Phosphatidylcholine Headgroup Research Articles

Do Cation-PI Interactions Occur in Lipid Bilayers Between Phosphatidylcholine Headgroups and Interfacially Localized Tryptophans?

Lipids can modulate membrane protein activity in many different ways. To understand the basic priciples governing this complex lattice of interactions between lipids and membrane proteins, simple model peptides have been created. Among those are the families of WALP and KALP peptides, which consist of a poly-(leucine-alanine) stretch flanked by tryptophans and lysines, respectively.Here we focused on studying how electrostatic interactions between these flanking residues and the lipid polar head groups can affect the behavior of the peptides and the lipids. We used 2H NMR on Ala-d4 labeled peptides to map changes in the orientation of the peptides in phosphatidylcholine bilayers in the absence and presence of the anionic lipid phosphatidylglycerol and we used 14N NMR to monitor changes in structure and dynamics of the phosphocholine head groups. Surprisingly, we found that WALP peptides, which are uncharged, are more sensitive to incorporation of negatively charged lipids, than their positively charged equivalents, the KALP peptides. As a possible explanation we raised the hypothesis that WALP peptides are sensitive to the concentration of phosphatidylcholine lipids in the membrane, due to favorable cation-pi interactions between the tryptophans and the choline moieties of the lipids. This hypothesis was supported by results from high resolution solid state NMR experiments, designed to monitor Trp-choline interactions. The existence of such a favorable interaction may shed new light on understanding the behavior of membrane proteins, in particular since in such proteins Trp frequently occurs as flanking residue at the lipid/water interface.

Study of the Cholesterol Umbrella Effect in DPPC and DOPC Bilayers by Molecular Dynamics Simulation

The instability of cholesterol clusters in dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) lipid bilayers was investigated via atomistic Molecular Dynamics (MD) simulation. Cholesterol clusters in phosphatidylcholine (PC) bilayers are found to be very unstable and to readily disperse into cholesterol monomers. The instability may result from the difficulty for the system to prevent water exposure to cholesterol's aggregated hydrophobic bodies in a cluster. The system responds to artificially arranged cholesterol clusters in several interesting manners: (i) Cholesterol clusters quickly form a “frustum” shape to reduce water penetration through cholesterol headgroups; (ii) Many clusters bury themselves deeper into the bilayer interior, causing local bilayer deformation; (iii) Cholesterol fluctuates rapidly, both laterally and vertically to the bilayer plane, in order to escape from clusters. These fluctuations result in the disintegration of clusters, and in one incidence, a highly unusual flip-flop event of a cholesterol across the DOPC bilayer occurs. Our results show that cholesterols have a strong tendency to avoid forming clusters in lipid bilayers and that the fundamental cholesterol-cholesterol interaction is unfavorable. Furthermore, the radial distribution functions of cholesterol hydroxyl oxygen to various headgroup atoms of PC reveal that the PC headgroups surrounding cholesterol have a clear tendency to reorient and extend toward cholesterol. The range of this “Umbrella Effect” can reach up to 2-3 nm, larger than previously reported.

Instability of Cholesterol Clusters in Lipid Bilayers and The Cholesterol’s Umbrella Effect

The instability of cholesterol clusters and the Umbrella effect of cholesterol in dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) lipid bilayers were investigated via atomistic molecular dynamics (MD) simulation. Cholesterol clusters in phosphatidylcholine (PC) bilayers are found to be very unstable and to readily disperse into cholesterol monomers. This instability results from the difficulty of the bilayer system in preventing water exposure to cholesterol's bulky hydrophobic bodies in a cluster. The system responds to artificially arranged cholesterol clusters in several interesting manners: (i) cholesterol clusters quickly form a "frustum" shape to reduce water penetration between cholesterol headgroups; (ii) many clusters bury themselves deeper into the bilayer interior, causing bilayer deformation; and (iii) cholesterol fluctuates rapidly, both laterally and vertically, to escape clusters. These fluctuations result in the disintegration of clusters and, in one incidence, a highly unusual flip-flop event of cholesterol across the DOPC bilayer. Our results show that cholesterols have a strong tendency to avoid forming clusters in lipid bilayers and that the fundamental cholesterol-cholesterol interaction is unfavorable. Furthermore, the radial distribution functions of cholesterol hydroxyl oxygen to various headgroup atoms of PC reveal that the PC headgroups surrounding cholesterol have a clear tendency to reorient and to extend toward cholesterol. This reorientation has a layered structure that extends 2-3 nm from the cholesterol molecule. This study demonstrates that the Umbrella hypothesis is valid in both saturated and unsaturated lipid bilayers.

