Temperature and pressure effects on structural and conformational properties of POPC/SM/cholesterol model raft mixtures—a FT-IR, SAXS, DSC, PPC and Laurdan fluorescence spectroscopy study

Abstract

We report on the effects of temperature and pressure on the structure, conformation and phase behavior of aqueous dispersions of the model lipid "raft" mixture palmitoyloleoylphosphatidylcholine (POPC)/bovine brain sphingomyelin (SM)/cholesterol (Chol) (1:1:1). We investigated interchain interactions, hydrogen bonding, conformational and structural properties as well as phase transformations of this system using Fourier transform-infrared (FT-IR) spectroscopy, small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), and Laurdan fluorescence spectroscopy. The IR spectral parameters in combination with the scattering patterns from the SAXS measurements were used to detect structural and conformational transformations upon changes of pressure up to 7-9 kbar and temperature in the range from 1 to about 80 degrees C. The generalized polarization function (GP) values, obtained from the Laurdan fluorescence spectroscopy studies also reveal temperature and pressure dependent phase changes. DSC and PPC were used to detect thermodynamic properties accompanying the temperature-dependent phase changes. In combination with literature fluorescence spectroscopy and microscopy data, a tentative p,T stability diagram of the mixture has been established. The data reveal a broad liquid-order/solid-ordered (lo+so) two-phase coexistence region below 8+/-2 degrees C at ambient pressure. With increasing temperature, a lo+ld+so three-phase region is formed, which extends up to approximately 27 degrees C, where a liquid-ordered/liquid-disordered (lo+ld) immiscibility region is formed. Finally, above 48+/-2 degrees C, the POPC/SM/Chol (1:1:1) mixture becomes completely fluid-like (liquid-disordered, ld). With increasing pressure, all phase transition lines shift to higher temperatures. Notably, the lo+ld (+so) phase coexistence region, mimicking raft-like lateral phase separation in natural membranes, extends over a rather wide temperature range of about 40 degrees C, and a pressure range, which extends up to about 2 kbar for T=37 degrees C. Interestingly, in this pressure range, ceasing of membrane protein function in natural membrane environments has been observed for a variety of systems.

Influence of High Pressure on the Dimerization of ToxR, a Protein Involved in Bacterial Signal Transduction

“…Typically GP values range from about Ϫ0.2 to 0.2 for purefluid-like, disordered phases of lipid bilayers up to Ϸ0.55 to 0.60 in all-solid-ordered, gel-like lipid phases (14). Upon pressurization up to 50 MPa, GP values of 0.43 are reached, which is close to the tight packing of membranes reached for allordered conformational states of lipid bilayers.…”

Phase separation is induced by phenothiazine derivatives in phospholipid/sphingomyelin/cholesterol mixtures containing low levels of cholesterol and sphingomyelin

“…Other feature of most commonly explored raft model systems is that cholesterol and sphingomyelin are present in high concentrations (approx. 30% of total lipid content, see [5] for most recent study in this field). It has been shown, however, that phase separation can be observed in model membranes containing much smaller cholesterol and sphingomyelin amounts.…”

Hallmarks of Reversible Phase Separation in Living, Unperturbed Cell Membranes

“…Cyclic changes in any one of these thermodynamic variables cause domains to reversibly appear and disappear in synthetic vesicles. 4,119 To test whether the domains in yeast vacuole membranes arise from demixing of a single liquid phase into two coexisting liquid phases, we subject yeast to rapid temperature cycles. Fig.…”