ ions.

<p>The investigated vesicles in the absence of Ca²⁺ ions were composed of DOPG and MO at a constant molar ratio of 30∶70 and the total lipid content was 7 wt%. Eight different rapid-mixing investigations with different volume ratios of the vesicles (volume A) to the buffer (volume B) are summarized. In addition, the final Ca²⁺ concentrations are given.</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002072#pone.0002072-Shearman1" target="_blank">[87]</a>, 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).</p