Two populations of phospholipids exist in sarcoplasmic reticulum and in recombined membranes containing Ca-ATPase

Phosphorus nuclear magnetic resonance spectra of sarcoplasmic reticulum membranes from rabbit muscle and of recombined membranes containing the calcium-dependent adenosinetriphosphatase (Ca-ATPase) of sarcoplasmic reticulum reveal two distinguishable, overlapping resonances. One resonance resembles a normal phospholipid bilayer resonance, and the other is much broader. The broader component is not seen in protein-free phospholipid vesicles. In recombined membranes of the Ca-ATPase, the intensity found in the broad component was proportional to the concentration of protein in the vesicles. The two-component spectra are interpreted to arise from at least two different domains of phospholipids, one of which is motionally restricted by the Ca-ATPase. Phospholipids exchange between these two domains at a rate less than 10(3) s-1. A model for protein-lipid interactions in membranes containing the Ca-ATPase is proposed in which some of the phospholipid head groups of the membrane interact directly with the protein.     

Maintenance of epithelial surface membrane lipid polarity: A role for differing phospholipid translocation rates

Summary Large differences in lipid composition of apical and basolateral membranes from epithelial cells exist. To determine the responsible mechanism(s), rat renal cortical brush border and basolateral membrane phospholipids were labeled using³²P and either [³H]-glycerol or [2-³H] acetate for incorporation and degradation studies, respectively. Brush border and basolateral membrane fractions were isolated simultaneously from the same cortical homogenate. Different phospholipid classes were degraded at variable rates with phosphatidylcholine having the fastest decay rate. Decay rates for individual phospholipid classes were, however, similar in both brush border and basolateral membrane fractions. In phospholipid incorporation studies again, large variations existed between individual phospholipid classes with phosphatidylcholine and phosphatidylinositol showing the most rapid rates of incorporation. Sphingomyelin and phosphatidylserine showed extremely slow incorporation rates and did not enter into the isotopic decay phase for 48 hr. In contrast to degradation studies, however, the same phospholipid class labeled the two surface membrane domains at highly variable rates. The difference in these rates, with the exception of phosphatidylinositol, were identical to the differences in phospholipid compositions between the two membranes. For example, phosphatidylcholine was incorporated into the basolateral membrane 2.5 × faster than into the brush border membrane and its relative composition was 2.5 × greater in the basolateral membrane. The opposite was true for sphingomyelin. These results indicate incorporation and not degradation rates of individual phospholipids play a major role in regulating the differing phospholipid composition of brush border and basolateral membranes.     

The glycophorin-phospholipid interface in recombined systems. A 31P-nuclear magnetic resonance study.

Glycophorin, the MN glycoprotein from the erythrocyte membrane, was recombined with egg phosphatidylcholine and with the total lipid extract from human erythrocyte membranes in a membranous form. 31P-nuclear magnetic resonance (NMR) spectra of the recombinants resembled spectra obtained from unsonicated phospholipid dispersions and biological membranes. The glycophorin/phospholipid ratio in these recombinants was varied from approximately 50:1 (lipid/protein) to 200:1, and 31P-NMR spectral intensities were obtained. Comparison of these intensities to that expected based on a pure phospholipid standard revealed that there were two phospholipid environments in the recombinants: one immobilized by the protein, and one slightly disordered and nonimmobilized. A relatively constant number of phospholipids were immobilized per glycophorin at all lipid/protein ratios studied.     

Intervesicular phospholipid exchange. An NMR study.

Phospholipid exchange between phosphatidylinositol and phosphatidylcholine vesicles has been studied by NMR spectroscopy with use of hydrophilic paramagnetic lanthanide probes (Pr³⁺ and Eu³⁺ ions). The dependence of the lanthanide induced shifts in the ¹H and ³¹P NMR spectra on the phospholipid composition of the vesicles could be used for its quantitative evaluation. The method has been proved to be applicable for studying phospholipid exchange stimulated by soluble proteins (postmicrosomal supernatant fraction) from rat liver. Furthermore it has been shown that the phospholipid molecules newly introduced by protein-stimulated exchange are predominantly incorporated into the outer monolayer of the vesicular bilayer membrane. This makes it possible to produce liposomes with asymmetric distribution of the phospholipids across the bilayer.     

