Insights into the complex association of bovine factor Va with acidic-lipid-containing synthetic membranes.

The mechanism of binding of blood coagulation cofactor factor Va to acidic-lipid-containing membranes has been addressed. Binding isotherms were generated at room temperature using the change in fluorescence anisotropy of pyrene-labeled bovine factor Va to detect binding to sonicated membrane vesicles containing either bovine brain phosphatidylserine (PS) or 1,2-dioleoyl-3-sn-phosphatidylglycerol (DOPG) in combination with 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC). The composition of the membranes was varied from 0 to 40 mol% for PS/POPC and from 0 to 65 mol % for DOPG/POPC membranes. Fitting the data to a classical Langmuir adsorption model yielded estimates of the dissociation constant (Kd) and the stoichiometry of binding. The values of Kd defined in this way displayed a maximum at low acidic lipid content but were nearly constant at intermediate to high fractions of acidic lipid. Fitting the binding isotherms to a two-process binding model (nonspecific adsorption in addition to binding of acidic lipids to sites on the protein) suggested a significant acidic-lipid-independent binding affinity in addition to occupancy of three protein sites that bind PS in preference to DOPG. Both analyses indicated that interaction of factor Va with an acidic-lipid-containing membrane is much more complex than those of factor Xa or prothrombin. Furthermore, a change in the conformation of bound pyrene-labeled factor Va with surface concentration of acidic lipid was implied by variation of both the saturating fluorescence anisotropy and the binding parameters with the acidic lipid content of the membrane. Finally, the results cannot support the contention that binding occurs through nonspecific adsorption to a patch or domain of acidic lipids in the membrane. Factor Va is suggested to associate with membranes by a complex process that includes both acidic-lipid-specific and acidic-lipid-independent sites and a protein structure change induced by occupancy of acidic-lipid-specific sites on the factor Va molecule.     

Membrane composition affects the reversibility of annexin A2t binding to solid supported membranes: a QCM study

By means of the quartz crystal microbalance (QCM) technique, we investigated the interaction of porcine heterotetrametric annexin A2t with solid supported lipid membranes. Dissociation and rate constants of annexin A2t binding to various lipid mixtures were determined as a function of Ca2+ concentrations in solution. In contrast to what has been observed for annexin A1, the binding affinity and kinetics of annexin A2t binding are not influenced by cholesterol. In the experimental setup chosen, the annexin A2t binding is strictly Ca2+-dependent and only affected by the amount of phosphatidylserine (PS) in the membrane and the Ca2+ concentration in solution. By Ca2+-titration experiments at constant annexin A2t concentration, we investigated the reversibility of annexin A2t adsorption and desorption. Surprisingly, Ca2+-titration curves display a significant hysteresis. Protein desorption curves starting from annexin A2t bound to the membrane at 1 mM CaCl2 exhibit high cooperativity with half-maximum Ca2+ concentrations in the submicromolar range. However, protein adsorption curves starting from an EGTA-containing solution with soluble annexin A2t always show two inflection points upon addition of Ca2+ ions. These two inflection points may be indicative of two protein populations differently bound to the solid-supported membrane. The ratio of these two annexin A2t populations depends on the amount of PS molecules and cholesterol in the membrane as well as on the Ca2+ concentration. We propose a model discussing the results obtained in terms of two binding sites differing in their affinity due to lipid rearrangement.     

Lupus antibody bivalency is required to enhance prothrombin binding to phospholipid

Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid-binding proteins including prothrombin. We have proposed that LA propagates coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall. A murine monoclonal anti-prothrombin Ab and three of three LA IgGs enhanced prothrombin binding to 75:25 phosphatidyl choline:phosphatidyl serine vesicles measured by either ultracentrifugation or right-angle light scattering. The assembly of prothrombin and LA IgG on phospholipid vesicles was estimated by surface plasmon resonance. The on rates for prothrombin and LA IgG were approximately the same as the on rate for prothrombin alone. In contrast, the off rates for prothrombin and LA IgG were 2- to 3-fold slower than the off rate for prothrombin. LA IgG bivalency was required for enhanced prothrombin binding to phospholipid vesicles, as Fab of the LA IgGs did not influence prothrombin binding at concentrations up to 40 microM. Modeling of the interactions of prothrombin, LA IgG and phospholipid vesicles indicated that augmentation of prothrombin binding to phospholipid vesicles by LA IgG could be accounted for by the bivalency of the LA IgG and the elevated microenvironmental concentration of prothrombin on the surface of phospholipid vesicles.     

