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.
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.
Kraineva
Julia
J
Department of Chemistry, Physical Chemistry I, University of Dortmund, Otto-Hahn Strasse 6, D-44221 Dortmund, Germany.
Narayanan
R Aravinda
RA
Kondrashkina
Elena
E
Thiyagarajan
Pappannan
P
Winter
Roland
R
D003460:Crystallization; D045304:Cytochromes c / Q000737:chemistry*; D005989:Glycerides / Q000737:chemistry*; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D044367:Phase Transition; D013696:Temperature; D014867:Water / Q000737:chemistry; D014961:X-Ray Diffraction / Q000379:methods
D005989:Glycerides; D008051:Lipid Bilayers; D014867:Water; D045304:Cytochromes c; C005953:monoolein