Cholesterol is well known to modulate the physical properties of biomembranes. Using modern x-ray scattering methods, we have studied the effects of cholesterol on the bending modulus K C , the thickness D HH , and the orientational order parameter S xray of lipid bilayers. We find that the effects are different for at least three classes of phospholipids characterized by different numbers of saturated hydrocarbon chains. Most strikingly, cholesterol strongly increases K C when both chains of the phospholipid are fully saturated but not at all when there are two monounsaturated chains.
We have undertaken a series of experiments to examine the behavior of individual components of cell membranes. Here we report an initial stage of these experiments, in which the properties of a chemically simple lipid mixture are carefully mapped onto a phase diagram. Four different experimental methods were used to establish the phase behavior of the 3-component mixture DSPC/DOPC/chol: (1) confocal fluorescence microscopy observation of giant unilamellar vesicles, GUVs; (2) FRET from perylene to C20:0-DiI; (3) fluorescence of dilute dyes C18:2-DiO and C20:0-DiI; and (4) wide angle X-ray diffraction. This particular 3-component mixture was chosen, in part, for a high level of immiscibility of the components in order to facilitate solving the phase behavior at all compositions. At 23 degrees C, a large fraction of the possible compositions for this mixture give rise to a solid phase. A region of 3-phase coexistence of {Lalpha+Lbeta+Lo} was detected and defined based on a combination of fluorescence microscopy of GUVs, FRET, and dilute C20:0-DiI fluorescence. At very low cholesterol concentrations, the solid phase is the tilted-chain phase Lbeta'. Most of the phase boundaries have been determined to be within a few percent of the composition. Measurements of the perturbations of the boundaries of this accurate phase diagram could serve as a means to understand the behaviors of a range of added lipids and proteins.
We used wide angle x-ray scattering (WAXS) from stacks of oriented lipid bilayers to measure chain orientational order parameters and lipid areas in model membranes consisting of mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol in fluid phases. The addition of 40% cholesterol to either DOPC or DPPC changes the WAXS pattern due to an increase in acyl chain orientational order, which is one of the main properties distinguishing the cholesterol-rich liquid-ordered (Lo) phase from the liquid-disordered (Ld) phase. In contrast, powder x-ray data from multilamellar vesicles does not yield information about orientational order, and the scattering from the Lo and Ld phases looks similar. An analytical model to describe the relationship between the chain orientational distribution and WAXS data was used to obtain an average orientational order parameter, S(x-ray). When 40% cholesterol is added to either DOPC or DPPC, S(x-ray) more than doubles, consistent with previous NMR order parameter measurements. By combining information about the average chain orientation with the chain-chain correlation spacing, we extended a commonly used method for calculating areas for gel-phase lipids to fluid-phase lipids and obtained agreement to within 5% of literature values.
Counterion atmospheres condensed onto charged biopolymers strongly affect their physical properties and biological functions, but have been difficult to quantify experimentally. Here, monovalent and divalent counterion atmospheres around DNA double helices in solution are probed using small-angle x-ray scattering techniques. Modulation of the ion scattering factors by anomalous (resonant) x-ray scattering and by interchanging ion identities yields direct measurements of the scattering signal due to the spatial correlation of surrounding ions to the DNA. The quality of the data permit, for the first time, quantitative tests of extended counterion distributions calculated from atomic-scale models of biologically relevant molecules.
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