Quantitative fluorescence analysis of the adsorption of lysozyme to phospholipid vesicles

Experiments directed to measure the interaction of lysozyme with liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) have been conducted by monitoring both protein and lipid fluorescence and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of analysis in which the fractional contribution at moderate binding conditions is determined from either total fluorescence decay or anisotropy decay curves of tryptophan at limiting binding conditions. In the energy transfer experiments PC and PS lipids labelled with two pyrene acyl chains served as energy acceptors of the excited tryptophan residues in lysozyme. The binding was strongly dependent on the molar fraction of negatively charged PS in neutral PC membranes and on the ionic strength. Changes in the tryptophan fluorescence decay characteristics were found to be connected with long correlation times, indicating conformational rearrangements induced by binding of the protein to these lipid membranes. The dynamics of membrane bound protein appeared to be dependent on the physical state of the membrane. Independent of protein fluorescence studies, formation of a protein-membrane complex can also be observed from the lipid properties of the system. The interaction of lysozyme with di-pyrenyl-labelled phosphatidylserine in anionic PS/PC membranes resulted in a substantial decrease of the intramolecular excimer formation, while the excimer formation of dipyrenyl-labelled phosphatidylcholine in neutral PC membranes barely changed in the presence of lysozyme.Abbreviations dipyr₄ sn-1,2-(pyrenylbutyl) - dipyr₁₀ sn-1,2-(pyrenyldecanoyl). - DMPC dimyristoyl-phosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - PC phosphatidylcholine - PS phosphatidylserine Correspondence to: A. J. W. G. Visser     

23Na-NMR studies of Na+ interaction with human red cell membranes from normotensives and hypertensives

Na+ interaction with unsealed human red cell ghosts has been studied by 23Na-NMR relaxation rate (R1) measurements. Data on a total of nine subjects including seven volunteer normotensives (NBP) and two untreated hypertensives (HBP) are presented. Qualitative treatment of the data gives information on the dynamic behavior of Na+ undergoing fast exchange between the free and bound states. The excess longitudinal relaxation rate (delta R)-1 plotted against total [Na+], known as the James-Noggle plot, exhibits different behavior for NBP and HBP ghosts, with a relatively low binding constant of approx. 100 M-1 for HBP (p less than 0.025) compared to a high constant of 500-1000 M-1 for NBP. To associate our NMR data with membrane-bound (Na+ + K+)-ATPase, 23Na relaxation rates were measured in the presence of 5 mM ouabain. James-Noggle plots constructed for ouabain-sensitive excess relaxation rates show the binding for NBP to be even high affinity (greater than 10(3) M-1) but low capacity. These data may suggest that for a given amount of intracellular Na+, the binding affinity could determine the distribution of Na+ between the membrane and cytoplasm, and that the (Na+ + K+)-ATPase which is primarily responsible for the Na+ affinity might assume an abnormal transport mechanism in HBP membranes.     

Translational diffusion of bovine prothrombin fragment 1 weakly bound to supported planar membranes: measurement by total internal reflection with fluorescence pattern photobleaching recovery.

Previous work has shown that bovine prothrombin fragment 1 binds to substrate-supported planar membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) in a Ca(2+)-specific manner. The apparent equilibrium dissociation constant is 1-15 microM, and the average membrane residency time is approximately 0.25 s-1. In the present work, fluorescence pattern photobleaching recovery with evanescent interference patterns (TIR-FPPR) has been used to measure the translational diffusion coefficients of the weakly bound fragment 1. The results show that the translational diffusion coefficients on fluid-like PS/PC planar membranes are on the order of 10(-9) cm2/s and are reduced when the fragment 1 surface density is increased. Control measurements were carried out for fragment 1 on solid-like PS/PC planar membranes. The dissociation kinetics were similar to those on fluid-like membranes, but protein translational mobility was not detected. TIR-FPPR was also used to measure the diffusion coefficient of the fluorescent lipid NBD-PC in fluid-like PS/PC planar membranes. In these measurements, the diffusion coefficient was approximately 10(-8) cm2/s, which is consistent with that measured by conventional fluorescence pattern photobleaching recovery. This work represents the first measurement of a translational diffusion coefficient for a protein weakly bound to a membrane surface.     

