Cholesterol dynamics in membranes.

Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of [13C4] labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of [13C4] labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased by the presence of PE. Both steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S perpendicular was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for tau perpendicular. In both the ROS disk membranes and the erythrocyte membranes, tau perpendicular was one-third to one-fifth of tau perpendicular in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior.     

Multiple binding sites for local anesthetics in membranes: characterization of the sites and their equilibria by deuterium NMR of specifically deuterated procaine and tetracaine

Anesthetics bound to model membranes were observed directly by means of deuterium nuclear magnetic resonance (NMR). The specifically deuterated local anesthetics procaine and tetracaine were synthesized, and their partition coefficients (water:phosphatidylcholine) and pKa values determined. The interaction of these anesthetics with lamellar dispersions of egg phosphatidylcholine was studied by 2H nuclear magnetic resonance and by electron spin resonance (ESR) of a spin-labelled phospholipid at low (5.5) and high (9.5) pH. The ESR experiments suggest that tetracaine intercalates in the membrane and that it equilibrates between water and the phospholipid bilayers of the multilamellar system. The NMR results are consistent with a model where the anesthetic is (1) free in water, (2) weakly bound, and (3) strongly bound to the membrane. A fast exchange exists between the two first sites, but exchange is slow with the third site. Binding of type 3 is observed only at high pH for procaine, whereas it is found both at low and high pH for tetracaine. Calculations of the partition coefficients for the charged and uncharged forms of tetracaine indicate that both sites, 2 and 3, are occupied by the charged form at low pH and by the uncharged form at high pH. The partition coefficient for the weakly bound species was estimated from an analysis of the dependence of line width on the lipid to water ratio. The NMR data suggest that the binding sites for the strongly bound charged and uncharged species are different, the former probably being closer to the membrane-water interface. Estimates of molecular order parameters for the strongly bound species indicate that it is located with its long molecular axis approximately parallel to the director for ordering of the fatty acyl chains. A small increase in lipid ordering by tetracaine is observed at low pH, as evidenced by 2H NMR of the deuterated N-methyl groups of phosphatidylcholine; the reverse occurs at high pH.     

Solution structure and interaction of cupiennin 1a, a spider venom peptide, with phospholipid bilayers

The solution structure of cupiennin 1a, a 35 residue, basic antibacterial peptide isolated from the venom of the spider Cupiennius salei, has been determined by nuclear magnetic resonance (NMR) spectroscopy. The peptide was found to adopt a helix-hinge-helix structure in a membrane mimicking solvent. The hinge may play a role in allowing the amphipathic N-terminal helix and polar C-terminal helix to orient independently upon membrane binding, in order to achieve maximal antibacterial efficacy. Solid-state 31P and 2H NMR was used to further study the effects of cupiennin 1a on the dynamic properties of lipid membranes, using zwitterionic chain deuterated dimyristoylphosphatidylcholine (d54-DMPC) and anionic dimyristoylphosphatidylglycerol (DMPG) multilamellar vesicles. In d54-DMPC alone, cupiennin 1a caused a decrease in the 31P chemical shift anisotropy, indicating some interaction with the lipid head groups, and a decrease in order over the entire acyl chain. In contrast, for the mixed (d54-DMPC/DMPG) lipid system cupiennin 1a appeared to induce lateral separation of the two lipids as evidenced by the 31P spectra, in which the peptide preferentially interacted with DMPG. Little effect was observed on the deuterated acyl chain order parameters in the d54-DMPC/DMPG model membranes. Furthermore, 31P NMR relaxation measurements confirmed a differential effect on the lipid motions depending upon the membrane composition. Therefore, subtle differences are likely in the mechanism by which cupiennin 1a causes membrane lysis in either prokaryotic or eukaryotic cells, and may explain the specific spectrum of activity.     

Location and dynamics of alpha-tocopherol in model phospholipid membranes with different charges.