Competition of annexin V and anticardiolipin antibodies for binding to phosphatidylserine containing membranes

Annexin V, an intracellular protein with a calcium-dependent high affinity for anionic phospholipid membranes, acts as an inhibitor of lipid-dependent reactions of the blood coagulation. Antiphospholipid antibodies found in the plasma of patients with antiphospholipid syndrome generally do not interact with phospholipid membranes directly, but recognize (plasma) proteins associated with lipid membranes, mostly prothrombin or beta(2)-glycoprotein I (beta(2)GPI). Previously, it has been proposed that antiphospholipid antibodies may cause thrombosis by displacing annexin V from procoagulant cell surfaces. We used ellipsometry to study the binding of annexin V and of complexes of beta(2)GPI with patient-derived IgG antibodies to beta(2)GPI, commonly referred to as anticardiolipin antibodies (ACA), to phospholipid bilayers composed of phosphatidylcholine (PC) and 20% phosphatidylserine (PS). More specifically, we investigated the competition of these proteins for the binding sites at these bilayers. We show that ACA-beta(2)GPI complexes, adsorbed to PSPC bilayers, are displaced for more than 70% by annexin V and that annexin V binding is unaffected by the presence of ACA-beta(2)GPI complexes. Conversely, annexin V preadsorbed to these bilayers completely prevents adsorption of ACA-beta(2)GPI complexes, and none of the preadsorbed annexin V is displaced by ACA-beta(2)GPI complexes. Using ellipsometry, we also studied the effect of ACA-beta(2)GPI complexes on the interaction of annexin V with the membranes of ionophore-activated blood platelets as a more physiological relevant model of cell membranes. The experiments with blood platelets confirm the high-affinity binding of annexin V to these membranes and unequivocally show that annexin V binding is unaffected by the presence of ACA-beta(2)GPI. In conclusion, our data unambiguously show that ACA-beta(2)GPI complexes are unable to displace annexin V from procoagulant membranes to any significant extent, whereas annexin V does displace the majority of preadsorbed ACA-beta(2)GPI complexes from these 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.     

Interaction of antibodies with liposomes bearing fluorescent haptens

Kinetic and equilibrium aspects of the recognition of antigenic model membranes by antibodies have been studied. Monoclonal anti-fluorescein IgG and its monovalent Fab fragment were allowed to interact with a fluorescein-lipid hapten that was incorporated into phospholipid vesicles. The binding was assayed in the nanomolar hapten concentration range by monitoring the quenching of hapten fluorescence by antibody. The rate and strength of the binding depended on the lipid composition of the vesicles; cholesterol enhanced both. The biphasic binding kinetics observed at high antibody concentrations for some compositions, plus additional spectroscopic evidence, led us to hypothesize that the hapten existed in a composition-dependent equilibrium between at least two conformations: (1) extended away from the membrane surface, available for binding, and (2) sequestered at or in the surface, unavailable for binding. The rate and strength of IgG binding were always greater than those of Fab, indicating bivalent binding by the IgG. This binding was intra-vesicular, since no agglutination of the vesicles was detected.     

Binding of peptides with basic residues to membranes containing acidic phospholipids.

