X-ray Kinematography of Phase Transformations of Three-Component Lipid Mixtures: A Time-Resolved Synchrotron X-ray Scattering Study Using the Pressure-Jump Relaxation Technique

Abstract

By using the pressure-jump relaxation technique in combination with time-resolved synchrotron small-angle X-ray diffraction (TRSAXS), the kinetics of lipid phase transformations of ternary lipid mixtures serving as model systems of heterogeneous raftlike membranes were investigated. To this end, we first established the temperature-pressure phase diagram of a model lipid raft mixture, 1,2-dioleoyl- sn-glycero-3-phosphatidylcholine (DOPC)/1,2-dipalmitoyl- sn-glycero-3-phosphatidylcholine (DPPC)/cholesterol (1:2:1), using Fourier transform infrared spectroscopy and SAXS, covering the pressure range from 1 bar to 10 kbar at temperatures in the range from 7 to 80 degrees C. We then studied the kinetics of interlamellar phase transitions of the ternary lipid system involving transitions from the fluidlike (liquid-disordered, l d) phase to the liquid-ordered (l o)/liquid-disordered (l d) two-phase coexistence region as well as between the two- and three-phase coexistence regions of the system, where also solid-ordered phases (s o) are involved. The phase transition from the all-fluid l d phase to the l o+l d two-phase coexistence region turns out to be rather rapid. Phases appear or disappear within the 25 ms time resolution of the technique, followed by a slow lattice relaxation process, which, depending on the pressure-jump amplitude, takes several seconds. Contrary to many one-component phospholipid phase transitions, the kinetics of the l d <--> l o+l d transition follows a similar time scale and mechanism for the pressurization and depressurization direction. A similar behavior is observed for the phase transition kinetics of the s o+l o+l d <--> l o+l d transformation and even for the s o+l o+l d <--> l d transformation, jumping across the l o+l d two-phase region. All transitions are fully reversible, and no intermediate states are populated. As indicated by the complex relaxation profiles observed, the overall rates observed seem to reflect the effect of coupling of various dynamical processes through the transformation, involving fast conformational changes in the sub-millisecond time regime and slow relaxation of the lattices growing, probably being largely controlled by the transport and redistribution of water into and in the new phases of the multilamellar vesicle assemblies.

Pressure effects on lipid membrane structure and dynamics

“…A further innovation of this system is the use of a high pressure reservoir on the pump side of the jumping valves; this allows larger amplitude pressure jumps to be performed. The specifications of this pressure system have set the bench mark for more recent soft condensed matter pressure cells and this system has facilitated some extremely exciting lipid experiments (Conn et al, , 2008Eisenblatter, 2006;Jeworrek et al, 2008;Kraineva et al, 2005;Squires et al, 2000Squires et al, , 2002.…”

Revealing conformational substates of lipidated N-Ras protein by pressure modulation

“…Here we employ hydrostatic pressure as a tool to shift the conformational equilibrium of N-Ras to sufficiently populate low-lying excited substates of Ras, which might be decisively important in signaling. To this end, we have studied the lipidated protein N-Ras HD/Far (hexadecyl/farnesyl) in its GDP and GTP analog (GppNHp) bound form (denoted as N-Ras (GDP) and N-Ras (GTP), respectively, hereafter) in the presence and absence of a canonical lipid raft membrane (29) by means of high pressure FTIR spectroscopy. Currently only few high pressure studies have been performed for probing conformational equilibria (6,7,11,21).…”

High-Pressure Biochemistry and Biophysics

“…Studies were also carried out on the phase behavior of cholesterol containing ternary lipid mixtures, generally containing an unsaturated lipid like a phosphatidylcholine and a saturated lipid-like sphingomyelin (SM) or DPPC. Such lipid systems are supposed to mimic distinct liquid-ordered lipid regions, called "rafts," which also seem to be present in cell membranes and are thought to be important for cellular functions such as signal transduction and the sorting and transport of lipids and proteins (Munro 2003;Janosch et al 2004;Nicolini et al 2005Nicolini et al , 2006Jeworrek et al 2008;Weise et al 2009;Kapoor et al 2012a,b). Lipid domain formation can be influenced by temperature, pH, calcium ions, protein adsorption, and may be expected to change upon pressurization as well.…”