Studies were made on the position and dynamics of the OH-group of alpha-tocopherol in phospholipid membranes. There was no difference in the spin-lattice (T1) relaxation times at the 5a-position of alpha-tocopherol labeled with 13C- or C19F3-determined from the nuclear magnetic resonance (NMR) spectra of liposomes positively charged with stearylamine (SA) and negatively charged with dicetylphosphate (DCP). The zeta-potentials of egg yolk phosphatidylcholine (EYPC) liposomes with and without SA or DCP were not affected by incorporation of 20 mol% alpha-tocopherol, though incorporation of 10 mol% ascorbyl-palmitate decreased the zeta-potentials of EYPC and EYPC-SA liposomes. The P==O stretching band (1235 cm-1) of the phosphate group and C==O stretching band (1734 cm-1) of the acyl ester linkage in dimyristoylphosphatidylcholine (DMPC) liposomes, measured by Fourier transform-infrared (FT-IR) spectroscopy, were not changed by incorporation of alpha-tocopherol. These results suggest that no specific interaction occurred between the OH-group of alpha-tocopherol and the polar interfacial region of the bilayer. The dynamic quenching effects of n-(N-oxy-4,4'-dimethyloxazolidine-2-yl)stearic acids (n-NSs) on the intrinsic fluorescence of alpha-tocopherol were in the order 5-NS > 7-NS = 12-NS > 16-NS. Acrylamide, a water-soluble fluorescence quencher with a very low capacity to penetrate through phospholipid bilayers, had very low quenching efficiency. These results indicate that the bulk of the chromanol moiety of alpha-tocopherol is located in a position close to that occupied by the nitroxide group of 5-NS in the membranes and is poorly exposed at the membrane surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deuterium nuclear magnetic resonance spectroscopic study of the fluorescent probe diphenylhexatriene in model membrane systems

We have investigated the deuterium (2H) nuclear magnetic resonance (NMR) spectra of two 2H-labeled fluorescence probes (trans,trans,trans-1,6-diphenylhexa-1,3,5-trienes, DPHs) incorporated into model lipid bilayer membrane systems at various temperatures. The membranes consisted of multilamellar bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) containing varying concentrations of cholesterol. The conventional one-order parameter approach often used in the analysis of the NMR data of lipid membranes does not explain the observed temperature variations of the spectral features. Consistent with the molecular symmetry, the results have thus been analyzed in terms of an ordering matrix with more than one independent element. The molecular order parameter (SNMR), the order along the long molecular axis, in the pure lipid system varies from 0.49 to 0.26 as the temperature is increased from 25 to 57 degrees C. These values are somewhat larger than the order parameters obtained from fluorescence depolarization (SFLU) on sonicated DMPC vesicles. Such discrepancies probably arise from the looser packing of the sonicated vesicles. Addition of cholesterol to the model membranes causes the order parameter of the probe molecules to increase. At 35 degrees C, SNMR increases from 0.38 (with no cholesterol) to 0.92 (in the presence of 50 mol % cholesterol). These values are about 10% larger than those obtained from fluorescence depolarization studies on sonicated vesicles. The SNMR for DPH are somewhat larger than those obtained in earlier NMR studies of 2H-labeled cholesterol. However, they compare well with those obtained for 2H-labeled DMPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron spin resonance study of the interaction of alpha-tocopherol with phospholipid model membranes.

The effect of up to 20 mol% incorporation of alpha-tocopherol on acyl chain order and dynamics in liquid crystalline phosphatidylcholine (PC) membranes was studied as a function of acyl chain unsaturation by electron spin resonance (ESR) of 5-, 7-, 12- and 16-doxyl spin labelled stearic acids intercalated into the membrane. Order parameters S in the upper portion of the chain (positions 5 and 7) and correlation times tau C in the lower portion (positions 12 and 16) determined from the ESR spectra indicate that in general alpha-tocopherol restricts acyl chain motion within the membrane. The magnitude of the increases in order appears to be dependent upon phospholipid molecular area, being the greatest (up to 15%) in saturated dimyristoylphosphatidylcholine (14:0-14:0 PC) which possesses a relatively small area per molecule as opposed to much smaller increases (less than 3%) in unsaturated PC membranes of larger molecular area. This behavior is interpreted as incompatible with the hypothesis of Lucy and coworkers (A.T. Diplock and J.A. Lucy (1973) FEBS Lett. 29, 205-210), who proposed that membranes are structurally stabilized by interactions between the phytyl side chain of alpha-tocopherol and the polyunsaturated chains of phospholipids.     