Temperature dependence of Na+/Ca2+ exchange activity in beef-heart sarcolemmal vesicles and proteoliposomes

Temperature dependence of Na+/Ca2+ exchange activity was studied in beef cardiac sarcolemmal vesicles in the absence and presence of the inhibitor amiloride and in proteoliposomes reconstituted with different lipid mixtures. Arrhenius plots for Na+/Ca2+ exchange activity in both control and amiloride-treated vesicles revealed an apparent energy of activation of 9665 +/- 585 (SE, n = 4) cal/mol, corresponding to a temperature coefficient (Q10) value of 1.70 +/- 0.05 (SE, n = 4) over the range 25-37 degrees C. When Na+/Ca2+ exchange was reconstituted into phosphatidylcholine (PC):phosphatidylserine (PS) (52:48, mol/mol), PC:PS:cholesterol (25:39:36, mol/mol), and PC:PS:distearoylphosphatidylcholine (DSPC) (31:48:21, mol/mol) proteoliposomes, the highest activity was found in PC:PS:cholesterol proteoliposomes. Arrhenius plots of Na+/Ca2+ exchange activity exhibited breakpoints at 23 degrees C (PC:PS), 33 degrees C (PC:PS:cholesterol), and 23 degrees C (PC:PS:DSPC). The increase in the thermotropic transition temperature with cholesterol could result from the condensing effect of this sterol, whereas the breaks observed with PC:PS and PC:PS:DSPC could be caused by a non-lipid-mediated membrane protein conformational change. These results indicate that the lipid microenvironment around the Na+/Ca2+ exchanger and the nature of the specific lipid-protein interactions influence the activity of this antiporter. Further evidence supporting the hypothesis that cholesterol behaves as a specific positive effector for the exchanger is also given.     

Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are metabolically related membrane aminophospholipids. In mammalian cells, PS is required for targeting and function of several intracellular signaling proteins. Moreover, PS is asymmetrically distributed in the plasma membrane. Although PS is highly enriched in the cytoplasmic leaflet of plasma membranes, PS exposure on the cell surface initiates blood clotting and removal of apoptotic cells. PS is synthesized in mammalian cells by two distinct PS synthases that exchange serine for choline or ethanolamine in phosphatidylcholine (PC) or PE, respectively. Targeted disruption of each PS synthase individually in mice demonstrated that neither enzyme is required for viability whereas elimination of both synthases was embryonic lethal. Thus, mammalian cells require a threshold amount of PS. PE is synthesized in mammalian cells by four different pathways, the quantitatively most important of which are the CDP-ethanolamine pathway that produces PE in the ER, and PS decarboxylation that occurs in mitochondria. PS is made in ER membranes and is imported into mitochondria for decarboxylation to PE via a domain of the ER [mitochondria-associated membranes (MAM)] that transiently associates with mitochondria. Elimination of PS decarboxylase in mice caused mitochondrial defects and embryonic lethality. Global elimination of the CDP-ethanolamine pathway was also incompatible with mouse survival. Thus, PE made by each of these pathways has independent and necessary functions. In mammals PE is a substrate for methylation to PC in the liver, a substrate for anandamide synthesis, and supplies ethanolamine for glycosylphosphatidylinositol anchors of cell-surface signaling proteins. Thus, PS and PE participate in many previously unanticipated facets of mammalian cell biology. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells.

