The influence of cholesterol on the phase behavior of glycerophospholipids and sphingomyelins was investigated by spin-label electron spin resonance (ESR) spectroscopy. 4-(4,4-Dimethyl-3-oxy-2-tridecyl-2-oxazolidinyl)butanoic acid (5-SASL) and 1-stearoyl-2-[4-(4,4-dimethyl-3-oxy-2-tridecyl-2-oxazolidinyl)butanoy l]-sn- glycero-3-phosphocholine (5-PCSL) spin-labels were employed for this purpose. The outer hyperfine splitting constants, Amax, measured from the spin-label ESR spectra as a function of temperature were taken as empirical indicators of cholesterol-induced changes in the acyl chain motions in the fluid state. The Amax values of 5-PCSL exhibit a triphasic dependence on the concentration of cholesterol for phosphatidylcholines and bovine brain sphingomyelin. We interpret this dependence as reflecting the existence of liquid-disordered, ld, liquid-ordered, lo, and coexistence regions, ld + lo. The phase boundary between the ld and the two-phase region and the boundary between the lo and the two-phase region in the phosphatidylcholine-cholesterol systems coalesce at temperatures 25-33 degrees C above the main-chain melting transition temperature of the cholesterol-free phosphatidylcholine bilayers. In the case of bovine brain sphingomyelin, the ld-lo phase coalescence occurs about 47 degrees C above the melting temperature of the pure sphingomyelin. The selectivity of interaction of cholesterol with glycerophospholipids of varying headgroup charge was studied by comparing the cholesterol-induced changes in the Amax values of derivatives of phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine spin-labeled at the fifth position of the sn-2 chain.(ABSTRACT TRUNCATED AT 250 WORDS)

The fluid-phase behavior of binary mixtures of cholesterol with phosphatidyichoilnes is investigated using magnetic resonance methods. Phospholipid biradicals provide the electron spin resonance spectroscopic resolution of two Immiscible fluid phases in the dlpalmitoylphosphatidylcholinecholesterol system. Isotropic chemical shifts of the phospholipid carbonyl carbons in binary mixtr with cholesterol measured using solid-state high-resolution nuclear magnetic resonance methods furnish evidence for a putative hydrogen bond between the 3P-hydroxyl of cholesterol and the sn-2 carbonyl of the phospholipid. The location in the bilayer of cholesterol in the two fluid phases is determined by measuring spin label-enhanced spin-lattice relaxation rates of the 13C nuclei of both the phospholipid and cholesterol molecules. These results suggest, in a time-averaged sense, that in the cholesterol-poor fluid phase the cholesterol molecule essentially spans the bilayer, whereas in the cholesterol-rich fluid phase the molecule is present in both monolayers of the bilayer. Fig. 1B. The phase diagrams for mixtures of cholesterol with other phospholipids, differing in both the acyl chain and the headgroup composition, have the same overall shape and consist of three one-phase regions and two two-phase regions as shown in Fig. 1B (8). The phases are denoted solid-ordered (so), liquid-ordered (4o), and liquiddisordered (4d). In the two-phase regions there is coexistence of the so and 4o phases or of the two fluid phases, 4 and eo.The occurrence of fluid-phase immiscibility has been suggested by a triphasic dependence on cholesterol concentration of the hyperfine interactions of phospholipid spin labels incorporated into cholesterol-containing phospholipid bilay- ers (6, 8).In this communication, we present magnetic resonance evidence further substantiating cholesterol-induced fluidphase immiscibility and suggesting the existence of hydrogen bonding between cholesterol and phospholipid molecules.We conclude by proposing a bilayer model specifying the time-averaged location of cholesterol in the Ed and 4o phases.MATERIALS AND METHODS Materials. Unlabeled phospholipids were purchased from Avanti Polar Lipids (Birmingham, AL). Phospholipid monoradicals and 'IC-enriched phospholipids were synthesized as described (8). The biradical was synthesized by acylation of sn-glycero-3-phosphocholine with the corresponding fatty acid spin labels (11).ESR Spectroscopy. The ESR spectra were recorded on an X-band Varian E-line spectrometer. The sample preparation protocols and the details of instrumentation are as described earlier (8). The monoradical spin label concentrations used were 1 mol % unless otherwise specified. The biradical concentrations were 0.5 mol %. NMR Spectroscopy. 13C NMR spectra were recorded at 90 MHz on a Nicolet NT-360B spectrometer equipped with a variable-temperature magic-angle spinning probe from Doty Scientific (Columbia, SC). The samples contained in zirconium rotors were spun at speeds between 1 and 3 kHz, whi...