Laser-assisted fluorescence microscopy for measuring cell membrane dynamics.

Membranes of living cells are characterized by laser-assisted fluorescence microscopy, in particular a combination of microspectrofluorometry, total internal reflection fluorescence microscopy (TIRFM), fluorescence lifetime imaging (FLIM) and Forster resonance energy transfer (FRET) spectroscopy. The generalized polarization (GP, characterizing a spectral shift which depends on the phase of membrane lipids) as well as the effective fluorescence lifetime (tau(eff)) of the membrane marker laurdan were revealed to be appropriate parameters for membrane stiffness and fluidity. GP decreased with temperature, but increased during cell growth and was always higher for the plasma membrane than for intracellular membranes. Microdomains of different fluorescence lifetimes tau(eff) were observed at temperatures above 30 degree C and disappeared during cell aging. Non-radiative energy transfer was used to detect laurdan selectively in close proximity to a molecular acceptor (DiI) and may present a possibility for measuring membrane dynamics in specific microenvironments.     

Extraction of lipid membrane incorporated vitamin E by sucrose polyesters

2H and 31P solid state NMR have been used to probe, at the molecular level, the interaction between structurally different sucrose polyesters and a phospholipid membrane into which alpha-tocopherol and specifically deuterated alpha-[5,7-(2)H(6)] tocopherol has been incorporated. Our results show that at high concentration (>or=10 mol%) sucrose octapalmitate (SOP) and sucrose hexapalmitate (SHxP) deplete bilayer-associated alpha-tocopherol in dipalmitoyl phosphatidalcholine (DPPC) multilamellar dispersions and preferentially sequester the alpha-tocopherol into a fluid sucrose polyesters (SPE) phase located proximal to the membrane surface. It is demonstrated that the ability of SPEs to function as a 'lipophilic sink' depends strongly on sucrose polyester concentration and degree of esterification.     

Elucidation of intermediate (mobile) and slow (solidlike) protein motions in bovine lens homogenates by carbon-13 NMR spectroscopy

The motional dynamics of lens cytoplasmic proteins present in calf lens homogenates were investigated by two 13C nuclear magnetic resonance (NMR) techniques sensitive to molecular motion to further define the organizational differences between the cortex and nucleus. For the study of intermediate (mobile) protein rotational reorientation motion time scales [rotational correlation time (tau 0) range of 1-500 ns], we employed 13C off-resonance rotating frame spin-lattice relaxation, whereas for the study of slow (solidlike) motions (tau 0 greater than or equal to 10 microseconds) we used the solid-state NMR techniques of dipolar decoupling and cross-polarization. The frequency dependence of the peptide bond carbonyl off-resonance rotating frame spectral intensity ratio of the lens proteins present in native calf nuclear homogenate (42% protein) at 35 degrees C indicates the presence of a polydisperse mobile protein fraction with a tau 0,eff (mean) value of 57 ns. This mean value is consistent with the average value calculated from the known water-soluble nuclear lens protein polydispersity assuming a cytoplasmic viscosity 3 times that of pure water. Lowering the temperature to 1 degree C, a temperature which produces the cold cataract, results in an overall decrease in tau 0,eff to 43 ns, suggesting a selective removal of beta H-, LM-, and possibly gamma s-crystallins from the mobile lens protein population. The presence of solidlike or motionally restricted protein species was established by dipolar decoupling and cross-polarization. The fraction of motionally restricted protein in the nuclear region varied from 0.35 to 0.45 in the temperature range of 35-1 degree C. For native cortical homogenate (25% protein), the off-resonances rotating frame spectral intensity ratio frequency-dependent curves for the protein carbonyl resonance yielded tau 0,eff values of 34 and 80 ns at 35 and 1 degree C, respectively. Both values were reconciled with the known lens cortex soluble protein polydispersity using an assumed cytoplasmic viscosity 1.5 times that of pure water at the same temperature. Comparison of proton dipolar-decoupled and nondecoupled 13C NMR spectra of native cortical homogenate at 20 degrees C indicates the absence of significant contributions from slowly tumbling, motionally restricted species. This interpretation was confirmed by the failure to detect significant lens protein 13C-1H cross-polarization at this temperature. However, at 1 degree C, the fraction of solidlike protein was 0.15. Concentrated cortical homogenates at 20 degrees C (42% protein), by contrast, gave cross-polarization spectra with maximum absolute signal intensities 50-70% of native nuclear homogenates, but with similar magnetization parameters...     