Membrane Heterogeneities and Fusogenicity in Phosphatidylcholine−Phosphatidic Acid Rigid Vesicles as a Function of pH and Lipid Chain Mismatch

The role of pH-dependent lipid heterogeneities on the fusogenicity of membranes was evaluated on model lipid bilayers in the form of unilamellar vesicles composed of lipid pairs at a fixed equimolar ratio of phosphatidylcholine (PC) and phosphatidic acid (PA) headgroups. The pH and the hydrophobic composition (lipid acyl tails) of membranes were systematically altered, and their effect on vesicle aggregation, membrane fusogenicity, content release, and content mixing was evaluated. Lowering pH increases the extent of protonated PA headgroups forming phase-separated PA-rich heterogeneities and giving rise to molecular packing defects originating at the phase boundaries. Phase boundaries within the membrane's hydrophobic portion are affected by the lipid acyl-tail dynamics (fluidity) and the matching or nonmatching lengths of the acyl tails of lipid pairs comprising the membrane. The aggregates' size increases with lowering pH and is independent of the membrane's hydrophobic composition. Contrary to aggregation, the initial rates of lipid mixing are proportional to pH and also depend on membrane's hydrophobic composition. The apparent lipid-mixing rate constants are greater for membranes containing lipid pairs with mismatched acyl-tail lengths, followed by pairs with matching acyl tails in the gel state and by pairs with matching tails where one lipid is close to its transition temperature. Addition of cholesterol conserves the independence of vesicle aggregation from the membrane's hydrophobic composition. However, it decreases the aggregation rates and inverts the tendency for fusion, among the three types of hydrophobic compositions, suggesting a role of cholesterol's preferential partition in different regions of membrane's heterogeneities. We propose a phenomenological model where the membrane phase boundaries containing molecular packing defects act as "sticking points" on the vesicle exterior via which vesicles aggregate upon contact followed by defect merging via intervesicle lipid exchange and mixing. Such heterogeneous bilayers in the form of drug encapsulating liposomes may potentially improve the therapeutic efficacy by fusing with endosomal membranes, thus increasing drug bioavailability.

Sphingomyelins as semi‐permanent capillary coatings for protein separations in CE and off‐line analysis with MALDI‐MS

Phosphatidylcholine (PC) is one of the major phospholipids that make up the biological cell membrane. It was previously reported to form capillary inner wall coatings for CE. The zwitterionic head group of PC produced a neutral net-charged bilayer, which was found effective in preventing wall adsorption of both cationic and anionic proteins [J. M. Cunliffe et al. Anal. Chem. 2002, 74, 776-783]. Another major membrane phospholipid that possesses a zwitterionic head group is sphingomyelin (SM). In this work, the novel characterization of SM on its effectiveness in capillary coating formation for CE separations of proteins and peptides was presented. Similar properties were observed between PC and SM, including their effects on the EOF, peak efficiencies, and migration time reproducibilities. SM appeared to be more readily soluble in aqueous solutions, and it was found equally effective as PC in facilitating protein separation. The main difference observed was their performances in delivering a peptide mixture for off-line analysis with MALDI-MS. Superior sample recovery was evident from the capillary coated with SM compared with that with PC. The number of peptides identified from a 1 ng/microL myglobin tryptic digest sample increased from 5 to 16 (42 and 69%, respectively in protein sequence coverage).

Synthesis and Biophysical Characterization of Chlorambucil Anticancer Ether Lipid Prodrugs

The synthesis and biophysical characterization of four prodrug ether phospholipid conjugates are described. The lipids are prepared from the anticancer drug chlorambucil and have C16 and C18 ether chains with phosphatidylcholine or phosphatidylglycerol headgroups. All four prodrugs have the ability to form unilamellar liposomes (86-125 nm) and are hydrolyzed by phospholipase A(2), resulting in chlorambucil release. Liposomal formulations of prodrug lipids displayed cytotoxicity toward HT-29, MT-3, and ES-2 cancer cell lines in the presence of phospholipase A(2), with IC(50) values in the 8-36 microM range.