Efficient phagocytosis of apoptotic cells is important for normal tissue development, homeostasis, and the resolution of inflammation. Although many receptors have been implicated in the clearance of apoptotic cells, the roles of these receptors in the engulfment process have not been well defined. We developed a novel system to distinguish between receptors involved in tethering of apoptotic cells versus those inducing their uptake. Our results suggest that regardless of the receptors engaged on the phagocyte, ingestion does not occur in the absence of phosphatidylserine (PS). Further, recognition of PS was found to be dependent on the presence of the PS receptor (PSR). Both PS and anti-PSR antibodies stimulated membrane ruffling, vesicle formation, and "bystander" uptake of cells bound to the surface of the phagocyte. We propose that the phagocytosis of apoptotic cells requires two events: tethering followed by PS-stimulated, PSR-mediated macropinocytosis.     

Exposure of Phosphatidylethanolamine on the Surface of Apoptotic Cells

In the early stages of apoptosis, phosphatidylserine (PS) is translocated from the inner side of the plasma membrane to the outer layer, which allows phagocytes to recognize and engulf the apoptotic cells. In this study we have analyzed the cell surface exposure of phosphatidylethanolamine (PE) in apoptotic CTLL-2 cells, a cytotoxic T cell line, using a tetracyclic polypeptide of 19 amino acids (Ro09-0198) which specifically recognizes the structure of PE and forms a tight equimolar complex with the phospholipid. Fluorescence microscopic analysis showed that the peptide, conjugated with fluorescence-labeled streptavidin (FL-SA-Ro), bound effectively to the cell surface of cells undergoing apoptosis in response to withdrawal of interleukin-2 from the culture media, but not to nonapoptotic cells. The binding of FL-SA-Ro to apoptotic cells was not uniform and the intense staining was observed on surface blebs of apoptotic cells. The FL-SA-Ro binding was inhibited specifically by liposomes containing PE, suggesting that PE is mainly exposed on the surface blebs of apoptotic cells. The specific binding of FL-SA-Ro to the apoptotic cells was also confirmed using a fluorescence-activated cell sorter and the time-dependent cell surface exposure of PE correlated well with the exposure of PS, as detected by the binding of annexin V. This study provides the first direct evidence that PE as well as PS is exposed on the cell surface during the early stages of apoptosis, resulting in the total loss of asymmetric distribution of aminophospholipids in the plasma membrane bilayer.     

Sodium and calcium binding to Panulirus interruptus hemocyanin as studied by 23Na nuclear magnetic resonance.

Addition of Panulirus hemocyanin to NaCl solutions produces marked changes in the 23Na relaxation parameters; they show that sodium ions interact with binding sites on the protein and exchange rapidly with the bulk. The observed non-lorentzian lineshapes and the non-exponential decay of the transverse magnetization indicate that non-extreme narrowing conditions apply and give information on the dynamics of the interaction. Panulirus hemocyanin has at least two classes of Na+ binding sites; the binding constant of the more strongly bound sodium ions is in the order of 1 X 10(2) M-1. Competition between Na+ and Ca2+ for protein binding sites is demonstrated by the effect of Ca2+ on the 23Na relaxation parameters. However, only the more strongly bound Na+ are displaced by Ca2+. The number of Ca2+ needed to displace these sodium ions is 3--5 per oxygen binding site. The 23Na relaxation parameters are influenced also by the state of oxygenation of the protein, indicating a linkage between Na+ and oxygen binding. The simplest interpretation of the data is that sodium ions bind more strongly to oxyhemocyanin in agreement with oxygen equilibrium experiments.     

TNT‐Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of Pro‐Phagocytic Signals From Apoptotic to Viable Cells