Dynamic properties of gramicidin A in phospholipid membranes

The flexibility of the tryptophan side chains of gramicidin A and the rotational diffusion of the peptide in methanolic solution and in three membrane systems were studied with deuterium nuclear magnetic resonance (NMR). Gramicidin A was selectively deuterated at the aromatic ring systems of its four tryptophan side chains. In methanolic solution, the tryptophan residues remained immobile and served as a probe for the overall rotation of the peptide. The experimentally determined rotational correlation time of tau c = 0.6 X 10(-9) s was consistent with the formation of gramicidin A dimers. For gramicidin A incorporated into bilayer membranes, quite different results were obtained depending on the chemical and physical nature of the lipids employed. When mixed with 1-palmitoyl-sn-glycero-3-phosphocholine (LPPC) at a stoichiometric lipid:peptide ratio of 4:1, gramicidin A induced the formation of stable bilayer membranes in which the lipids were highly fluid. In contrast, the gramicidin A molecules of this membrane remained completely static over a large temperature interval, suggesting strong protein-protein interactions. The peptide molecules appeared to form a rigid two-dimensional lattice in which the interstitial spaces were filled with fluidlike lipids. When gramicidin A was incorporated into bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) above the lipid phase transition, the deuterium NMR spectra were motionally narrowed, indicating large-amplitude rotational fluctuations. From the measurement of the quadrupole echo relaxation time, a rotational correlation time of 2 X 10(-7) s was estimated, leading to a membrane viscosity of 1-2 P if the rotational unit was assumed to be a gramicidin A dimer. (ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of phloretin with membranes: on the mode of action of phloretin at the water-lipid interface

 The interaction of phloretin with single lipid bilayers on a spherical support and with multilamellar vesicles was studied by differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR). The results indicated that phloretin interacts with the lipid layer and changes its structural parameters. In DSC experiments, phloretin in its neutral form strongly decreased the lipid phase transition temperature and slightly reduced the cooperativity of the phase transition within the lipid layer. In NMR measurements, phloretin led to an increase of the transverse relaxation time constant but had no effect on the spin-lattice relaxation time constant. The overall dipole moment of phloretin was experimentally determined and was found to be roughly 40% lower than has been published previously. This result suggested that the size of the dipole moment of phloretin does not provide such a high contribution to the effect of phloretin on the dipole potential of monolayers and bilayers as has been published previously. To understand the discrepancy between phloretin adsorption and dipole potential change, we performed computational conformational analysis of phloretin in the gas phase. The results showed that a wide distribution of the dipole moments of phloretin conformers exists, which mainly depends on the orientation of the OH moieties. The adsorption of phloretin as determined from its binding to solid supported bilayers differed from the one determined from dipole potential measurements on black lipid membranes. The difference between the phloretin dissociation constants of both types of experiments suggested a change of its dipole moment normal to the membrane surface in a concentration-dependent manner, which was in agreement with the results of the computational conformational analysis. Received: 21 June 1999 / Revised version: 7 January 2000 / Accepted: 31 March 2000     

Deuterium NMR study of the interaction of alpha-tocopherol with a phospholipid model membrane

