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Background  Awad et al.  [1]  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 ∼140 Å (L α -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°C a direct transition from the vesicles to the H 2  phase was observed, whereas at ambient temperature (20°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.

Calcium Triggered Lα-H2 Phase Transition Monitored by Combined Rapid Mixing and Time-Resolved Synchrotron SAXS