Nuclear magnetic resonance studies of amphiphile hydration: Effects of cholesterol on phosphatidyl choline hydration

Water which remains unfrozen at ?25 °C in the presence of phosphatidyl choline (PC) gives rise to a proton magnetic resonance signal which can be used to measure the hydration of single-walled vesicles and multilamellar liposomes of PC. The proton magnetic resonance signal of the unfrozen water in these systems is strongly dependent upon the nature of the molecular domain in which the water is situated. For example, at cholesterol to PC molar ratios below 35 mol%, the vesicle hydration signal consists of a relatively narrow symmetric peak (line width, ~150 Hz). At higher molar ratios, however, rather broad asymmetric signals appear (line widths, ~300–1000 Hz) which indicate that when significant quantities of cholesterol are packed in the bilayer there must be regions in which there is a preferred direction for motion of the unfrozen water. It is possible to solubilize significant quantities of cholesterol by sonicating it in concentrated solutions of sodium dodecyl sulfate. Addition of cholesterol to PC vesicles via these sodium dodecyl sulfate-cholesterol complexes caused hydration changes in the PC, which, at high cholesterol to PC molar ratios, paralleled the effects of cholesterol on PC hydration in homogeneous vesicles in which the cholesterol and PC were simply cosonicated.     

The effects of solutes on the freezing properties of and hydration forces in lipid lamellar phases.

Quantitative deuterium nuclear magnetic resonance is used to study the freezing behavior of the water in phosphatidylcholine lamellar phases, and the effect upon it of dimethylsulfoxide (DMSO), sorbitol, sucrose, and trehalose. When sufficient solute is present, an isotropic phase of concentrated aqueous solution may coexist with the lamellar phase at freezing temperatures. We determine the composition of both unfrozen phases as a function of temperature by using the intensity of the calibrated free induction decay signal (FID). The presence of DMSO or sorbitol increases the hydration of the lamellar phase at all freezing temperatures studied, and the size of the increase in hydration is comparable to that expected from their purely osmotic effect. Sucrose and trehalose increase the hydration of the lamellar phase, but, at concentrations of several molal, the increase is less than that which their purely osmotic effect would be expected to produce. A possible explanation is that very high volume fractions of sucrose and trehalose disrupt the water structure and thus reduce the repulsive hydration interaction between membranes. Because of their osmotic effect, all of the solutes studied reduced the intramembrane mechanical stresses produced in lamellar phases by freezing. Sucrose and trehalose at high concentrations produce a greater reduction than do the other solutes.     

Biological Ion Exchanger Resins: II. QUERP Water and Ion Exchange Selectivity

Biological selectivity is shown to vary with medium osmotic strength and temperature. Selectivity reversals occur at 4°C and at an external osmolality of 0.800 indicating that intracellular hydration and endosolvent (intracellular water) structure are important determinants in selectivity. Magnetic resonance measurements of line width by steady-state nuclear magnetic resonance (NMR) indicate a difference in the intracellular water signal of 16 Hz between the K form and Na form of Escherichia coli, providing additional evidence that changes in the ionic composition of cells are accompanied by changes in endosolvent structure. The changes were found to be consistent with the thermodynamic and magnetic resonance properties of aqueous electrolyte solutions. Calculation of the dependence of ion-pairing forces on medium dielectric reinforces the role of endosolvent structure in determining ion exchange selectivity.     

Water binding and phase structures for different Acholeplasma laidlawii membrane lipids studied by deuteron nuclear magnetic resonance and x-ray diffraction

Water binding capability and phase structures for different lipid species extracted from Acholeplasma laidlawii A membranes have been studied using deuteron nuclear magnetic resonance and low-angle X-ray diffraction.The dominating membrane lipids are monoglucosyldiglyceride and diglucosyldiglyceride and each of them takes up limited amounts of water (bound plus trapped), i.e., up to 13% (w/w), whereas the phospholipids and phosphoglycolipids have larger hydration capacities.Addition of magnesium and calcium ions, but not sodium ions, to the diglucosyldiglyceride increases the hydration capability. This increase is accompanied by the formation of a metastable liquid crystalline phase and a hysteresis effect for the transition temperature.Large differences in water deuteron quadrupole splitting were observed between mono- and diglucosyldiglyceride. Both ²H nuclear magnetic resonance and low-angle X-ray diffraction studies on lipids containing biosynthetically incorporated ω-d₃-palmitic acid clearly indicate the existence of a reverse hexagonal phase structure for the monoglucosyldiglyceride and lamellar structures for the diglucosyldiglyceride and the other membrane lipids.The low hydration capability of the large diglucosyldiglyceride polar head is discussed in terms of polar head configuration.Both mono- and diglucosyldiglyceride have several physical properties similar to those of phosphatidylethanolamine.     