The Influence of Membrane Lipids in Staphylococcus aureus Gamma-Hemolysins Pore Formation

The natural target of Staphylococcus aureus bicomponent gamma-hemolysins are leucocyte cell membranes. Because a proteinaceous receptor has not been found yet, we checked for the importance of the different membrane lipid compositions by measuring the activity of the toxin on several pure lipid model membranes. We investigated the effect of membrane thickness, fluidity, and presence of nonbilayer lipids and found that the toxin pore-forming ability increased in the presence of phosphocholines with short saturated acyl chains or with unsaturated chains even though not short. An increase of activity was also evident in the presence of cone-shaped lipids like phosphatidylethanolamine or diphytanoylphosphatidylcholine, whereas cylindrical lipids, like sphingomyelin, did not favor the activity. All these results suggest that gamma-hemolysins could bind to the bilayer only if the phosphatidylcholine (PC) head is freely accessible. This condition is satisfied by the concurrent presence of cholesterol and certain lipids, as highlighted by the so-called umbrella model (J. Huang and G. W. Feigenson, Biophys J 76:2142-2157, 1999). According to this model, cholesterol could help to a better exposition of PC head groups only if acyl chains are short or unsaturated. In fact, phosphatidylcholines with more than 13 carbon atoms acyl chains can cover cholesterol molecules; in this way, PC head groups pack tightly, rendering them inaccessible to the toxin, which thus shows a reduced pore-forming ability.

Interaction of imatinib with liposomes: Voltammetric and AFM characterization

The interaction of imatinib with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 2-oleoyl-1-stearoyl-sn-glycero-3-phosphocholine (OSPC) liposomes and the adsorption of DPPC and OSPC were studied using atomic force microscopy (AFM) at highly oriented pyrolytic graphite (HOPG) and differential pulse voltammetry at glassy carbon electrode (GCE). The HOPG induces the rupture of the liposomes and allows the lipids to adsorb along one of the three axes of symmetry of the HOPG basal planes, forming well-ordered lamellar structures. After interaction, both DPPC monolayers and DPPC–imatinib complexes are adsorbed onto HOPG. The OSPC–imatinib complexes self-organize only into ordered but larger domains of parallel stripes that maintain the threefold symmetry of the HOPG, due to an easier imatinib penetration into the unsaturated OSPC liposome bilayers. The voltammetric results show that upon interaction, the electrochemical active moiety of imatinib is incorporated into the lipid bilayer becoming unavailable to the GCE surface for oxidation, leading to local structural modifications of the lipid bilayer which were also electrochemically detected. A model is proposed for the liposome–imatinib interaction considering that imatinib interacts primarily by van der Waals and hydrogen bonds with the phosphatidylcholine headgroups, leading to defects in the liposome bilayer and allowing further incorporation of imatinib into the liposome lamellae.

Environmentally Friendly Formulations of Alachlor and Atrazine: Preparation, Characterization, and Reduced Leaching

Atrazine and alachlor formulations were designed by encapsulating the herbicide molecules into phosphatidylcholine (PC) vesicles, which subsequently were adsorbed on montmorillonite. PC and montmorillonite are classified as substances of minimal toxicological risk by the U.S. EPA. PC enhanced alachlor and atrazine solubilities by 15- and 18-fold, respectively. A 6 mM PC:5 g/L clay ratio was found as optimal for PC adsorption on the clay. Active ingredient contents of the PC-clay formulations ranged up to 8.6% for atrazine and 39.5% for alachlor. Infrared spectroscopy showed hydrophobic interactions of herbicide molecules with the alkyl chains of PC, in addition to hydrophilic interactions with the PC headgroup. Release experiments in a sandy soil showed a slower rate from the PC-clay formulations than the commercial ones. Soil column experiments under moderate irrigation and bioactivity experiments indicate that a reduction in the recommended dose of alachlor and atrazine can be accomplished by using PC-clay formulations.

Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

Sphingomyelin (SM) is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II (EqtII). The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup. Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp(112) and Tyr(113) exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins.

