Kinetics of Lamellar-to-Cubic and Intercubic Phase Transitions of Pure and Cytochrome c Containing Monoolein Dispersions Monitored by Time-Resolved Small-Angle X-ray Diffraction

Abstract

We investigated the effect of incorporation of a small aqueous peripheral membrane protein (cyt c) into the three-dimensional periodic nanochannel structures formed by the lipid monoolein (MO) on its rich phase behavior as a function of temperature, pressure, and protein concentration using synchrotron X-ray small-angle diffraction. By simultaneous use of the pressure-jump relaxation technique and time-resolved synchrotron X-ray diffraction, we also studied the kinetics of various lipid mesophase transformations of the system for understanding the mechanistic pathways of their formation influenced by the protein-lipid interactions. Cyt c incorporated into the bicontinuous cubic phase Ia3d of MO has a significant effect on the lipid structure and the pressure stability of the system already at low protein concentrations. Concentrations higher than 0.2 wt % of cyt c led to an increase in interfacial curvature due to interaction of the protein with the lipid headgroups. This promotes the formation of a new, probably partially micellar cubic phase of crystallographic space group P4(3)32. Upon pressurization, the P4(3)32 phase undergoes a phase transition to a cubic Pn3m phase with smaller partial specific volume. Increase in protein concentration increases the pressure stability of the P4(3)32 phase. The formation of this phase from the cubic phase Pn3m is a slow process taking many seconds and having a time lag in the beginning. It seems to occur as a two-state process without ordered intermediate states. At temperatures above 60 degrees C, the P4(3)32 phase is unable to accommodate the unfolded protein and transforms to a bicontinuous cubic Ia3d phase. Time-resolved small-angle X-ray scattering studies show that the L(alpha) --> Ia3d transition in pure MO dispersions under limited hydration conditions occurs within a time interval of 1 s at 35 degrees C preceded by a lag phase of 1.5 s. The Ia3d cubic phase initially forms with a much larger lattice constant due to hydration and experiences an initially lower curvature that relaxes within about 1 s. Interestingly, no other cubic phases are involved as intermediates in the transition, i.e., the gyroid cubic phase is able to form directly from the L(alpha) phase. The mechanism behind the L(alpha) --> Ia3d transition in pure MO dispersions has been discussed within the framework of recent stalk models for membrane fusion. In the presence of cyt c, the L(alpha) --> Ia3d transition is much slower. The rather long relaxation times of the order of seconds are probably due to a kinetic trapping of the system and limitation by the transport and redistribution of water and lipid in the evolving new lipid phases. We also studied the transition from the pure lamellar L(alpha) phase to the Ia3d-P4(3)32 two phase region and observed a rather complex transition behavior with transient lamellar and cubic intermediate states.

Dynamics of Structural Transformations between Lamellar and Inverse Bicontinuous Cubic Lyotropic Phases

“…These dynamical features are not exclusive to ME; both the decrease in lattice parameter of the lamellar phase and the existence of an intermediate swollen cubic phase have been previously noted for several other amphiphiles including monomyristolein (unpublished data), 1-monoolein (MO) [24], and 2:1 Lauric Acid (LA) and Di-laurylphosphatidylcholine (DLPC) (2LA-DLPC) mixtures [16]. Additionally, for the systems monomyristolein in excess water and monoelaidin and monoolein under limited water conditions (unpublished data) we again observe a highly specific final lattice parameter of the lamellar phase and a strong dependence of the rate of shrinking on pressurejump amplitude.…”

High Pressure X-ray Studies of Lipid Membranes and Lipid Phase Transitions

“…Pressure cells based on this design have been developed by several groups [16,18,26,27], however the robust and versatile system designed by Woenckhaus [20] is particularly noteworthy as it represents a benchmark for more recent cell development and it has facilitated a wide range of exciting high pressure and dynamic experiments on lipid systems [28][29][30][31][32][33][34][35][36]. This system can perform both static high pressure and pressure jump experiments between atmospheric pressure and 0.7 GPa (7 kbar) at temperatures from −40 to 100 ∘ C. This cell employs 0.8 mm thick diamond as X-ray windows which offers a transmission of over 80% for 17 keV X-rays.…”

“…A number of soft matter X-ray pressure systems have been developed or used extensively at major synchrotron SAXS beamlines including beamline ID2 at the European Synchrotron Radiation Facility [20,28,49], beamline A2 at the Deutsche Synchrotron [32], the Austrian SAXS beamline at Elettra [18], BL9 at DELTA [22,50], station G1 at the Cornell High Energy Synchrotron Source [21], beamline 18ID at the Advanced Photon Source, Argonne and beamline I22 at Diamond Light Source [23].…”

“…A wide range of lyotropic lipid phase transitions have been investigated in this way, including: gel - [76], -II [77], -bicontinuous cubic [28], II -bicontinuous cubic [33]. and bicontinuous cubic -bicontinuous cubic [29,32] X-ray diffraction has proved extremely useful in identifying relatively short lived structural intermediates in lipid phase transitions. For example [28], during the transition between the The rates at which lipid structural transitions take place can be quantified by tracking the relative intensities of diffraction peaks in a series of time resolved X-ray patterns.…”

Melanoma therapy with transdermal mitoxantrone cubic phases

“…The MTO cubic phases was a viscous semi-solid and appeared to be blackish-blue in color. The structure of the cubic phases can be identified through the PLM and SAXS methods (Kraineva et al, 2005). In the PLM images, the lamellar phase exhibited some reflected light due to its anisotropy ( Figure 1A).…”