Nuclear magnetic resonance relaxation time and self-diffusion coefficient measurements of water in frog ovarian eggs (Rana pipiens)

Self-diffusion coefficient measurements of water in untreated ovarian eggs of Rana pipiens using nuclear magnetic resonance indicate that cytoplasmic water has reduced translational mobility compared with pure water. Using a simple two-state model, we find that ～67% is “relatively immobile.” Consideration of the nuclear magnetic resonance spin-lattice and spin-spin relaxation times indicates that the decreased mobility can largely be ascribed to hydration. Our value for the self-diffusion coefficient (6.8 × 10^?6 cm²/sec) is lower than those reported by other investigators using isotopic water exchange techniques on frog eggs chemically treated to remove the membrane. However, the results reported here are in agreement with unpublished data on untreated frog eggs implying that chemical treatment has modified the cytoplasm in some manner.     

Direct Evidence from Nuclear Magnetic Resonance Studies for Bound Sodium in Frog Skeletal Muscle

The recent work of Cope on ²³Na magnetic resonance studies of frog muscle has been repeated with the view of investigating certain objections which can be raised concerning the original studies. The present work leads to the conclusion that Cope's results concerning bound sodium are essentially correct in that a large fraction of the ²³Na present does not contribute normally to a detectable nuclear magnetic resonance (NMR) signal. This “missing” signal can be detected at high radio-frequency intensity however, and a signal-saturation study distinctly reveals its presence.     

The major carotenoid pigment of a psychrotrophic Micrococcus roseus strain: purification, structure, and interaction with synthetic membranes.

The major carotenoid pigment of a psychrotrophic Micrococcus roseus strain was purified to homogeneity from methanol extracts of dried cells by reverse-phase liquid chromatography and was designated P-3. On the basis of the UV-visible, infrared, mass, and 1H nuclear magnetic resonance spectra of P-3, it was identified as bisdehydro-beta-carotene-2-carboxylic acid. The pigment interacted with synthetic membranes of phosphatidylcholine and dimyristoyl phosphatidylcholine and stabilized the membranes. These results also indicate that P-3 is different from canthaxanthin, the major carotenoid pigment from a mesophilic M. roseus strain.     

Interaction of the membrane-bound D-lactate dehydrogenase of Escherichia coli with phospholipid vesicles and reconstitution of activity using a spin-labeled fatty acid as an electron acceptor: a magnetic resonance and biochemical study.

The interaction with phospholipid vesicles of the membrane-bound respiratory enzyme D-lactate dehydrogenase of Escherichia coli has been studied. Proteolytic digestion studies show that D-lactate dehydrogenase is protected from trypsin digestion to a larger extent when it interacts with phosphatidylglycerol than with phosphatidylcholine vesicles. Wild-type D-lactate dehydrogenase and mutants in which an additional tryptophan is substituted in selected areas by site-specific oligonucleotide-directed mutagenesis have been labeled with 5-fluorotryptophan. 19F nuclear magnetic resonance studies of the interaction of these labeled enzymes with small unilamellar phospholipid vesicles show that Trp 243, 340, and 361 are exposed to the lipid phase, while Trp 384, 407, and 567 are accessible to the external aqueous phase. Reconstitution of enzymatic activity in phospholipid vesicles has been studied by adding enzyme and substrate to phospholipid vesicles containing a spin-labeled fatty acid as an electron acceptor. The reduction of the doxyl group of the spin-labeled fatty acid has been monitored indirectly by nuclear magnetic resonance and directly by electron paramagnetic resonance. These results indicate that an artificial electron-transfer system can be created by mixing D-lactate dehydrogenase and D-lactate together with phospholipid vesicles containing spin-labeled fatty acids.     