The effect of cholesterol on the acyl chain order of three glycerophosphocholines with 14, 16, and 18 carbons per acyl chain, namely, di(14:0)PC, di(16:0)PC, and di(18:0)PC, above the gel to liquid-crystalline phase transition temperature was investigated by using 2H nuclear magnetic resonance spectroscopy. Average acyl chain lengths were calculated from the segmental order parameters (Smol) for the sn-1 and the sn-2 chains in the absence of cholesterol and at 3:1, 2:1, and 1:1 mole ratios of phospholipid-cholesterol. The three binary mixtures of cholesterol with phosphatidylcholines are in the liquid-ordered (lo) phase. For all the three phosphatidylcholine-cholesterol systems, the distance from the carbonyl groups to the terminal methyl groups is shorter than the length of the cholesterol molecule. A molecular model for the lo phase consistent with these observations has in a statistical sense a part of each cholesterol molecule in one monolayer extending into the other monolayer. This results in a packing arrangement akin to that in interdigitated systems. On the basis of the effect of cholesterol on phospholipid acyl chain orientational order, it is suggested that the liquid-disordered (ld) phase at low cholesterol concentrations corresponds to a packing mode in which the cholesterol molecule spans the entire transbilayer hydrophobic region. A molecular mechanism is proposed in which increasing the concentration of cholesterol has the effect of stretching the acyl chains of phospholipids by increasing the population of trans conformers up to a stage where the hydrophobic length is considerably longer than the cholesterol molecule. Beyond this concentration, the partially interdigitated phase forms.(ABSTRACT TRUNCATED AT 250 WORDS)

The average sizes of fluid and gel domains in the two-component, two-phase system formed from mixtures of dimyristoyl phosphatidylcholine and distearoyl phosphatidylcholine were determined from an analysis of the electron spin resonance spectral lineshapes of a dimyristoyl phosphatidylcholine-nitroxide spin label as a function of spin label concentration. The ratio, R, of the intensities measured at two magnetic field strengths was found to be diagnostic of a statistical distribution of spin labels in disconnected domains. R is defined as V'/2Vpp, where Vpp is the maximum intensity and V' is the intensity at a position in the wings of a first derivative electron spin resonance line that is a constant multiple of the peak-to-peak linewidth. The intensity ratio for Gaussian or Voigt lineshapes is less than or equal to the value for a Lorentzian lineshape. The intensity ratio was found to be greater than the value for a Lorentzian line when spectra from disconnected domains containing a statistical distribution of spin labels undergoing spin-spin interactions were summed. The intensity ratio, R, calculated by spectral simulations as a function of the average number of labels per domain, N, was found to increase to a maximum with increasing N and then to decrease. The dependence on spin label concentration of the experimentally measured intensity ratios paralleled this predicted behavior. A method is presented to calculate the average number of lipids per fluid or gel domain based on a knowledge of R, and of the distribution of the spin label between the fluid and gel phases determined from the phase diagram. The results demonstrate that the number of lipids per domain increases linearly from a fixed number of nucleation sites, as the fraction of the phase that is disconnected increases. At any given mole fraction of the particular phase, the gel domains are bigger than the fluid domains because they have a lower nucleation density. The results also suggest that the disconnected domains are, in most cases, nonrandomly distributed in the plane of the bilayer.

Electron spin resonance (ESR) spectroscopy and chemical binding assays were used to study the interaction of bovine spinal cord myelin basic protein (MBP) with dimyristoylphosphatidylglycerol (DMPG) membranes. Increasing binding of MBP to DMPG bilayers resulted in an increasing motional restriction of P G spin-labeled at the C-5 atom position in the acyl chain, up to a maximum degree of association of 1 MBP molecule per 36 lipid molecules. ESR spectra of PG spin-labels labeled at other positions in the sn-2 chain showed a similar motional restriction, while still preserving the chain flexibility gradient characteristic of fluid lipid bilayers. In addition, labels at the C-12 and C-14 atom positions gave twocomponent spectra, suggesting a partial hydrophobic penetration of the M B P into the bilayer. Spectral subtractions were used to quantitate the membrane penetration in terms of the stoichiometry of the lipid-protein complexes. Approximately 50% of the spin-labeled lipid chains were directly affected at saturation protein binding. The salt and pH dependence of the ESR spectra and of the protein binding demonstrated that electrostatic interaction of the basic residues of the MBP with the PG headgroups is necessary for an effective association of the MBP with phospholipid bilayers. Binding of the protein, and concomitant perturbation of the lipid chain mobility, was reduced as the ionic strength increased, until at salt concentrations above 1 M NaCl the protein was no longer bound. The binding and ESR spectral perturbation also decreased as the protein charge was reduced by pH titration to above the p l of the protein at approximately p H 10.