Spin label ESR and 31P-NMR studies of the cubic and inverted hexagonal phases of dimyristoylphosphatidylcholine/myristic acid (1:2, mol/mol) mixtures

Abstract

The thermotropic phase behaviour and chain dynamics of the dimyristoylphosphatidylcholine/myristic acid (1:2, mol/mol) mixture at pH 4.0 have been studied with 31P-NMR and spin-label ESR. Broadline proton-dipolar decoupled 31P-NMR spectra show that the system undergoes a transition from a lamellar phase to a non-lamellar phase at 47 degrees C. The 31P-NMR spectrum collapses, from a lamellar powder pattern with apparent chemical shift anisotropy delta sigma = -38 ppm below 47 degrees C, to an isotropic spectrum with superimposed hexagonal powder pattern of delta sigma = +13 ppm above 47 degrees C. With increasing temperature, the hexagonal component grows at the expense of the isotropic component, finally transforming to a single hexagonal phase at approximately 70 degrees C. The ESR spectra from both fatty acid and phosphatidylcholine probes spin-labelled in the hydrocarbon chain have been used to study the ordering and mobility of the lipid chains through the lamellar-non lamellar transition. An abrupt increase in the chain mobility observed at approx. 47 degrees C demonstrates that the lamellar-non lamellar phase transition coincides with chain melting. The apparent order parameter and polarity profiles, deduced from the ESR spectra of 11 different chain-labelled positional isomers, demonstrate flexibility and polarity gradients characteristic of a lyotropic liquid crystalline phase. The temperature dependence of the order parameters does not show any changes in the fluid phase, indicating that the conversion from the isotropic (cubic) phase to the inverted hexagonal phase is not accompanied by large changes in chain order. A comparison of the apparent order parameters and polarity profiles of the phosphatidylcholine and fatty acid spin labels indicates that the fatty acid spin labels are intercalated approximately one CH2 unit more deeply in the hydrocarbon region than are the positionally isomeric phosphatidylcholine spin labels.

Polarity dependence of EPR parameters for TOAC and MTSSL spin labels: Correlation with DOXYL spin labels for membrane studies

“…Practical considerations for the measurement of a N o from anisotropic spin-label EPR spectra in biological systems are given in Refs. [12,13,17,18]. For aprotic environments, the necessary relations are established by a common effective dielectric constant.…”

“…1) [9,10]. The influence of lipid composition on these transbilayer profiles has also been studied [9,[11][12][13][14]. The polarity exhibits a trough-like profile across the membrane, which is registered by the hyperfine couplings and g-values of the DOXYL spin labels.…”

“…This polarity profile not only constitutes the permeability barrier of the membrane and determines the energetics of insertion and transmembrane-anchoring of integral proteins, it also provides a means by which the location of spin-labelled peptides and proteins may be determined in membranes. Determining the vertical location within the membrane of other spinlabelled lipid species by this method is straightforward (see e.g., [12,13,15]). However, the DOXYL nitroxides that are used with lipids are not those normally employed in the spin-labelling of peptides or proteins for EPR studies.…”

Effects of unsaturated fatty acids and triacylglycerols on phosphatidylethanolamine membrane structure

“…Some works have provided evidence;. e.g., spin label electron paramagnetic resonance experiments on 1,2-dimyristoyl-sn-glycero-3-phosphocholine/myristic acid (1:2 molar ratio) mixtures showed that the FA acyl chain was located approximately one methylene group deeper than the sn-2 chain of the PC in the H II phase (46). FAs labeled with spectroscopic probes showed differences between the depth of FAs and the PC fatty acyl chains (44).…”

Pivotal surfaces in inverse hexagonal and cubic phases of phospholipids and glycolipids

“…Table 3 gives the dependence on lipid chain length of the dimensions of the H II -phases that are formed by 1:2 mol/mol mixtures of disaturated phosphatidylcholines with fatty acids of the corresponding chain lengths. The fatty acids are in the protonated (uncharged) state and thus have a strong tendency to form inverted phases on admixture with phosphatidylcholines (Marsh and Seddon, 1982;Rama Krishna and Marsh, 1990;Heimburg et al, 1990). Data are given at a temperature that is 10 • above the transition to the H II -phase for the respective chain lengths.…”