The exposure of phosphatidylserine (PS) on the surface membrane of apoptotic cells triggers the recruitment of phagocytic receptors and subsequently results in uptake by phagocytes. Here we describe how apoptotic cells can use intercellular membrane nanotubes to transfer exposed PS to neighboring viable cells, and thus deposit an “eat‐me” tag on the viable cells. Tunneling nanotubes (TNTs) connected UV‐treated apoptotic rat pheochromocytoma PC12 cells with neighboring untreated cells. These TNTs were composed of PS‐exposed plasma membrane and facilitated the transfer of the membrane from apoptotic to viable cells. Other pro‐phagocytic signals, such as oxidized phospholipids and calreticulin, were also transferred to viable cells. In addition, anti‐phagocytic signal CD47 presenting on the plasma membrane of viable cells was masked by the transferred PS‐membrane. Confocal imaging revealed an increase of phagocytosis of viable PC12 cells by murine RAW264.7 macrophages when the viable PC12 cells were cocultured with UV‐treated PC12 cells. Treatment with 50 nM cytochalasin D would abolish TNTs and correspondingly inhibit this phagocytosis of the viable cells. Our study indicates that exposed‐PS membrane is delivered from apoptotic to viable cells through TNTs. This transferred membrane may act as a pro‐phagocytic signal for macrophages to induce phagocytosis of viable cells in a situation where they are in the vicinity of apoptotic cells. J. Cell. Physiol. 232: 2271–2279, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.     

Fluorescence decay curves under pulsed excitation: distinction between free and bound NAD(P)H on isolated living cells

Under pulsed excitation the decay curve of the intrinsic cellular fluorescence has been recorded using an original equipment. Cell by cell experiments are feasible so that the NAD(P)H bound/free ratio and the relaxation time characteristic of bound NAD(P)H can be used in order to build up histograms characterizing the cell population.     

Sodium-23 NMR relaxation times in nucleated red blood cells and suspensions of nuclei.

The relaxation behavior of intracellular 23Na in suspensions of chicken erythrocytes and of their nuclei was investigated. The transverse magnetization was found to decay biexponentially. The average relaxation rates for the nucleated chicken erythrocytes are considerably shorter than the average relaxation rates obtained for dog and human nonnucleated red blood cells. Of particular significance is the twofold decrease in the short component of T2. Calculations based on the measured 23Na NMR relaxation rates in suspensions of nuclei indicate that most of the difference between the relaxation rates in the mammalian as compared to the chicken erythrocytes, can be accounted for by the contribution of the nuclei in the latter.     

Nuclear magnetic resonance of tissue Na correlation time

Shporer and Civan (Biochim. Biophys. Acta (1974) 354, 291–304) reported the effect of magnetic-field strength on the NMR relaxation times of ²³Na in frog skeletal muscle. From these data, they estimated the correlation time τ_c for bound ²³Na whose tumbling is severely restricted, and they suggested that the fraction of bound ²³Na does not exceed some few percent of the total ²³Na population. However, a step in their theoretical approach seems oversimplified. With an improved approach, we obtained an effective τ_c of 4–9 ns for bound ²³Na. This value is some 10 times shorter than the corresponding value estimated by them from the same data. On the other hand, their conclusion concerning the amount of bound ²³Na seems to remain valid. The origin of the observed difference between the two transverse relaxation times of tissue ²³Na is also discussed.     

Monitoring drug induced apoptosis and treatment sensitivity in non-small cell lung carcinoma using dielectrophoresis

Non-invasive real time methods for characterizing biomolecular events that contribute towards apoptotic kinetics would be of significant importance in the field of cancer biology. Effective drug-induced apoptosis is an important factor for establishing the relationship between cancer genetics and treatment sensitivity. The objective of this study was to develop a non-invasive technique to characterize cancer cells that are undergoing drug-induced apoptosis. We used dielectrophoresis to determine apoptotic cells as early as 2 h post drug treatment as compared to 24 h with standard flow cytometry method using non-small cell lung cancer (NSCLC) adenocarcinoma cell line (HCC1833) as a study model. Our studies have shown significant differences in apoptotic cells by chromatin condensation, formation of apoptotic bodies and exposure of phosphatidylserine (PS) on the extracellular surface when the cells where treated with a potent Bcl-2 family inhibitor drug (ABT-263). Time lapse dielectrophoretic studies were performed over 24 h period after exposure to ABT-263 at clinically relevant concentrations. The dielectrophoretic studies were compared to Annexin-V FITC flow assay for the detection of PS in mid-stage apoptosis using flow cytometry. As a result of physical and biochemical changes, inherent dielectric properties of cells undergoing varying stages of apoptosis showed amplified changes in their cytoplasmic and membrane capacitance. In addition, zeta potential of these fixed isolated cells was measured to obtain direct correlation to biomolecular events.     