The effects of 5, 10, and 20 mol % incorporation of alpha-tocopherol (vitamin E) on 50 wt % aqueous multilamellar dispersions of sn-2-substituted [2H31]palmitoylphosphatidylcholine (PC-d31), a saturated, deuterated phospholipid prepared from egg lysophosphatidylcholine, have been studied by deuterium nuclear magnetic resonance (2H NMR) and differential scanning calorimetry (DSC). Moment analysis of the 2H NMR spectra as a function of temperature and DSC heating curves demonstrate that the main gel to liquid-crystalline phase transition is progressively broadened and its onset temperature lowered by increasing concentrations of alpha-tocopherol. Below the transition temperature (40 degrees C) for PC-d31 bilayers, the 2H NMR spectra indicate that acyl chain motion is increased by addition of alpha-tocopherol and that this effect extends to lower temperatures with higher alpha-tocopherol content. Above the transition, average carbon-deuterium bond order parameters calculated from the first spectral moment establish that alpha-tocopherol increases acyl chain ordering within the PC-d31 bilayer by as much as 17% at 20 mol % incorporation. Profiles of order parameter vs. chain position, constructed from 2H NMR spectra following application of the depaking technique, show that despite higher order the general form of the profile is not significantly altered by alpha-tocopherol.     

NMR studies of melanins: characterization of a soluble melanin free acid from Sepia ink.

This paper deals with the nuclear magnetic resonance characterization of a soluble derivative (melanin free acid) of Sepia melanin obtained by a peroxidative treatment of the parent (insoluble) species. High resolution 13C and 15N solid state NMR spectroscopies allow the assessment of the chemical changes occurring in the macromolecule upon solubilization. 1H and 13C NMR solution spectra are discussed in light of the results obtained from the solid state spectra. Furthermore, the coordination properties of melanin have been investigated through 27Al NMR spectroscopy and proton relaxation enhancement studies of the paramagnetic gadolinium complex of melanin free acid. Through these experiments it has been possible to evaluate the molecular reorientational time tau R (and from it an estimated molecular weight close to 20 KDa) and the strength of the metal-macromolecule interaction.     

Aluminum binding to phosphatidylcholine lipid bilayer membranes: 27Al and 31P NMR spectroscopic studies

27Al and 31P nuclear magnetic resonance (NMR) spectroscopies were used to investigate aluminum interactions at pH 3.4 with model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). A solution state 27Al NMR difference assay was developed to quantify aluminum binding to POPC multilamellar vesicles (MLVs). Corresponding one-dimensional (1D) fast magic angle spinning (MAS) 31P NMR spectra showed that aluminum induced the appearance of two new isotropic resonances for POPC shifted to -6.4 ppm and -9.6 ppm upfield relative to, and in slow exchange with, the control resonance at -0.6 ppm. Correlation of the (27)Al and (31)P NMR binding data revealed a 1:2 aluminum:phospholipid stoichiometry in the aluminum-bound complex at -9.6 ppm and a 1:1 aluminum:phospholipid stoichiometry in that at -6.4 ppm. Slow MAS 31P NMR spectra demonstrated shifts in the anisotropic chemical shift tensor components of the aluminum-bound POPC consistent with a close coordination of aluminum with phosphorus. A model of the aluminum-bis-phospholipid complex is proposed on the basis of these findings.     

Dynamics and orientation of glycolipid headgroups by 2H-NMR: gentiobiose

Deuterium nuclear magnetic resonance has been used to investigate the dynamics and determine the orientation of the headgroup of the glycolipid 1,2-di-O-tetradecyl-3-O-(6-O-beta-D-glucopyranosyl-beta-D-glucopyranosyl )-sn- glycerol (beta-DTDGL), in aqueous multilamellar dispersions. In addition, its anomeric analog, having an alpha glucose-glycerol linkage, was prepared and examined. The lipids were labelled with deuterium at specific positions in the disaccharide moiety. Analysis of the deuterium quadrupolar splittings for the first glucose ring (glycerol-linked) gave segmental order parameters of 0.43 and 0.35 for the beta and alpha isomers, respectively. Both isomers had similar orientations of the sugar ring relative to the bilayer surface, as determined for lipid in the liquid-crystalline phase. 2H-NMR results for the lipid labelled at C-6' are consistent with a single conformation about the C-5'-C-6' bond of the first glucose residue, with a dihedral angle (O-5'-C-5'-C-6'-O-6') of -17 degrees. The results obtained for the second sugar ring suggest that two conformers may be present, which are in slow exchange on the 2H-NMR timescale. Measurements of longitudinal relaxation times, T1z, gave similar values for both sugar moieties in the headgroup, suggesting that the disaccharide does not exhibit the flexibility expected about the 1----6 linkage. Since T1z for 2H in these compounds decreases with increasing temperature and increases with magnetic field strength, the motion(s) dominating relaxation is in the long-correlation-time regime [omega 0 tau c)2 greater than 1). Thus, the gentiobiosyl headgroup undergoes the slowest motion of the glycolipid headgroups studied to date.     

