Rigidification of neutral lipid bilayers in the presence of salts

Abstract

We studied the influence of sodium and calcium chloride on the global and local membrane properties of fluid palmitoyl-oleoyl phosphatidylcholine bilayers, applying synchrotron small-angle x-ray diffraction, spin-labeling electron paramagnetic resonance spectroscopy, and differential scanning calorimetry, as well as simultaneous density and acoustic measurements. The salt concentration was varied over a wide range from 0 to 5 M. We found that NaCl leads to a continuous swelling of the bilayers, whereas the behavior of the bilayer separation dW in the presence of CaCl2 is more complex, showing an initial large dW value, which decreased upon further addition of salt and finally increased again in the high concentration regime. This can be understood by a change of balance between electrostatic and van der Waals interactions. We were further able to show that both salts lead to a significant increase of order within the lipid bilayer, leading to a decrease of bilayer elasticity and shift of main phase transition temperature. This effect is more pronounced for Ca2+, and occurs mainly in the high salt-concentration regime. Thus, we were able to reconcile previous controversies between molecular dynamics simulations and x-ray diffraction experiments regarding the effect of salts on neutral lipid bilayers.

FIGURE 1

Diffraction patterns of POPC at 300 K in the presence of NaCl (A) and CaCl₂ (B). Numbers adjacent to the data denote the respective salt concentrations in M. Solid lines give the best fits obtained using the global analysis technique. Arrows indicate the position of additional first-order Bragg peaks due to phase separation.

FIGURE 2

Dependence of the lamellar repeat distance (A), membrane thickness (B), bilayer separation (C), area/lipid (D), and bending fluctuations (E) on the concentration of NaCl (•) and CaCl₂ (□).

FIGURE 3

EPR spectra of POPC at 300 K without salt (A) and in the presence of 5 M NaCl (B) and 5 M CaCl₂ (C). Arrows indicate the contribution from increased restricted motions.

FIGURE 4

Average order parameter (A) and free rotational space (B) for POPC at 300 K as a function of NaCl (•) and CaCl₂ (□) concentrations. Bars indicate distribution of values obtained from the GHOST condensation technique (40).

FIGURE 5

Dependence of the specific volume (A), sound velocity number (B), and specific adiabatic compressibility (C) on the concentrations of NaCl (•) and CaCl₂ (□).

FIGURE 6

(A) Heat capacity of POPC in pure water and in the presence of 1 M, 3 M, and 5 M NaCl. (B) Thermograms obtained in the presence of 1 M, 3 M, and 5 M CaCl₂. Transition temperatures are summarized in Table 1.

FIGURE 7

Temperature dependence of the d-spacing of POPC in the presence of 3 M (▵) and 5 M CaCl₂ (▾). The dashed line indicates the T_m for both salt solutions observed by DSC (Fig. 6 and Table 1).