PMID: 18839945;PMCID: PMC2756786;Abstract:
Sphingomyelin is a lipid that is abundant in the nervous systems of mammals, where it is associated with putative microdomains in cellular membranes and undergoes alterations due to aging or neurodegeneration. We investigated the effect of varying the concentration of cholesterol in binary and ternary mixtures with N-palmitoylsphingomyelin (PSM) and 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC) using deuterium nuclear magnetic resonance (2H NMR) spectroscopy in both macroscopically aligned and unoriented multilamellar dispersions. In our experiments, we used PSM and POPC perdeuterated on the N-acyl and sn-1 acyl chains, respectively. By measuring solid-state 2H NMR spectra of the two lipids separately in mixtures with the same compositions as a function of cholesterol mole fraction and temperature, we obtained clear evidence for the coexistence of two liquid-crystalline domains in distinct regions of the phase diagram. According to our analysis of the first moments M1 and the observed 2H NMR spectra, one of the domains appears to be a liquid-ordered phase. We applied a mean-torque potential model as an additional tool to calculate the average hydrocarbon thickness, the area per lipid, and structural parameters such as chain extension and thermal expansion coefficient in order to further define the two coexisting phases. Our data imply that phase separation takes place in raftlike ternary PSM/POPC/cholesterol mixtures over a broad temperature range but vanishes at cholesterol concentrations equal to or greater than a mole fraction of 0.33. Cholesterol interacts preferentially with sphingomyelin only at smaller mole fractions, above which a homogeneous liquid-ordered phase is present. The reasons for these phase separation phenomena seem to be differences in the effects of cholesterol on the configurational order of the palmitoyl chains in PSM-d31 and POPC-d31 and a difference in the affinity of cholesterol for sphingomyelin observed at low temperatures. Hydrophobic matching explains the occurrence of raftlike domains in cellular membranes at intermediate cholesterol concentrations but not saturating amounts of cholesterol. © 2008 American Chemical Society.
Abstract:
In this work we have calculated spin-lattice (T1) relaxation time expressions for the homo- and heteronuclear dipolar relaxation of lipid bilayers, in addition to the heteronuclear Overhauser enhancement (NOE), using correlation functions derived previously. The results can be applied to the analysis of the 1H and 13C T1 times of lipid bilayers and the 13C-1H NOE. Three different models for the segmental fluctuations of the membraneous lipid molecules have been considered: (i) a simple diffusion-type model for the local segmental motions; (ii) a noncollective model in which relatively slow bilayer fluctuations are described by a single correlation time; and (iii) a collective model for the slow motions characterized by a continuous distribution of correlation times. For the diffusion model, the dependence on the bilayer orientation, order parameters 〈P2〉 and 〈P4〉, and the diffusion tensor anisotropy have been included in a general manner. Depending on the degree of segmental ordering and the anisotropy of the diffusion tensor, the maximum 13C-1H NOE can be either greater or less than the value of 2.988 obtained in the absence of an ordering potential. The various relaxation models were fit to 13C T1 data recently obtained for vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at seven different magnetic field strengths, i.e., resonance frequencies, in the liquid crystalline state. A simple diffusion-type model (i) based on analogies to paraflinic liquids provides a very poor fit to the above 13C T1 data as a function of temperature and frequency, even for extreme values of the ordering and diffusion tensor anisotropy,and thus can be rejected at the present time. The 13C results can be fit satisfactorily over the range 15.0-126 MHz by models which include contributions from relatively slow bilayer fluctuations. A noncollective model (ii) with three or four adjustable parameters or a collective model (iii) with two parameters both describe the data to within experimental error. At present, the 13C T1 results suggest that the relaxation of the bilayer hydrocarbon region, in the liquid crystalline state, can best be accounted for by a collective model with a relaxation expression of the type T1-1≅Aτf(2)+BCH2ω1 as concluded from a similar analysis of the 2H T1 data for DPPC multilamellar dispersions. In the above expression, τf(2) is the correlation time for the local motions, SCH(=SCD) is the observed bond segmental order parameter, ω1 is the resonance frequency, and A and B are constants which depend on the nucleus considered. Thus, the observed relaxation rate includes contributions from fast or local-type motions, in addition to cooperative fluctuations of a more long-range character. For the collective model, extrapolation of the 13C T1-1 values obtained for the DPPC vesicles to infinite frequency yields estimates of τf(2) which agree with those calculated from the frequency-independent T1-1 rates of n-hexadecane at the same absolute temperature, suggesting that the segmental microviscosities of the two systems are similar, in agreement with 2H NMR studies. © 1984 American Institute of Physics.
PMID: 10366845;Abstract:
Surface plasmon resonance (SPR) has become a popular method for investigating biomolecular interactions. A new variant of this technique, coupled plasmon-waveguide resonance (CPWR) spectroscopy, allows the characterization of anisotropic biological membranes. Plasmon resonance can therefore be used to study the molecular events involved in a wide variety of membrane processes, including energy conversion and signal transduction.