NMR structural studies of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes

The beta-barrels found in the outer membranes of prokaryotic and eukaryotic organisms constitute an important functional class of proteins. Here we present solid-state NMR spectra of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes. We show that OmpX is folded in both glass-supported oriented lipid bilayers and in lipid bicelles that can be magnetically oriented with the membrane plane parallel or perpendicular to the direction of the magnetic field. The presence of resolved peaks in these spectra demonstrates that OmpX undergoes rotational diffusion around an axis perpendicular to the membrane surface. A tightly hydrogen-bonded domain of OmpX resists exchange with D2O for days and is assigned to the transmembrane beta-barrel, while peaks at isotropic resonance frequencies that disappear rapidly in D2O are assigned to the extracellular and periplasmic loops. The two-dimensional 1H/15N separated local field spectra of OmpX have several resolved peaks, and agree well with the spectra calculated from the crystal structure of OmpX rotated with the barrel axis nearly parallel (5 degrees tilt) to the direction of the magnetic field. The data indicate that it will be possible to obtain site-specific resonance assignments and to determine the structure, tilt, and rotation of OmpX in membranes using the solid-state NMR methods that are currently being applied to alpha-helical membrane proteins.     

Nuclear magnetic resonance studies of amino acids and proteins. Deuterium nuclear magnetic resonance relaxation of deuteriomethyl-labeled amino acids in crystals and in Halobacterium halobium and Escherichia coli cell membranes

We have obtained deuterium (2H) Fourier transform nuclear magnetic resonance (NMR) spectra of zwitterionic L-[beta-2H3]alanine, DL-[gamma-2H6]valine, DL-[beta, gamma-2H4]threonine, L-[delta-2H3]leucine, and L-[alpha, beta, gamma, gamma', delta-2H10]isoleucine in the crystalline solid state and have determined the deuteriomethyl group spin-lattice relaxation rates as a function of temperature. The results yield the Arrhenius activation energies (delta E) for methyl rotation, and through use of a suitable mathematical model, rotational correlation times, tau c. For alanine, valine, threonine, leucine, and isoleucine at 37 degrees C, tau c and delta E values are 780, 100, 40, 38, and 18 ps and 22, 14.0, 17.6, 15.5, and 8.6 kJ, respectively. For L-[beta-2H3]alanine in the zwitterionic lattice, a spin-lattice relaxation time (T1) minimum of 2.1 +/- 0.3 ms is observed (at 0 degree C), in excellent agreement with the 1.92-ms prediction of the mathematical model. Similar tau c and delta E measurements are reported for bacteriorhodopsin in the purple membrane of Halobacterium halobium R1 and for Escherichia coli cell membranes. Overall, our results demonstrate a great similarity between the dynamics in amino acid crystals and in membrane proteins. However, threonine exhibits a nonlinear Arrhenius behavior in bacteriorhodopsin, and in the valine-, leucine-, and isoleucine-labeled membrane samples at higher temperatures (approximately greater than 37 degrees C), there is evidence of an additional slow side-chain motion. The lipid phase state in E. coli does not appear to influence, on the average, the dynamics of the valine side chains. These results indicate that the sensitivity of the deuterium NMR technique is now adequate to study in moderate detail the dynamics of most types of amino acids in a membrane protein and that adequate sensitivity, in some instances, should be available for the study of individual amino acids in suitably labeled membrane proteins.     

Vitamin E: inhibition of retinol-induced hemolysis and membrane-stabilizing behavior.