There are clusters of basic amino acids on many cytoplasmic proteins that bind transiently to membranes (e.g., protein kinase C) as well as on the cytoplasmic domain of many intrinsic membrane proteins (e.g., glycophorin). To explore the possibility that these basic residues bind electrostatically to monovalent acidic lipids, we studied the binding of the peptides Lysn and Argn (n = 1-5) to bilayer membranes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). We made electrophoretic mobility measurements using multilamellar vesicles, fluorescence and equilibrium binding measurements using large unilamellar vesicles, and surface potential measurements using monolayers. None of the peptides bound to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but all bound to vesicles formed from PC/PS or PC/PG mixtures. None of the peptides exhibited specificity between PS and PG. Each lysine residue that was added to Lys2 decreased by one order of magnitude the concentration of peptide required to reverse the charge on the vesicle; equivalently it increased by one order of magnitude the binding affinity of the peptides for the PS vesicles. The simplest explanation is that each added lysine binds independently to a separate PS with a microscopic association constant of 10 M-1 or a free energy of approximately 1.4 kcal/mol. Similar, but not identical, results were obtained with the Argn peptides. A simple theoretical model combines the Gouy-Chapman theory (which accounts for the nonspecific electrostatic accumulation of the peptides in the aqueous diffuse double layer adjacent to the membrane) with mass action equations (which account for the binding of the peptides to greater than 1 PS). This model can account qualitatively for the dependence of binding on both the number of basic residues in the peptides and the mole fraction of PS in the membrane.     

Flexible charged macromolecules on mixed fluid lipid membranes: theory and Monte Carlo simulations

Fluid membranes containing charged lipids enhance binding of oppositely charged proteins by mobilizing these lipids into the interaction zone, overcoming the concomitant entropic losses due to lipid segregation and lower conformational freedom upon macromolecule adsorption. We study this energetic-entropic interplay using Monte Carlo simulations and theory. Our model system consists of a flexible cationic polyelectrolyte, interacting, via Debye-Hückel and short-ranged repulsive potentials, with membranes containing neutral lipids, 1% tetravalent, and 10% (or 1%) monovalent anionic lipids. Adsorption onto a fluid membrane is invariably stronger than to an equally charged frozen or uniform membrane. Although monovalent lipids may suffice for binding rigid macromolecules, polyvalent counter-lipids (e.g., phosphatidylinositol 4,5 bisphosphate), whose entropy loss upon localization is negligible, are crucial for binding flexible macromolecules, which lose conformational entropy upon adsorption. Extending Rosenbluth's Monte Carlo scheme we directly simulate polymer adsorption on fluid membranes. Yet, we argue that similar information could be derived from a biased superposition of quenched membrane simulations. Using a simple cell model we account for surface concentration effects, and show that the average adsorption probabilities on annealed and quenched membranes coincide at vanishing surface concentrations. We discuss the relevance of our model to the electrostatic-switch mechanism of, e.g., the myristoylated alanine-rich C kinase substrate protein.     

Adsorption of gamma-aminobutyric acid to phosphatidylserine membranes.

The interaction of the negatively-charged phosphatidylserine (PS) and gamma-Aminobutyric acid (GABA) is examined in black lipid membranes (BLM) and inverse micelles. GABA does not permeate through PS membranes and, in concentrations of 10(-5)-10(-4) M, it reduces the negative potential at the membrane-aqueous solution interface. The effect is owing to the adsorption of the GABA cationic species and the consequent decrease of the negative surface charge density of the membrane. When the intrinsic pH of the membrane-solution interface is considered, the Gouy-Chapman-Stern theory describes the GABA screening effect and makes it possible to calculate the GABA-PS binding constant. This value is compared with that obtained measuring the partition of 14C-GABA between an organic phase containing PS and the aqueous solution. The results presented strongly suggest that the electrostatic force plays a major role in GABA-PS interaction.     

Adsorption of bovine prothrombin to spread phospholipid monolayers

The interaction of bovine prothrombin with phospholipids was measured, using as the lipid source monolayers spread at the air-buffer interface. Fluorescence spectroscopy was implemented to determine the equilibrium concentration of free prothrombin in the aqueous subphase of the protein-monolayer suspensions, in a continuous assay system. The increase in surface pressure (pi) from the protein-monolayer adsorption was also measured and, with values of the adsorbed protein concentration (c[s]), was used to calculate dc(s)/d(pi). At a particular phosphatidylserine (PS) content of liquid-expanded (LE) phosphatidylcholine (PC)/PS monolayers, dc(s)/d(pi) was independent of the initial surface pressure (pi[i]), when this latter value exceeded 30 mN/m. However, dc(s)/d(pi) varied significantly with the relative PS content of the monolayer. Values of the equilibrium dissociation constants calculated from the concentration dependence of delta(pi) indicated that the affinity of prothrombin for LE monolayers was higher at higher PS contents and lower packing densities. The affinity of prothrombin for liquid-condensed (LC) PC/PS monolayers was found to be much weaker relative to LE monolayers of similar phospholipid composition. This approach, employing spread monolayers to study prothrombin-phospholipid binding, coupled with a simple and accurate method to determine the free protein concentration in protein-monolayer suspensions, offers significant advantages for the investigation of protein-membrane interaction. The equilibrium characteristics that describe the interaction of prothrombin with the different phospholipid monolayers under various conditions also provide support for previous results which indicated that hydrophobic interactions are involved in the adsorption of vitamin K-dependent coagulation and anticoagulation proteins to model membrane systems.     