Perturbations of phospholipid head groups by membrane proteins in biological membranes and recombinants.

P-31 nuclear magnetic resonance (NMR) spin-lattice relaxation times (T1) have been used to probe the behavior of phospholipid head groups in the presence of membrane proteins. Measurements have been made on rabbit muscle sarcoplasmic reticulum and recombinants of the Ca2+ Mg2+ ATPase, rod outer segment disk membranes and recombinants of rhodopsin, and human erythrocyte ghosts and recombinants of human erythrocyte glycophorin. Recombined membranes with lipid/protein ratios greater than or equal to that found in biological membranes showed T1 behavior similar to the biological membranes and pure phosphatidylcholine. However, recombined membranes with a low lipid/protein ratio exhibited a T1 that was dramatically shorter than any of the other systems. Analysis of the relaxation mechanism and the factors contributing to it implicate a phospholipid head group conformation change at high protein content. It is suggested that this is due to trapping of phospholipid between proteins and is not the same phenomenon as motional restriction at the lipid-protein interface at higher lipid contents.     

Membrane order perturbation in the presence of antimicrobial peptides by H solid-state NMR spectroscopy

The ²H solid-state NMR spectra of deuterated fatty acyl chains provide direct access to the order of the hydrophobic membrane interior. From the deuterium order parameter profiles of perdeuterated fatty acyl chains the membrane hydrophobic thickness can be calculated. Here we show data obtained from POPC, POPE and mixed POPE/POPG bilayers, representative of bacterial membranes, in the presence of cholesterol or ergosterol and antimicrobial peptaibols. Whereas sterols have a strong ordering effect also on these membranes, the peptides exhibit neutral or disordering effects. By comparing with data from the literature it becomes obvious that cationic amphipathic peptides that probably reside within the interface of phospholipid membranes tend to strongly disorder the packing of the fatty acyl chains, an effect that has been correlated to antimicrobial and DNA transfection activities. In contrast transmembrane sequences or hydrophobic peptides that probably partition deeply into the membrane tend to have only modest disordering activities. The ²H solid-state NMR approach has also been used to monitor the lateral separation of domains rich in anionic phospholipids in the presence of cationic peptides and has thereby provided important insights into their mechanisms of action.     

A 2H solid-state NMR study of the effect of antimicrobial agents on intact Escherichia coli without mutating

Solid-state nuclear magnetic resonance (NMR) is a useful tool to probe the organization and dynamics of phospholipids in bilayers. The interactions of molecules with membranes are usually studied with model systems; however, the complex composition of biological membranes motivates such investigations on intact cells. We have thus developed a protocol to deuterate membrane phospholipids in Escherichia coli without mutating to facilitate ²H solid-state NMR studies on intact bacteria. By exploiting the natural lipid biosynthesis pathway and using perdeuterated palmitic acid, our results show that 76% deuteration of the phospholipid fatty acid chains was attained. To verify the responsiveness of these membrane-deuterated E. coli, the effect of known antimicrobial agents was studied. ²H solid-state NMR spectra combined to spectral moment analysis support the insertion of the antibiotic polymyxin B lipid tail in the bacterial membrane. The use of membrane-deuterated bacteria was shown to be important in cases where antibiotic action of molecules relies on the interaction with lipopolysaccharides. This is the case of fullerenol nanoparticles which showed a different effect on intact cells when compared to dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol membranes. Our results also suggest that membrane rigidification could play a role in the biocide activity of the detergent cetyltrimethyammonium chloride. Finally, the deuterated E. coli were used to verify the potential antibacterial effect of a marennine-like pigment produced by marine microalgae. We were able to detect a different perturbation of the bacteria membranes by intra- and extracellular forms of the pigment, thus providing valuable information on their action mechanism and suggesting structural differences.     