For the elucidation of the mechanism of membrane stabilization by vitamin E, the effects of alpha-tocopherol and its model compounds on either retinol-induced hemolysis of rabbit erythrocytes or the permeability and fluidity of liposomal membranes have been studied. Retinol-induced rabbit erythrocyte hemolysis has been found not to be caused by the oxidative disruption of erythrocyte membrane lipids initiated by retinol oxidation, but rather to arise from physical damage of the membrane micelle induced by penetration of retinol molecules. In suppressing hemolysis, alpha-tocopherol was more effective than other naturally occurring tocopherols. alpha-Tocopheryl acetate, nicotinate, and 6-deoxy-alpha-tocopherol were more effective than alpha-tocopherol itself. The inhibitory effects of alpha-tocopherol model compounds having side chains with at least two isoprene units or a long straight chain instead of the isoprenoid side chain were similar to those of alpha-tocopherol. These data suggest that for protection of membranes against retinol-induced damage, the hydroxyl group of alpha-tocopherol is not critical, but rather the chroman ring, three methyl groups on the aromatic ring, and the long side chain are necessary. To verify the mechanism of the inhibitory effect on hemolysis, not only the effect of vitamin E and its model compounds on the membrane permeability and fluidity, but also the mobility of alpha-tocopherol molecule in membranes has been investigated using bilayer liposomes as the model membranes. Addition of alpha-tocopherol to membranes produced a greater decrease in the permeability and fluidity of rat liver phosphatidylcholine liposomes compared with egg yolk phosphatidylcholine liposomes. In dipalmitoylphosphatidylcholine liposomes, however, alpha-tocopherol was less effective, that is, the more unsaturated the lipids, the more they interact with alpha-tocopherol. 2,2,5,7,8-Pentamethyl-6-chromanol with no isoprenoid side chain and phytol without the chromanol moiety had no effect. The measurement of 13C NMR relaxation times revealed that the mobility of methyl groups on the aromatic ring of alpha-tocopherol in membranes is significantly restricted. In contrast, the methyl groups at positions 4'a and 8'a on the isoprenoid side chain have high degrees of motional freedom in the lipid core of membranes. Furthermore, it was found that alpha-tocopherol in membranes interacts with chromate ions added as potassium chromate outside the membranes, resulting in an increase in membrane fluidity. These results are compatible with those of the inhibitory effect on retinol-induced erythrocyte hemolysis. On the basis of the results obtained here, a possible mechanism for membrane stabilization by vitamin E is proposed.     

NMR, calorimetric, spin-label, and optical studies on a trifluoromethyl-substituted styryl molecular probe in dimyristoylphosphatidylcholine vesicles and multilamellar suspensions: a model for location of optical probes

NMR, calorimetric, and optical spectroscopic studies have been performed on a trifluoromethyl-substituted styryl molecular probe bound to vesicles and multilamellar suspensions formed from dimyristoylphosphatidylcholine (DMPC). In the fluorine NMR spectrum at 35 degrees C there are two partially resolved resonances, but these collapse to an apparently single resonance at temperatures above 60 degrees C. However, a line-shape analysis is not consistent with exchange between two sites on an NMR time scale, and the two resonances are assumed to be due to probe sites in the inner and outer leaflets of the vesicles. Two fluorescence lifetimes, each associated with one of these sites, characterize the decay curves for the molecular probe bound to DMPC vesicles. The shift reagent Eu(FOD)3 and several nitroxide spin labels covalently bound to lipophilic structures strongly attenuate the lower frequency component of the fluorine NMR spectrum and also shift the other resonance to higher frequencies. The effect of two spin labels on the probe fluorine T2 relaxation time has been used to estimate the distance between the spin label unpaired electron and the trifluoromethyl group. The location of the spin label site in the membrane was determined from the effect of the unpaired electron on the lipid 13C linewidths. A model for the location of the probe in the bilayer was developed from the above information and refined using molecular mechanics calculations on a probe-DMPC lipid complex. The long axis of the probe parallels the bilayer normal; the styryl-group portion of the optical chromophore is located slightly below the glycerol backbone, and the remainder of the chromophore extends well into the hydrophobic region of the bilayer. Therefore, the optical properties of the probe should not be significantly influenced by alterations of the membrane surface charge density. Parameters derived from DSC studies in the gel-to-lipid crystal phase transition of DMPC are extremely sensitive to the probe. Even at 0.0001 mol fraction of probe, the transition is substantially broadened, and the delta H for the transition has increased, just as one predicts for the formation of a tight complex described above.