Specificity of Na+ binding to phosphatidylserine vesicles from a 23Na NMR relaxation rate study.

23Na NMR relaxation rate measurements show that Na+ binds specifically to phosphatidylserine vesicles and is displaced partially from the binding site by K+ and Ca2+ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na+ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na+-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na+) fall in the range 0.4--1.2 M-1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na+ at 0.1 M Na+ concentration.     

Analysis of sodium-23 nuclear magnetic resonance spin-lattice relaxation for the study of the intracellular sodium state.

Sodium-23 Nuclear Magnetic Resonance relaxation spectroscopy has been used to investigate the state of intracellular Na+ in control and CCl4-treated rat livers. The analysis of spin-lattice relaxation rates at 1.88 and 7.07 Tesla based on a two-site exchange model led to estimates of pertinent modulation times. Also it has been found that a relatively high quantity of Na+ (PB = 1.59 x 10(-2)) is bound to charged sites of intracellular macromolecules or membranes. The degree of binding strongly decreases in CCl4 treated rat livers.     

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic ‘eat-me' signals: cleavage and inhibition of phagocytosis by Lp-PLA2

Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H₂O₂ was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A₂ (Lp-PLA₂), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA₂ was blocked by a selective inhibitor of Lp-PLA₂, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic ‘eat-me'' signal that participates in the engulfment of cells undergoing intrinsic apoptosis.     

Staphylococcal superantigen-like protein 10 binds to phosphatidylserine and apoptotic cells

Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins that have structural similarities to staphylococcal superantigens. Although SSLs do not have superantigenic activity, some of them have been reported to bind to host immune related molecules and they have been implicated in immune evasion by S. aureus. In this study, we showed that SSL10 is capable of binding to phospholipids. SSL10 bound to phosphatidylserine (PS) containing liposome, but not to phosphatidylcholine liposome. SSL10, but not SSL7, bound to PS containing liposome, suggesting that SSL10 specifically binds to PS. Analysis of PS binding ability among recombinant truncated SSL10 fragments revealed that the β-barrel in the N-terminal oligonucleotide/oligosaccharide-binding (OB)-fold domain contributes to PS binding capacity. Fluorescein isothiocyanate labeled OB-fold of SSL10 stained hydrogen peroxide treated Jurkat cells. Annexin V is widely utilized for detection of apoptosis. Unlike annexin V, the OB-fold domain of SSL10 also bound to apoptotic cells in the presence of EDTA, suggesting that the OB-fold of SSL10 recognizes PS and apoptotic cells in a Ca(2+) independent manner. These findings suggest SSL10 and its derived peptides may be a novel detection tool for apoptotic cells.     

Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages.

During normal tissue remodeling, macrophages remove unwanted cells, including those that have undergone programmed cell death, or apoptosis. This widespread process extends to the deletion of thymocytes (negative selection), in which cells expressing inappropriate Ag receptors undergo apoptosis, and are phagocytosed by thymic macrophages. Although phagocytosis of effete leukocytes by macrophages has been known since the time of Metchnikoff, only recently has it been recognized that apoptosis leads to surface changes that allow recognition and removal of these cells before they are lysed. Our data suggest that macrophages specifically recognize phosphatidylserine that is exposed on the surface of lymphocytes during the development of apoptosis. Macrophage phagocytosis of apoptotic lymphocytes was inhibited, in a dose-dependent manner, by liposomes containing phosphatidyl-L-serine, but not by liposomes containing other anionic phospholipids, including phosphatidyl-D-serine. Phagocytosis of apoptotic lymphocytes was also inhibited by the L isoforms of compounds structurally related to phosphatidylserine, including glycerophosphorylserine and phosphoserine. The membranes of apoptotic lymphocytes bound increased amounts of merocyanine 540 dye relative to those of normal cells, indicating that their membrane lipids were more loosely packed, consistent with a loss of membrane phospholipid asymmetry. Apoptotic lymphocytes were shown to express phosphatidylserine (PS) externally, because PS on their surfaces was accessible to derivatization by fluorescamine, and because apoptotic cells expressed procoagulant activity. These observations suggest that apoptotic lymphocytes lose membrane phospholipid asymmetry and expose phosphatidylserine on the outer leaflet of the plasma membrane. Macrophages then phagocytose apoptotic lymphocytes after specific recognition of the exposed PS.     