Reconstitution of an electrically active conformational transition in rhodopsin-containing membranes

An electrically active event that has been observed in native rod outer segment disk membranes can be reconstituted into membrane vesicles containing purified rhodopsin and defined phospholipids. The magnitude of this charge-transfer event, as estimated using spin-labeled derivatives of hydrophobic ions, is a function of the phospholipid composition. In reconstituted membranes containing rhodopsin and egg phosphatidylcholine, the charge transferred during this event is approximately 10% that measured in the native system. The addition of 20 mol% egg phosphatidylethanolamine, phosphatidic acid or brain phosphatidylserine returns the magnitude of the charge transfer to within 60 to 100% of the native activity. The response seen in the reconstituted membrane system is consistent with a previously proposed interfacial charge-transfer mechanism.     

Spin-label ESR studies of lipid-protein interactions in thylakoid membranes

Lipid-protein interactions in thylakoid membranes, and in the subthylakoid membrane fractions containing either photosystem 1 or photosystem 2, have been studied by using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyldiacylglycerol, phosphatidylglycerol, and phosphatidylcholine. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted membrane lipids interacting directly with the integral membrane proteins. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with the membrane proteins and to determine the selectivity between the different lipid classes for the lipid-protein interaction. The fractions of motionally restricted lipid in the thylakoid membrane are 0.36, 0.39, and 0.53, for the spin-labeled monogalactosyldiacylglycerol, phosphatidylcholine, the phosphatidylglycerol, respectively. Spin-labeled monogalactosyldiacylglycerol exhibits very little preferential interaction over phosphatidylchline, which suggests that part of the role of monogalactosyldiacylglycerol in thylakoid membranes is structural, as is the case for phosphatidylcholine in mammalian membranes. Spin-labeled phosphatidylglycerol shows a preferential interaction over the corresponding monogalactosyldiacylglycerol and phosphatidylcholine analogues, in contrast to the common behavior of this lipid in mammalian systems. This pattern of lipid selectivity is preserved in both the photosystem 1 and photosystem 2 enriched subthylakoid membrane fractions.     

31P nuclear magnetic resonance studies of the phospholipid-protein interface in cell membranes.

Both native and recombined membrane systems from the human erythrocyte membrane and the rabbit sarcoplasmic reticulum have been studied with 31P Nuclear Magnetic Resonance (NMR). We compare intensities of the anisotropic 31P resonance exhibited by these membranes with the intensity expected from the known phospholipid content of the membranous sample. In a recombinant with human erythrocyte glycophorin, a component of the phospholipid is "missing" from the 31P NMR resonance, apparently due to a severe broadening of the resonance of that component. Approximately 29 phospholipid molecules were found immobilized per glycophorin molecule in the membrane, regardless of the phospholipid:protein ratio. Cholesterol may inhibit the immobilization of phospholipids by glycophorin. Recombinants with band three from the human erythrocyte membrane contain an immobilized phospholipid component, analogous to the results with glycophorin. 31P NMR data from the native sarcoplasmic reticulum membrane also revealed an immobilized phospholipid component whose magnitude is independent of temperature between 30 degrees C and 45 degrees C. Extensive papain proteolysis of the membrane completely digests the Ca++ Mg++ ATPase and removes the immobilization of phospholipids noted in the intact membrane. Limited trypsin cleavage, however, does not completely remove the immobilized component; salt reduces the immobilized component.     

The interaction of neuropeptide Y with negatively charged and zwitterionic phospholipid membranes

The interaction of the 36 amino acid neuropeptide Y (NPY) with liposomes was studied using the intrinsic tyrosine fluorescence of NPY and an NPY fragment comprising amino acids 18–36. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the experimentally measured binding curves, the standard Gibbs free energy for the peptide transfer from aqueous solution to the lipid membrane was calculated to be around ?30 kJ/mol for membrane mixtures containing physiological amounts of acidic lipids at pH 5. The effective charge of the peptide depends on the pH of the buffer and is about half of its theoretical net charge. The results were confirmed using the fluorescence of the NPY analogue [Trp³²]-NPY. Further, the position of NPY’s α-helix in the membrane was estimated from the intrinsic tyrosine fluorescence of NPY, from quenching experiments with spin-labelled phospholipids using [Trp³²]-NPY, and from ¹H magic-angle spinning NMR relaxation measurements using spin-labelled [Ala³¹, TOAC³²]-NPY. The results suggest that the immersion depth of NPY into the membrane is triggered by the membrane composition. The α-helix of NPY is located in the upper chain region of zwitterionic membranes but its position is shifted to the glycerol region in negatively charged membranes. For membranes composed of phosphatidylcholine and phosphatidylserine, an intermediate position of the α-helix is observed.     