Heterogeneous Domains and Membrane Permeability in Phosphatidylcholine−Phosphatidic Acid Rigid Vesicles As a Function of pH and Lipid Chain Mismatch

Heterogeneous lipid membranes tuned by pH were evaluated at 37 degrees C in the form of PEGylated vesicles composed of lipid pairs with dipalmitoyl ( n = 16) and distearoyl ( n = 18) chain lengths. One lipid type was chosen to have the titratable moiety phosphatidic acid on its headgroup, and the other lipid type was chosen to have a phosphatidylcholine headgroup. The effect of pH on the formation of lipid heterogeneities and on membrane permeability was studied on vesicles composed of lipid pairs with matching and nonmatching chain lengths. The formation of lipid heterogeneities increases with decreasing pH in membranes composed of lipid pairs with either matching or nonmatching chain lengths. Increased permeability with decreasing pH was exhibited only by membranes composed of lipid pairs with nonmatching chain lengths. Permeability rates correlate strongly with the predicted extent of interfacial boundaries of heterogeneities, suggesting defective packing among nonmatching acyl chains of lipids. In heterogeneous mixtures with one lipid type in the fluid state ( n = 12), the dependence of membrane permeability on pH is weaker. In the presence of serum proteins, PEGylated gel-phase vesicles containing lipid pairs with nonmatching chain lengths exhibit faster release rates with decreasing pH compared to measured release rates in phosphate buffer, suggesting a second mechanism of formation of separated phases. PEGylated vesicles composed of lipid pairs with nonmatching chain lengths labeled with internalizing anti-HER2/neu antibodies that target overexpressed antigens on the surface of SKOV3-NMP2 ovarian cancer cells exhibit specific cancer cell targeting, followed by extensive internalization (more than 84% of bound vesicles) and fast release of contents intracellularly. These PEGylated vesicles composed of rigid membranes for long blood circulation times that exhibit pH-dependent release of contents intracellularly could become potent drug delivery carriers for the targeted therapy of solid tumors.

Coarse-Grained MD Simulations of Membrane Protein-Bilayer Self-Assembly

Complete determination of a membrane protein structure requires knowledge of the protein position within the lipid bilayer. As the number of determined structures of membrane proteins increases so does the need for computational methods which predict their position in the lipid bilayer. Here we present a coarse-grained molecular dynamics approach to lipid bilayer self-assembly around membrane proteins. We demonstrate that this method can be used to predict accurately the protein position in the bilayer for membrane proteins with a range of different sizes and architectures.

Structural determinants of water permeability through the lipid membrane.

Despite intense study over many years, the mechanisms by which water and small nonelectrolytes cross lipid bilayers remain unclear. While prior studies of permeability through membranes have focused on solute characteristics, such as size, polarity, and partition coefficient in hydrophobic solvent, we focus here on water permeability in seven single component bilayers composed of different lipids, five with phosphatidylcholine headgroups and different chain lengths and unsaturation, one with a phosphatidylserine headgroup, and one with a phosphatidylethanolamine headgroup. We find that water permeability correlates most strongly with the area/lipid and is poorly correlated with bilayer thickness and other previously determined structural and mechanical properties of these single component bilayers. These results suggest a new model for permeability that is developed in the accompanying theoretical paper in which the area occupied by the lipid is the major determinant and the hydrocarbon thickness is a secondary determinant. Cholesterol was also incorporated into DOPC bilayers and X-ray diffuse scattering was used to determine quantitative structure with the result that the area occupied by DOPC in the membrane decreases while bilayer thickness increases in a correlated way because lipid volume does not change. The water permeability decreases with added cholesterol and it correlates in a different way from pure lipids with area per lipid, bilayer thickness, and also with area compressibility.

A comparative molecular dynamics analysis of the amyloid β-peptide in a lipid bilayer

Because the amyloid β-peptide (Aβ) functions as approximately half of the transmembrane domain of the amyloid precursor protein and interaction of Aβ with membranes is proposed to result in neurotoxicity, the association of Aβ with membranes likely is important in the etiology of Alzheimer’s disease. Atomic details of the interaction of Aβ with membranes are not accessible with most experimental techniques, but computational methods can provide this information. Here, we present the results of ten 100-ns molecular dynamics (MD) simulations of the 40-residue amyloid β-peptide (Aβ 40) embedded in a dipalmitoylphosphatidylcholine (DPPC) bilayer. The present study examines the effects of insertion depth, protonation state of key residues, and ionic strength on Aβ 40 in a DPPC bilayer. In all cases, a portion of the peptide remained embedded in the bilayer. In the case of deeper insertion depth, Aβ 40 adopted a near-transmembrane orientation, drawing water molecules into the bilayer to associate with its charged amino acids. In the case of shallower insertion, the most widely-accepted construct, the peptide associated strongly with the membrane–water interface and the phosphatidylcholine headgroups of the bilayer. In most cases, significant disordering of the extracellular segment of the peptide was observed, and the brief appearance of a β-strand was noted in one case. Our results compare well with a variety of experimental and computational findings. From this study, we conclude that Aβ associated with membranes is dynamic and capable of adopting a number of conformations, each of which may have significance in understanding the progression of Alzheimer’s disease.