Membrane binding of peptides containing both basic and aromatic residues. Experimental studies with peptides corresponding to the scaffolding region of caveolin and the effector region of MARCKS

We have studied the binding of peptides containing both basic and aromatic residues to phospholipid vesicles. The peptides caveolin(92-101) and MARCKS(151-175) both contain five aromatic residues, but have 3 and 13 positive charges, respectively. Our results show the aromatic residues insert into the bilayer and anchor the peptides weakly to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC). Incorporation of a monovalent acidic lipid (e.g., phosphatidylserine, PS) into the vesicles enhances the binding of both peptides via nonspecific electrostatic interactions. As predicted from application of the Poisson-Boltzmann equation to atomic models of the peptide and membranes, the enhancement is larger (e.g., 10(4)- vs 10-fold for 17% PS) for the more basic MARCKS(151-175). Replacing the five Phe with five Ala residues in MARCKS(151-175) decreases the binding to 10:1 PC/PS vesicles only slightly (6-fold). This result is also consistent with the predictions of our theoretical model: the loss of the attractive hydrophobic energy is partially compensated by a decrease in the repulsive Born/desolvation energy as the peptide moves away from the membrane surface. Incorporating multivalent phosphatidylinositol 4, 5-bisphosphate (PIP(2)) into PC vesicles produces dramatically different effects on the membrane binding of the two peptides: 1% PIP(2) enhances caveolin(92-101) binding only 3-fold, but increases MARCKS(151-175) binding 10(4)-fold. The strong interaction between the effector region of MARCKS and PIP(2) has interesting implications for the cellular function of MARCKS.     

Binding of imipramine to phospholipid bilayers using radioligand binding assay

Binding of the tricyclic antidepressant imipramine (IMI) to neutral and negatively charged lipid membranes was investigated using a radioligand binding assay combined with centrifugation or filtration. Lipid bilayers were composed of brain phosphatidylcholine (PC) and phosphatidylserine (PS). IMI binding isotherms were measured up to IMI concentration of 0.5 mmol/l. Due to electrostatic attraction, binding between the positively charged IMI and the negatively charged surfaces of PS membranes was augmented compared to binding to neutral PC membranes. After correction for electrostatic effects by means of the Gouy-Chapman theory, the binding isotherms were described both by surface partition coefficients and by binding parameters (association constants and binding capacities). It was confirmed that binding of IMI to model membranes is strongly affected by negatively charged phospholipids and that the binding is heterogeneous; in fact, weak surface adsorption and incorporation of the drug into the hydrophobic core of lipid bilayer can be seen and characterized. These results support the hypothesis suggesting that the lipid part of biological membranes plays a role in the mechanism of antidepressant action.     

Phosphoinositide-specific phospholipase C-delta 1 binds with high affinity to phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate.

We studied the binding of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta) to vesicles containing the negatively charged phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS). PLC-delta did not bind significantly to large unilamellar vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but bound strongly to vesicles formed from mixtures of PC and PIP2. The apparent association constant for the putative 1:1 complex formed between PLC-delta and PIP2 was Ka congruent to 10(5) M-1. The binding strength increased further (Ka congruent to 10(6) M-1) when the vesicles also contained 30% PS. High-affinity binding of PLC-delta to PIP2 did not require Ca2+. PLC-delta bound only weakly to vesicles formed from mixtures of PC and either PS or phosphatidylinositol (PI); binding increased as the mole fraction of acidic lipid in the vesicles increased. We also studied the membrane binding of a small basic peptide that corresponds to a conserved region of PLC. Like PLC-delta, the peptide bound weakly to vesicles containing monovalent negatively charged lipids; unlike PLC-delta, it did not bind strongly to vesicles containing PIP2. Our data suggest that a significant fraction of the PLC-delta in a cell could be bound to PIP2 on the cytoplasmic surface of the plasma membrane.     