2H and 31P NMR study of pentalysine interaction with headgroup deuterated phosphatidylcholine and phosphatidylserine

The interaction of cationic pentalysine with phospholipid membranes was studied by using phosphorus and deuterium Nuclear Magnetic Resonance (NMR) of headgroup deuterated dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS). In the absence of pentalysine, some of the deuterium and phosphorus spectra of DMPC/DMPS 5:1 (m:m) membranes gave lineshapes similar to those of partially-oriented bilayers with the planes of the bilayers being parallel to the magnetic field. The deuterium NMR data show that the quadrupolar splittings of the deuterated methylenes of the DMPC headgroup are not affected by adsorption of pentalysine on the PC/PS membranes. By contrast, the pentalysine produces significant changes in the quadrupolar splittings of the negatively charged DMPS headgroup. The results are discussed in relation to previous ²H NMR investigations of phospholipid headgroup perturbations arising from bilayer interaction with cationic molecules.Abbreviations NMR nuclear magnetic resonance - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DMPS 1,2-dimyristoyl-sn-glycero-3-phosphoserine - POPC 1-palmitoyl, 2-oleyl-sn-glycero-3-phosphocholine - POPG 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol - PC phosphatidylcholine - PS phosphatidyl serine - PG phosphatidylglycerol - HEPES N-(2-hydroxy-ethyl)piperazine-N-2-ethanesulfonic acid - TRIS tris-(hydroxymethyl)aminoethane - EDTA ethylenediamine-tetra-acetic acid     

Cholesterol and Clioquinol modulation of Aβ(1-42) interaction with phospholipid bilayers and metals

The β-sheet plaques that are the most obvious pathological feature of Alzheimer's disease are composed of amyloid-β peptides and are highly enriched in the metal ions Zn, Fe and Cu. The interaction of the full-length amyloid peptide, Aβ(1-42), with phospholipid lipid bilayers was studied in the presence of the metal-chelating drug, Clioquinol (CQ). The effect of cholesterol and metal ions was also determined using solid-state ³¹P and ²H NMR. CQ modulated the effect of metal ions on the integrity of the bilayer and although CQ perturbed the phospholipid membrane, the bilayer integrity was maintained. Model membranes enriched in cholesterol were studied under conditions of peptide association and incorporation. Solid-state NMR showed that the bilayer integrity was preserved in cholesterol-enriched membranes in comparison to phosphatidylcholine-phosphatidylserine bilayers. Changes in peptide structure, consistent with an increase in β-sheet, were observed using specifically ¹³C-labelled Aβ(1-42) by magic angle spinning NMR. Results using aligned phosphatidylcholine bilayers and completely ¹⁵N-labelled peptide indicated that the peptide aggregated. The results are consistent with oligomeric β-sheet structured peptides only partially penetrating the bilayer and cholesterol reducing the membrane disruption.     

Relation between lipid polymorphism and transbilayer movement of lipid in rat liver microsomes

We have studied the effects of trinitrophenylation on the transbilayer movement of phosphatidylcholine and the macroscopic lipid structure in rat liver microsomal membranes. The transbilayer movement of phosphatidylcholine was investigated using the PC-specific transfer protein. ³¹P-NMR was employed to monitor the phospholipid organization in intact microsomal vesicles. The results indicate that modification of microsomes with trinitrobenzenesulfonic acid enhances the transbilayer movement of phosphatidylcholine at 4°C. Furthermore, phosphatidylethanolamine headgroup trinitrophenylation in microsomes increases the isotropic component in the ³¹P-NMR spectra even at 4°C, possibly representing the appearance of intermediate non-bilayer lipid structures. The observed parallel between these data suggests that phosphatidylethanolamine molecules in the microsomal membrane, probably in combination with a protein component, are able to destabilize the bilayer organization, thereby facilitating the transmembrane movement of phospholipids.     