1H NMR investigation on interaction between ibuprofen and lipoproteins

A large number of studies indicate that oxidative modification of plasma lipoproteins, especially low-density lipoprotein (LDL), is a critical factor in initiation and progression of atherosclerosis. We have previously found that ibuprofen (IBP), a potential antioxidant drug to inhibit LDL oxidation, interacted with lipoproteins in intact human plasma. In the present study, we compare the binding affinities of IBP to LDL and HDL (high-density lipoprotein) by 1H NMR spectroscopy. When IBP is added into the HDL and LDL samples, the N +(CH 3) 3 moieties of phosphatidylcholine (PC) and sphingomyelin (SM) in lipoprotein particles experience the chemical shift up-field drift. Intermolecular cross-peaks observed in NOESY spectra imply that there are direct interactions between ibuprofen and lipoproteins at both hydrophobic and hydrophilic (ionic) regions. These interactions are likely to be important in the solubility of ibuprofen into lipoprotein particles. Ibuprofen has higher impact on the PC and SM head group ( N +(CH 3) 3) and (CH 2) n group in HDL than that in LDL. This could be explained by either IBP has higher binding affinity to HDL than to LDL, or IBP induces orientation of the phospholipid head group at the surface of the lipoprotein particles.

Action of α-cyclodextrin on phospholipid assemblies

Interactions of α-cyclodextrin (α-CD) with dimyristoylphosphatidylcholine (DMPC) and Egg phosphatidylcholine (Egg-PC) were studied (i) by analyzing surface pressure-area isotherms and surface tension of phospholipid monolayers formed at the interface between air and α-CD aqueous solutions and (ii) by X-ray diffraction performed on fully hydrated α-CD/phospholipid binary mixtures. The cyclodextrin molecules strongly interact with the two-dimension phospholipid assembly. Their addition into the aqueous sub-phase leads to the removal of part of the phospholipids from the air-water interface: the higher the α-CD concentration, the higher the phospholipid depletion. This should preferentially involve interactions between cyclodextrin and the phosphatidylcholine head group as α-CD is water-soluble and not surface-active. At the three-dimension level, the bilayer packing of the phospholipid lamellar phase appears not affected by the presence of cyclodextrin as shown by X-ray scattering at small angles whereas wide-angle diffraction patterns reveal the formation of a crystalline phase organized in a pseudo-hexagonal lattice usually characteristic of α-CD dimers. These results point out that α-CD should interact with bilayer-forming phospholipid molecules but likely according to a process that would preserve intact at least a part of the multilamellar assembly.

Interaction of mammalian Hsp22 with lipid membranes

Hsp22/HspB8 is a member of the small heat-shock protein family, whose function is not yet completely understood. Our immunolocalization studies in a human neuroblastoma cell line, SK-N-SH, using confocal microscopy show that a significant fraction of Hsp22 is localized to the plasma membrane. We therefore investigated its interactions with lipid vesicles in vitro. Intrinsic tryptophan fluorescence is quenched in the presence of lipid vesicles derived from either bovine brain lipid extract or purified lipids. Time-resolved fluorescence studies show a decrease in the lifetimes of the tryptophan residues. Both of these results indicate burial of some tryptophan residues of Hsp22 upon interaction with lipid vesicles. Membrane interactions also lead to increase in fluorescence polarization of Hsp22. Gel-filtration chromatography shows that Hsp22 binds stably with lipid vesicles; the extent of binding depends on the nature of the lipid. Hsp22 binds more strongly to vesicles made of lipids containing a phosphatidic acid, phosphatidylinositol or phosphatidylserine headgroup (known to be present in the inner leaflet of plasma membrane) compared with lipid vesicles made of a phosphatidylcholine head-group alone. Far-UV CD spectra reveal conformational changes upon binding to the lipid vesicles or in membrane-mimetic solvent, trifluoroethanol. Thus our fluorescence, CD and gel-filtration studies show that Hsp22 interacts with membrane and this interaction leads to stable binding and conformational changes. The present study therefore clearly demonstrates that Hsp22 exhibits potential membrane interaction that may play an important role in its cellular functions.