The binding of lipopolysaccharide from Escherichia coli to mammalian cell membranes and its effect on liposomes.

The kinetics of the absorption of 32P- or 14C-labelled lipopolysaccharide from Escherichia coli NCTC 8623, serotype 0 125, chemotype XII, to erythrocytes, leukocytes, peritoneal macrophages and peritoneal lymphocytes was examined. Under variable conditions maximal levels of binding were found due to saturation of receptor sites on the cell membrane or steric hindrance by bound lipopolysaccharide. During adsorption slight leakage of haemoglobin was found but complete lysis of erythrocytes was ruled out after noting the effect of lipopolysaccharide on artificial lipid bilayers. The affinit of lipopolysaccharide to cell membranes revealed a consistent pattern of cyclic fluctuation between adsorption and desorption. A model was proposed to explain this cyclic fluctuation in binding based on membrane reorganization. It was significant that the cycle of lipopolysaccharide adsorption-desorption proceeded to completion even if the process was interrupted. The indication was that, once triggered, membrane reorganization occurred independently without influence from the test environment.     

A combinatorial code for the interaction of alpha-synuclein with membranes

Considerable genetic and pathological evidence has implicated the small, soluble protein alpha-synuclein in the pathogenesis of familial and sporadic forms of Parkinsons disease (PD). However, the precise role of alpha-synuclein in the disease process as well as its normal function remain poorly understood. We recently found that an interaction with lipid rafts is crucial for the normal, pre-synaptic localization of alpha-synuclein. To understand how alpha-synuclein interacts with lipid rafts, we have now developed an in vitro binding assay to rafts purified from native membranes. Recapitulating the specificity observed in vivo, recombinant wild type but not PD-associated A30P mutant alpha-synuclein binds to lipid rafts isolated from cultured cells and purified synaptic vesicles. Proteolytic digestion of the rafts does not disrupt the binding of alpha-synuclein, indicating an interaction with lipid rather than protein components of these membranes. We have also found that alpha-synuclein binds directly to artificial membranes whose lipid composition mimics that of lipid rafts. The binding of alpha-synuclein to these raft-like liposomes requires acidic phospholipids, with a preference for phosphatidylserine (PS). Interestingly, a variety of synthetic PS with defined acyl chains do not support binding when used individually. Rather, the interaction with alpha-synuclein requires a combination of PS with oleic (18:1) and polyunsaturated (either 20:4 or 22:6) fatty acyl chains, suggesting a role for phase separation within the membrane. Furthermore, alpha-synuclein binds with higher affinity to artificial membranes with the PS head group on the polyunsaturated fatty acyl chain rather than on the oleoyl side chain, indicating a stringent combinatorial code for the interaction of alpha-synuclein with membranes.     

Kinetics of adsorption of beta-amyloid peptide Abeta(1-40) to lipid bilayers

The Alzheimer's disease-related peptide beta-amyloid (Abeta) is toxic to neurons. The toxicity of the peptide appears to require conversion of the monomeric form to an aggregated fibrillar species. The interaction of Abeta with cell membranes has attracted interest as one plausible mechanism by which the peptide exerts its toxic activity. We developed two methods to measure the adsorption of fresh (monomeric) and aged (aggregated) Abeta to lipid bilayers. In one method, the kinetics of Abeta adsorption and desorption to liposomes deposited onto a dextran-coated surface was measured using surface plasmon resonance. In the other method, Abeta was contacted with liposome-coated magnetic beads; adsorbed Abeta was separated from solution-phase peptide by use of a magnetic field. Monomeric Abeta adsorbed quickly but reversibly to lipid bilayers with low affinity, while aggregated Abeta adsorbed slowly but irreversibly. These two methods provide complementary means of quantifying the adsorption of aggregating proteins to membranes. The results correlate strongly with previous observations that fibrillar, but not monomeric, Abeta restricts the motion of acyl tails in phospholipid bilayers. The methods should be useful for further elucidation of the role of membrane adsorption in mediating Abeta toxicity, and in the search for inhibitors of toxicity.     