Proton and carbon-13 nuclear magnetic resonance studies of rhodopsin-phospholipid interactions

Proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra of rhodopsin-phospholipid membrane vesicles and sonicated disk membranes are presented and discussed. The presence of rhodopsin in egg phosphatidylcholine vesicles results in homogeneous broadening of the methylene and methyl resonances. This effect is enhanced with increasing rhodopsin content and decreased by increasing temperature. The proton NMR data indicate the phospholipid molecules exchange rapidly (less than 10(-3) s) between the bulk membrane lipid and the lipid in the immediate proximity of the rhodopsin. These interactions result in a reduction in either or both the frequency and amplitude of the tilting motion of the acyl chains. The 13C NMR spectra identify the acyl chains and the glycerol backbone as the major sites of protein lipid interaction. In the disk membranes the saturated sn-1 acyl chain is significantly more strongly immobilized than the polyunsaturated sn-2 acyl chain. This suggest a membrane model in which the lipid molecules preferentially solvate the protein with the sn-1 chain, which we term an edge-on orientation. The NMR data on rhodopsin-asolectin membrane vesicles demonstrate that the lipid composition is not altered during reconstitution of the membranes from purified rhodopsin and lipids in detergent.     

Spin-label electron spin resonance study of bacteriophage M13 coat protein incorporation into mixed lipid bilayers

The major coat protein of bacteriophage M13 was incorporated in mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (80/20 w/w) vesicles probed with different spin-labeled phospholipids, labeled on the C-14 atom of the sn-2 chain. The specificity for a series of phospholipids was determined from a motionally restricted component seen in the electron spin resonance (ESR) spectra of vesicles with the coat protein incorporated. At 30 degrees C and pH 8, the fraction of motionally restricted phosphatidic acid spin-label is 0.36, 0.52, and 0.72 for lipid/protein ratios of 18, 14, and 9 mol/mol, respectively. The ESR spectra, analyzed by digital subtraction, resulted in a phospholipid preference following the pattern cardiolipin = phosphatidic acid greater than stearic acid = phosphatidylserine = phosphatidylglycerol greater than phosphatidylcholine = phosphatidylethanolamine. The specificities found are related to the composition of the target Escherichia coli cytoplasmic membrane.     

Interaction between glycophorin and phospholipids in recombined systems

Both the MN-glycoprotein from human erythrocytes and the hydrophobic fragment from the protein isolated with trypsin treatment, T(is), have been recombined with egg phosphatidylcholine in bilayers at various phospholipid/protein ratios. In order to investigate the effect of the protein on the phospholipid headgroups, ³¹P nuclear magnetic resonance spectra were obtained with the MN-glycoprotein recombined with egg phosphtidylcholine, which revealed two classes of phospholipid enviroments, one immobilized and one not immobilized. Electron spin resonance (ESR) of fatty acid methyl ester spin labels provided supporting evidence. Computer analysis of the ESR spectra indicate that 4–5 moles of phospholipid are immobilized per mole of protein over a wide range of lipid-to-protein ratios. The immobilization of the phospholipids appears mediated by both the polar headgroups and the hydrocarbon tails of the phospholipid.     

Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy

Rhodopsin is a canonical member of class A of the G protein-coupled receptors (GPCRs) that are implicated in many of the drug interventions in humans and are of great pharmaceutical interest. The molecular mechanism of rhodopsin activation remains unknown as atomistic structural information for the active metarhodopsin II state is currently lacking. Solid-state ²H NMR constitutes a powerful approach to study atomic-level dynamics of membrane proteins. In the present application, we describe how information is obtained about interactions of the retinal cofactor with rhodopsin that change with light activation of the photoreceptor. The retinal methyl groups play an important role in rhodopsin function by directing conformational changes upon transition into the active state. Site-specific ²H labels have been introduced into the methyl groups of retinal and solid-state ²H NMR methods applied to obtain order parameters and correlation times that quantify the mobility of the cofactor in the inactive dark state, as well as the cryotrapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent ²H NMR line shapes for selectively deuterated methyl groups of rhodopsin in aligned membranes enables determination of the average ligand conformation within the binding pocket. The relaxation data suggest that the β-ionone ring is not expelled from its hydrophobic pocket in the transition from the pre-activated metarhodopsin I to the active metarhodopsin II state. Rather, the major structural changes of the retinal cofactor occur already at the metarhodopsin I state in the activation process. The metarhodopsin I to metarhodopsin II transition involves mainly conformational changes of the protein within the membrane lipid bilayer rather than the ligand. The dynamics of the retinylidene methyl groups upon isomerization are explained by an activation mechanism involving cooperative rearrangements of extracellular loop E2 together with transmembrane helices H5 and H6. These activating movements are triggered by steric clashes of the isomerized all-trans retinal with the β4 strand of the E2 loop and the side chains of Glu¹²² and Trp²⁶⁵ within the binding pocket. The solid-state ²H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism.     

Principles of multiparametric optimization for phospholipidomics by 31P NMR spectroscopy

Phospholipids have long been known to be the principal constituents of the bilayer matrix of cell membranes. While the main function of cell membranes is to provide physical separation between intracellular and extracellular compartments, further biological and biochemical functions for phospholipids have been identified more recently, notably in cell signaling, cell recognition and cell–cell interaction, but also in cell growth, electrical insulation of neurons and many other processes. Therefore, accurate and efficient determination of tissue phospholipid composition is essential for our understanding of biological tissue function. ³¹P NMR spectroscopy is a quantitative and fast method for analyzing phospholipid extracts from biological samples without prior separation. However, the number of phospholipid classes and subclasses that can be quantified separately and reliably in ³¹P NMR spectra of tissue extracts is critically dependent on a variety of experimental conditions. Until recently, little attention has been paid to the optimization of phospholipid ³¹P NMR spectra. This review surveys the basic physicochemical properties that determine the quality of phospholipid spectra, and describes an optimization strategy based on this assessment. Notably, the following experimental parameters need to be controlled for systematic optimization: (1) extract concentration, (2) concentration of chelating agent, (3) pH value of the aqueous component of the solvent system, and (4) temperature of the NMR measurement. We conclude that a multiparametric optimization approach is crucial to obtaining highly predictable and reproducible ³¹P NMR spectra of phospholipids.     

Effects of potassium on lipid-protein interactions in light sarcoplasmic reticulum

This study shows the effect of K+ on phospholipid-protein interactions in light sarcoplasmic reticulum (LSR) as measured by 31P NMR. In the presence of 110 mM K+, a substantial effect of the membrane protein on the behavior of the phospholipids was detected. Subtracting the spectrum of the LSR lipid extract from the spectrum of the intact LSR membrane produced a difference spectrum of much greater breadth than the normal phospholipid bilayer powder pattern. This powder pattern is indicative of a phospholipid domain considerably more motionally restricted than the phospholipids in a normal phospholipid bilayer. The apparent axially symmetric powder pattern is consistent with axial diffusion. In a reconstituted membrane containing the calcium pump protein at a lipid/protein ratio much less than in the light sarcoplasmic reticulum, the broad component was more prominent. The relative resonance intensity of the broad component appeared to be proportional to the lipid/protein ratio of the membrane. In 10 mM K+, no broad powder pattern is observed in the corresponding difference spectrum. Thus, in the absence of potassium, the membrane protein has much less influence on the phospholipid of the membrane, as measured by 31P NMR. In addition to the effects of K+ on the membrane structure of the sarcoplasmic reticulum, K+ modulated the function of the calcium pump. The rate of calcium-dependent ATP hydrolysis increased in light sarcoplasmic reticulum when [K+] increased from 10 to 110 mM. The rate of calcium transport was also stimulated by an increase in K+.