Human Opioid Peptide Met-Enkephalin Binds to Anionic Phosphatidylserine in High Preference to Zwitterionic Phosphatidylcholine: Natural-Abundance 13C NMR Study on the Binding State in Large Unilamellar Vesicles

A human opioid neuropeptide, Met-enkephalin (M-Enk: Tyr1-Gly2-Gly3-Phe4-Met5), having no net charge binds to anionic phosphatidylserine (PS) in high preference to zwitterionic phosphatidylcholine (PC). The binding mechanism in the PS and PC bilayers was studied on the basis of the inter- and intramolecular interaction data obtained by natural-abundance 13C nuclear magnetic resonance (NMR) of the peptide. Prominent upfield changes of the 13C resonance were observed in the C-terminal residue upon binding to PS, whereas no such marked change was observed upon binding to PC. The upfield chemical shift changes with their characteristic carbon site dependence are ascribed to the electrostatic binding between the peptide C-terminal CO2- and the PS headgroup NH3+. Despite the net negative charge of the PS bilayer surface, M-Enk thus anchors the negatively charged C-terminus. In the N-terminal residue, on the other hand, marked downfield chemical shift changes are observed upon binding to both the PS and PC bilayers, the magnitude of the changes being much larger in the PS system. The downfield changes with their characteristic carbon site dependence are ascribed to the electrostatic binding between the peptide N-terminal NH3+ and the lipid headgroup negative charge(s) (CO2- or PO4- in PS, PO4- in PC). Perturbation on the signal half-widths due to membrane binding also indicates the preferential and deeper binding of M-Enk on the PS membrane surface than on the PC membrane surface. Local charge cancellation takes place efficiently between M-Enk termini and the PS headgroups and compensates for the strong electrostatic hydration of the ionic groups. Distribution of the charged (positive and negative) and uncharged sites in the headgroups along the bilayer normal is responsible for the marked difference between PS and PC headgroups in controlling the binding state of the zwitterionic M-Enk.

Lipid–protein interactions in human plasma LDL evidenced by magnetic resonance

Low density lipoprotein (LDL) particles exhibit extremely complex three-dimensional structural organization which is still not understood at the molecular level. The aim of this study was to provide the experimental evidence of a direct non-covalent interaction of the protein part with the lipid matrix. The approach was based on the combination of 1H NMR (600 MHz) spectroscopy with thiol-specific spin labeling of the protein (apoB). It is shown that the spectral peaks assigned to the methyl head groups of phosphatidylcholine and sphingomyelin in the 1H spectra of LDL exhibit line broadening when otherwise free thiol groups of apoB are covalently modified by methanethiosulfonate spin label. The effect is similar in the presence of water soluble paramagnetic compound. These results indicate that fragments of apoB, which are part of the receptor binding region, are directly in contact with the solvated phospholipid head groups of the lipid matrix.

Phospholipid-Dependent Regulation of Cytochrome c3-Mediated Electron Transport across Membranes

Cytochrome c 3 (cyt c 3) can mediate electron transport across phosphatidylcholine (PC)/cardiolipin (CL) and PC/phosphatidylglycerol (PG) membranes. A two-molecule process is involved in the electron transport across PC/CL membranes in the liquid-crystalline state. In contrast, a single-molecule process dominates the electron transport across PC/CL membranes in the gel state and PC/PG membranes in the liquid-crystalline and gel states. Namely, the electron transport mechanism differs with the phospholipid composition and membrane fluidity. The rate-limiting step of the two-molecule process was lateral diffusion of cyt c 3 in membranes. The rate constants for the three single-molecule process cases were similar to each other. To elucidate these reaction processes, interactions between cyt c 3 and phosphate groups and between cyt c 3 and the glycerol backbones of phospholipid bilayers were investigated by means of 31P and 2H solid-state NMR, respectively, for CL and PC/CL membranes. The results showed that the polar headgroups of both phosphatidylcholine and CL are involved in the binding of cyt c 3. Also, cyt c 3 penetrates into membranes, which would induce distortion of the lipid bilayer. The molecular mechanisms underlying the single- and two-molecule processes are discussed in terms of membrane structure.