PS exposure increases the susceptibility of cells to fusion with DOTAP liposomes

Cationic liposomes are used as efficient carriers for gene delivery into mammalian cells due to their ability to bind nucleic acids, adsorb onto the cell surface and fuse with negatively charged membranes. This last property enables the release and escape of their cargo from endosomal compartments. The efficiency of this fusion mainly depends on the surface charge of the target membranes. Here, we report that cells of two different lines, epithelial adenocarcinoma HeLa and lymphocytic leukemia Jurkat T, which externalize PS, are more susceptible to fusion with DOTAP liposomes than control cells. We compared the ability to undergo fusion of untreated and apoptotic cells. Apoptosis was induced by various pro-apoptotic agents and treatments, namely: incubation in the presence of MnCl(2), cytostatic drugs fludarabine and mitoxantrone, staurosporine and serum depletion in the case of HeLa cells. Jurkat T cells were treated similarly except apoptosis was additionally induced by incubation in the presence of 4% EtOH. Epithelial cells fused with the highest efficiencies of lipid mixing, when pretreated with staurosporine. Jurkat T cells were less susceptible to fusion, but they also displayed an increase in fusion efficiency after the induction of apoptosis. Alternatively, we treated the cells with metabolic inhibitors causing ATP-depletion in order to inactivate aminophospholipid translocase. After ATP-depletion, HeLa and Jurkat T cells fused with DOTAP liposomes with higher efficiencies than control cells. Our conclusion is that the lipid asymmetry of natural membranes may limit fusion with cationic liposomes.     

The cytoplasmic domains of phospholamban and phospholemman associate with phospholipid membrane surfaces

Phospholamban (PLB) and phospholemman (PLM, also called FXYD1) are small transmembrane proteins that interact with P-type ATPases and regulate ion transport in cardiac cells and other tissues. This work has investigated the hypothesis that the cytoplasmic domains of PLB and PLM, when not interacting with their regulatory targets, are stabilized through associations with the surface of the phospholipid membrane. Peptides representing the 35 C-terminal cytoplasmic residues of PLM (PLM(37-72)), the 23 N-terminal cytoplasmic residues of PLB (PLB(1-23)), and the same sequence phosphorylated at Ser-16 (P-PLB(1-23)) were synthesized to examine their interactions with model membranes composed of zwitterionic phosphatidylcholine (PC) lipids alone or in admixture with anionic phosphatidylglycerol (PG) lipids. Wide-line 2H NMR spectra of PC/PG membranes, with PC deuterated in the choline moiety, indicated that all three peptides interacted with the membrane surface and perturbed the orientation of the choline headgroups. Fluorescence and 31P magic-angle spinning (MAS) NMR measurements indicated that PLB(1-23) and P-PLB(1-23) had a higher affinity for PC/PG membranes, which carry an overall negative surface charge, than for PC membranes, which have no net surface charge. The 31P MAS NMR spectra of the PC/PG membranes in the presence of PLM(37-72), PLB(1-23), and P-PLB(1-23) indicated that all three peptides induced clustering of the lipids into PC-enriched and PG-enriched regions. These findings support the theory that the cytoplasmic domains of PLB and PLM are stabilized by interacting with lipid headgroups at the membrane surface, and it is speculated that such interactions may modulate the functional properties of biological membranes.