The self-diffusion of water in Artemia cysts

The self-diffusion coefficient of water molecules has been measured by nuclear magnetic resonance in cysts of Artemia over a wide range of hydration. Compared to the value for bulk water, the diffusion coefficient is reduced by a factor of 7 at the highest hydration and by approximately 100 at the lowest hydration. The results are used to evaluate various models that have been proposed to account for the reduction of water self-diffusion coefficients in complex systems.     

Conformational changes of the phosphatidylcholine headgroup due to membrane dehydration. A 2H-NMR study

The influence of hydration on the orientation of the phosphocholine dipole in bilayer membranes was studied with nuclear magnetic resonance. The phosphocholine headgroup of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was deuterated at the two methylene segments. Phosphorus-31 and deuterium nuclear magnetic resonance measurements were made as a function of hydration in the range of 10-70 wt.% H2O revealing a distinct change in the alignment of the phosphocholine headgroup. With decreasing hydration the N+ end of the phosphocholine head group dipole moves closer to the hydrocarbon layer. The conformational change induced by the loss of water molecules at the membrane surface is qualitatively similar to that observed upon addition of polyhydroxyl compounds.     

Interstitial sodium nuclear magnetic resonance relaxation times in perfused hearts.

Separation of intracellular and extracellular sodium nuclear magnetic resonance (NMR) signals would enable nondestructive monitoring of intracellular sodium. It has been proposed that differences between the relaxation times of intracellular and extracellular sodium be used either directly or indirectly to separate the signal from each compartment. However, whereas intracellular sodium relaxation times have been characterized for some systems, these times were unknown for interstitial sodium. In this study, the interstitial sodium NMR relaxation times have been measured in perfused frog and rat hearts under control conditions. This was achieved by eliminating the NMR signal from the extracardiac (perfusate) sodium, and then quantifying the remaining cardiac signal. The intracellular signal was measured to be 8% (frog) or 22% (rat) of the cardiac signal and its subtraction was found to have a negligible effect on the cardiac relaxation times. Therefore this cardiac signal is considered to provide a good estimate of interstitial relaxation behavior. For perfused frog (rat) hearts under control conditions, this signal was found to have a T1 of 31.6 +/- 3.0 ms (27.3 +/- 1.6 ms) and a biexponential T2 of 1.9 +/- 1.0 ms (2.1 +/- 0.3 ms) and 25.2 +/- 1.3 ms (26.3 +/- 3.2 ms). Due to the methods used to separate cardiac signal from perfusate signal, it is possible that this characterized only a part of the signal from the interstitium. The short T2 component attributable to the interstitial signal indicates that separation of the NMR signals from each compartment on the basis of relaxation times alone may be difficult.     

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.     

Freezing stresses and hydration of isolated cell walls

The hydration of the cell walls of the giant alga Chara australis was measured as a function of temperature using quantitative deuterium nuclear magnetic resonance (NMR) of samples hydrated with D2O. At temperatures 23-5K below freezing, the hydration ratio (the ratio of mass of unfrozen water in microscopic phases in the cell wall to the dry mass) increases slowly with increasing temperature from about 0.2 to 0.4. It then rises rapidly with temperature in the few Kelvin below the freezing temperature. The linewidth of the NMR signal varies approximately linearly with the reciprocal of the hydration ratio, and with the freezing point depression or water potential. These empirical relations may be useful in estimating cell wall water contents in heterogeneous samples.     

Physicochemical studies of the protein-lipid interactions in melittin-containing micelles

Complexes of melittin with detergents and phospholipids have been characterized by fluorescence, circular dichroism, ultracentrifugation, quasi-elastic light scattering and 1H nuclear magnetic resonance (NMR) experiments. By ultracentrifugation and quasi-elastic light-scattering measurements it is shown that melittin forms stoichiometrically well-defined complexes with dodecylphosphocholine micelles consisting of one melittin molecule and approximately forty detergent molecules. Evidence from fluorescence, circular dichroism and 1H nuclear magnetic resonance experiments indicates that the conformation of melittin bound to micelles of various detergents or of diheptanoyl phosphatidylcholine is largely independent of the type of lipid and furthermore appears to be quite closely related to the conformation of melittin bound to phosphatidylcholine bilayers. 1H NMR is used to investigate the conformation of micelle-bound melittin in more detail and to compare certain aspects of the melittin conformation in the micelles with the spatial structures of monomeric and self-aggregated tetrameric melittin in aqueous solution. The experience gained with this system demonstrates that high resolution NMR of complexes of membrane proteins with micelles provides a viable method for conformational studies of membrane proteins.     