Interaction of human gamma-carboxyglutamic acid domainless prothrombin with phospholipids

The conversion of prothrombin into thrombin occurs at the surface of stimulated platelets. In order to see the influence of gamma-carboxyglutamic acid in the interaction of prothrombin with phospholipid, we investigated the direct interaction of the peptide 1-41 and prothrombin (des 1-44) with phospholipid monolayers of various compositions. Adsorption of the labeled proteins was determined by surface radioactivity measurements. Penetration of the proteins into the lipid layers was inferred from capacitance variation of the monolayer, measured by a.c. polarography. Prothrombin (des 1-44) was found both to adsorb and to penetrate the lipid monolayers, in the presence and in the absence of Ca++. The effects are higher on 100% PS than on 25% PS. This protein was also found to increase the permeability of vesicles containing 25% PS to T1+ ions, in the presence and in the absence of Ca++. Comparison with prothrombin shows that Gla residues are clearly involved in the interaction at 25% PS; nevertheless, the peptide 1-41 does not penetrate. A model of interaction of prothrombin with phospholipid, including both adsorption of prothrombin by Gla residues and its penetration by another domain, is proposed.     

The adsorption of prothrombin to phosphatidylserine multilayers quantitated by ellipsometry

We investigated by means of an automated ellipsometer the adsorption of prothrombin from a buffer solution by multilayers of 14:0/14:0- and 18:1/18:1-phosphatidylserine (PS) stacked on chromium slides. In this instrument thickness and refractive index of the adsorbed phospholipid and proteins are monitored continuously. Two equations are derived to relate the mass of stacked phospholipids and the mass of protein adsorbed to the thickness and refractive index. These equations are based upon the Lorentz-Lorenz relation among the molar refractivities, refractive indices, and the densities of binary mixtures. Experimental validation of these equations is performed by measuring stacked multilayers of known mass of phosphatidylserine and the adsorption of [125I] albumin and [3H]prothrombin on these multilayers. Using these equations we measured the dissociation constants Kd and the number of binding sites nb of prothrombin. Values of Kd = 0.15 x 10(-8) M and nb = 122 molecules of PS/molecule of prothrombin were observed for di C14:0 PS and values of Kd = 0.45 x 10(-8) M and nb = 54 molecules of PS/molecule of prothrombin for di C18:1 PS. These data compare well to data obtained by other methods available in the literature.     

The omega-loop region of the human prothrombin gamma-carboxyglutamic acid domain penetrates anionic phospholipid membranes

The hydrophobic omega-loop within the prothrombin gamma-carboxyglutamic acid-rich (Gla) domain is important in membrane binding. The role of this region in membrane binding was investigated using a synthetic peptide, PT-(1-46)F4W, which includes the N-terminal 46 residues of human prothrombin with Phe-4 replaced by Trp providing a fluorescent probe. PT-(1-46)F4W and PT-(1-46) bind calcium ions and phospholipid membranes, and inhibit the prothrombinase complex. PT-(1-46)F4W, but not PT-(1-46), exhibits a blue shift (5 nm) and red-edge excitation shift (28 nm) in the presence of phosphatidylserine (PS)-containing vesicles, suggesting Trp-4 is located within the motionally restricted membrane interfacial region. PS-containing vesicles protect PT-(1-46)F4W, but not PT-(1-46), fluorescence from potassium iodide-induced quenching. Stern-Volmer analysis of the quenching of PT-(1-46)F4W in the presence and absence of 80% phosphatidylcholine/20% PS vesicles suggested that Trp-4 is positioned within the membrane and protected from aqueous quenching agents whereas Trp-41 remains solvent-accessible in the presence of PS-containing vesicles. Fluorescence quenching of membrane-bound PT-(1-46)F4W is optimal with 7- and 10-doxyl-labeled lipids, indicating that Trp-4 is inserted 5 to 7 A into the bilayer. This report demonstrates that the omega-loop region of prothrombin specifically interacts with PS-containing membranes within the interfacial membrane region.