The hydration of phospholipids and phospholipid-cholesterol complexes

The hydration characteristics of phosphatidylcholines and the effect of cholesterol on these were studied with differential thermal analysis and water vapour adsorption experiments. Also the water adsorption of egg phosphatidylethanolamine and the effect of cholesterol on this was studied and compared with corresponding qualities of phosphatidylcholine.The differential thermal analysis study showed that the monohydrates of egg, dipalmitoyl, and dioleoyl phosphatidylcholine tightly bind ～9 molecules of water per phosphatidylcholine molecule. Cholesterol is proved to somewhat increase the water binding of the phospholipids. Cholesterol is also shown to decrease the heat change of the chain melting transition of dioleoyl phosphatidylcholine, but not to abolish it completely.The water adsorption experiments indicate that the hydration of phosphatidylcholines takes place in two steps; a strong initial water binding and a second phase of weak binding. The adsorption isotherm of egg phosphatidylethanolamine is strikingly different from that of egg phosphatidylcholine. Cholesterol is shown, also by this method, to increase the hydration of phospholipids especially that of dipalmitoylphosphatidylcholine.The results in this study are in good agreement with those presented by many other authors. Starting with the accumulated information of the hydration characteristics of phosphatidylcholines the organization of the bound water around the polar group is discussed and the most probable model is evaluated.     

Assignments of the paramagnetically shifted heme methyl nuclear magnetic resonance peaks of cyanometmyoglobin by selective deuteration

Three of the four paramagnetically shifted heme methyl nuclear magnetic resonance peaks of cyanometmyoglobin could be assigned by comparing the proton nuclear magnetic resonance spectra of myoglobins reconstituted from selectively deuterated hemes. These spectra indicate that the fourth methyl nuclear magnetic resonance peak has to be looked for outside the region ?9 to ?43 parts per million.     

The effect of hydration on the dynamics of trimethoprim bound to dihydrofolate reductase. A deuterium NMR study.

To determine the effect of hydration on the dynamics of a protein complex, we used deuterium nuclear magnetic resonance (NMR) techniques to examine a trimethoprim (TMP)/E. coli dihydrofolate reductase (DHFR) complex in its lyophilized, partially hydrated, polycrystalline, and ammonium sulfate-precipitated states. The results indicate that TMP is rigid in the lyophilized powder state. The dynamic behavior could be restored by partial rehydration. At 30 wt% hydration the deuterium spectrum of the partially hydrated sample was indistinguishable from that of the polycrystalline and ammonium sulfate-precipitated samples, suggesting that the structure of the protein/TMP complex is similar in the three physical states. Furthermore, we found that the para- and meta-methoxyl groups have very different dynamical behavior.     

Orientations of the tryptophan 9 and 11 side chains of the gramicidin channel based on deuterium nuclear magnetic resonance spectroscopy.

Deuterium nuclear magnetic resonance spectroscopy was used to investigate the orientations of the indole rings of Trp9 and Trp11 in specific indole-d5-labeled samples of gramicidin A incorporated into dimyristoyl phosphatidylcholine bilayers in the beta 6.3 channel conformation. The magnitudes and signs of the deuterium quadrupolar splittings were fit to the rings and assigned to specific ring bonds, using a full rotation search of the chi 1 and chi 2 angles of each Trp and a least-squares method. Unique assignments were obtained. The data and assignments are in close agreement with four sets of (chi 1, chi 2) angles for each Trp in which the indole N-H is oriented toward the membrane's exterior surface. (Four additional sets of (chi 1, chi 2) angles with the N-H's pointing toward the membrane interior are inconsistent with previous observations.) One of the sets of (chi 1, chi 2) angles for each Trp is consistent with the corresponding Trp orientation found by Arsen'ev et al. (1986. Biol. Membr. 3:1077-1104) for gramicidin in sodium dodecyl sulfate micelles. Together, the 1H and 2H nuclear magnetic resonance methods suggest that the Trp9 and Trp11 side chain orientations could be very similar in dimyristoyl phosphatidylcholine membranes and in sodium dodecyl sulfate micelles. The data for Trp11 could be fit using a static quadrupolar coupling constant of 180 kHz under the assumption that the ring is essentially immobile. By contrast, Trp9 could be fit only under the assumption that the quadrupolar splittings for ring 9 are reduced by approximately 14% due to motional averaging. Such a difference in motional averaging between rings 11 and 9 is also consistent with the 15N data of Hu et al. (1993. Biochemistry. 32:7035-7047).     

The interaction of 1-anilino-8-naphthalenesulfonate with lipids: A nuclear magnetic resonance study

The proton magnetic resonance (PMR) and phosphorus magnetic resonance (PhMR) spectra of egg phosphatidylcholine in the presence of 1-anilino-8-naphthalenesulfonate (ANS) have been studied. At low ratios of ANS to phospholipid, the spectra indicate that ANS molecules are in the lipid interface region where they interact with the head-group protons. ANS also penetrates into the hydrocarbon region to some extent. As the ANS/phospholipid ratio approaches one, a significant splitting of the head-group signal occurs. This splitting is associated with head-group signals from inner and outer molecules of the phospholipid vesicles. As the ANS/phospholipid ratio is further increased, a gel phase often occurs. The spectra for this gel phase suggest a highly mobile head-group. Further ANS addition results in a PMR spectrum suggestive of ANS—phospholipid micelle formation. The results for a phospholipid—cholesterol complex and for the total lipid extract from a cell membrane show that the ANS effect is more complicated in these cases.     

The mitochondrial precursor protein apocytochrome c strongly influences the order of the headgroup and acyl chains of phosphatidylserine dispersions. A 2H and 31P NMR study

Deuterium and phosphorus nuclear magnetic resonance techniques were used to study the interaction of the mitochondrial precursor protein apocytochrome c with headgroup-deuterated (dioleoylphosphatidyl-L-[2-2H1]serine) and acyl chain deuterated (1,2-[11,11-2H2]dioleoylphosphatidylserine) dispersions. Binding of the protein to dioleoylphosphatidylserine liposomes results in phosphorus nuclear magnetic resonance spectra typical of phospholipids undergoing fast axial rotation in extended liquid-crystalline bilayers with a reduced residual chemical shift anisotropy and an increased line width. 2H NMR spectra on headgroup-deuterated dioleoylphosphatidylserine dispersions showed a decrease in quadrupolar splitting and a broadening of the signal on interaction with apocytochrome c. Addition of increasing amounts of apocytochrome c to the acyl chain deuterated dioleoylphosphatidylserine dispersions results in the gradual appearance of a second component in the spectra with a 44% reduced quadrupolar splitting. Such large reduction of the quadrupolar splitting has never been observed for any protein studied yet. The lipid structures corresponding to these two components could be separated by sucrose gradient centrifugation, demonstrating the existence of two macroscopic phases. In mixtures of phosphatidylserine and phosphatidylcholine similar effects are observed. The induction of a new spectral component with a well-defined reduced quadrupolar splitting seems to be confined to the N-terminus since addition of a small hydrophilic amino-terminal peptide (residues 1-38) also induces a second component with a strongly reduced quadrupolar splitting. A chemically synthesized peptide corresponding to amino acid residues 2-17 of the presequence of the mitochondrial protein cytochrome oxidase subunit IV also has a large perturbing effect on the order of the acyl chains, indicating that the observed effects may be a property shared by many mitochondrial precursor proteins. In contrast, binding of the mature protein, cytochrome c, to acyl chain deuterated phosphatidylserine dispersions has no effect on the deuterium and phosphorus nuclear magnetic resonance spectra, thereby demonstrating precursor-specific perturbation of the phospholipid order. The inability of holocytochrome c to perturb the phospholipid order is due to folding of this protein, since unfolding of cytochrome c by heat or urea treatment results in similar effects on dioleoylphosphatidylserine bilayers, as observed for the unfolded precursor. Implications of these data for the import of apocytochrome c into mitochondria will be discussed.