Fluorescence study of protein-lipid complexes with a new symmetric squarylium probe

The novel symmetric squarylium derivative SQ-1 has been synthesized and tested for its sensitivity to the formation of protein-lipid complexes. SQ-1 binding to the model membranes composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin (CL) in different molar ratios was found to be controlled mainly by hydrophobic interactions. Lysozyme (Lz) and ribonuclease A (RNase) exerted an influence on the probe association with lipid vesicles resulting presumably from the competition between SQ-1 and the proteins for bilayer free volume and modification of its properties. The magnitude of this effect was much higher for lysozyme which may stem from the amphipathy of protein alpha-helix involved in the membrane binding. Varying membrane composition provides evidence for the dye sensitivity to both hydrophobic and electrostatic protein-lipid interactions. Fluorescence anisotropy studies uncovered the restriction of SQ-1 rotational mobility in lipid environment in the presence of Lz and RNase being indicative of the incorporation of the proteins into bilayer interior. The results of binding, fluorescence quenching and kinetic experiments suggested lysozyme-induced local lipid demixing upon protein association with negatively charged membranes with threshold concentration of CL for the lipid demixing being 10 mol%.     

Cytotoxicity of bovine seminal ribonuclease: monomer versus dimer

Bovine seminal ribonuclease (BS-RNase) is a homologue of bovine pancreatic ribonuclease (RNase A). Unlike RNase A, BS-RNase has notable toxicity for human tumor cells. Wild-type BS-RNase is a homodimer linked by two intermolecular disulfide bonds. This quaternary structure endows BS-RNase with resistance to inhibition by the cytosolic ribonuclease inhibitor protein (RI), which binds tightly to RNase A and monomeric BS-RNase. Here, we report on the creation and analysis of monomeric variants of BS-RNase that evade RI but retain full enzymatic activity. The cytotoxic activity of these monomeric variants exceeds that of the wild-type dimer by up to 30-fold, indicating that the dimeric structure of BS-RNase is not required for cytotoxicity. Dimers of these monomeric variants are more cytotoxic than wild-type BS-RNase, suggesting that the cytotoxicity of the wild-type enzyme is limited by RI inhibition following dissociation of the dimer in the reducing environment of the cytosol. Finally, the cytotoxic activity of these dimers is less than that of the constituent monomers, indicating that their quaternary structure is a liability. These data provide new insight into structure-function relationships of BS-RNase. Moreover, BS-RNase monomers described herein are more toxic to human tumor cells than is any known variant or homologue of RNase A including Onconase, an amphibian homologue in phase III clinical trials for the treatment of unresectable malignant mesothelioma.     

Structure-cytotoxicity relationships in bovine seminal ribonuclease: new insights from heat and chemical denaturation studies on variants

Bovine seminal ribonuclease (BS-RNase), a homodimeric protein displaying selective cytotoxicity towards tumor cells, is isolated as a mixture of two isoforms, a dimeric form in which the chains swap their N-termini, and an unswapped dimer. In the cytosolic reducing environment, the dimeric form in which the chains swap their N-termini is converted into a noncovalent dimer (termed NCD), in which the monomers remain intertwined through their N-terminal ends. The quaternary structure renders the reduced protein resistant to the ribonuclease inhibitor, a protein that binds most ribonucleases with very high affinity. On the other hand, upon selective reduction, the unswapped dimer is converted in two monomers, which are readily bound and inactivated by the ribonuclease inhibitor. On the basis of these considerations, it has been proposed that the cytotoxic activity of BS-RNase relies on the 3D structure and stability of its NCD derivative. Here, we report a comparison of the thermodynamic and chemical stability of the NCD form of BS-RNase with that of the monomeric derivative, together with an investigation of the thermal dissociation mechanism revealing the presence of a dimeric intermediate. In addition, we report that the replacement of of Arg80 by Ser significantly decreases the cytotoxic activity of BS-RNase and the stability of the NCD form with respect to the parent protein, but does not affect the ribonucleolytic activity or the dissociation mechanism. The data show the importance of Arg80 for the cytotoxicity of BS-RNase, and also support the hypothesis that the reduced derivative of BS-RNase is responsible for its cytotoxic activity.     

Population shift vs induced fit: the case of bovine seminal ribonuclease swapping dimer

Bovine seminal ribonuclease (BS-RNase) is a unique member of the pancreatic-like ribonuclease superfamily. This enzyme exists as two conformational isomers with distinctive biological properties. The structure of the major isomer is characterized by the swapping of the N-terminal segment (MxM BS-RNase). In this article, the crystal structures of the ligand-free MxM BS-RNase and its complex with 2'-deoxycitidylyl(3',5')-2'-deoxyadenosine derived from isomorphous crystals have been refined. Interestingly, the comparison between this novel ligand-free form and the previously published sulfate-bound structure reveals significant differences. In particular, the ligand-free MxM BS-RNase is closer to the structure of MxM BS-RNase productive complexes than to the sulfate-bound form. These results reveal that MxM BS-RNase presents a remarkable flexibility, despite the structural constraints of the interchain disulfide bridges and the swapping of the N-terminal helices. These findings have important implications to the ligand binding mechanism of MxM BS-RNase. Indeed, a population shift rather than a substrate-induced conformational transition may occur in the MxM BS-RNase ligand binding process.     

Comprehensive comparison of the cytotoxic activities of onconase and bovine seminal ribonuclease

Onconase (ONC) and bovine seminal ribonuclease (BS-RNase) are homologs of bovine pancreatic ribonuclease (RNase A). Unlike RNase A, ONC and BS-RNase can evade the cytosolic ribonuclease inhibitor protein and are potent cytotoxins. Here, the endogenous cytotoxic activities of ONC and BS-RNase are compared in a wide variety of assays. Injections of ONC into one or both testes of mice and rats evokes a stronger aspermatogenic activity than does the injection of BS-RNase. Epididymides exposed to ONC lose mass and all sperm. Testicular tissue is gradually colonized by immunite and fibrocytic cells. Yet, Leydig cells are always present and functional in the ligamented parts of testicles injected with ONC or BS-RNase. ONC is likewise more toxic to mouse embryos than is BS-RNase, both in vitro and in vivo. The antiproliferative effect of ONC on human tumor cell line ML-2 and lymphocytes in a mixed lymphocyte culture is also more pronounced than is that of BS-RNase. The number of granulocyte-macrophage colony-forming units is repressed almost completely by ONC, whereas a five-fold higher dose of BS-RNase does not cause substantial inhibition. In mice, ONC is less immunogenic than BS-RNase but more immunogenic than RNase A. Together, these data indicate that ONC is a pluripotent cytotoxin, and serves as the benchmark with which to gauge the cytotoxicity of other ribonucleases.     

A role for the intersubunit disulfides of seminal RNase in the mechanism of its antitumor action.

The dimeric structure of seminal ribonuclease (BS-RNase) is maintained by noncovalent interactions and by two intersubunit disulfide bridges. Another unusual feature of this enzyme is its antitumour action, consisting in a cytotoxic activity selective for malignant cells. This cytotoxic action is exerted when the protein reaches the cytosol of the affected cells, where it degrades ribosomal RNA, thus blocking protein synthesis and leading cells to death. The current model proposed for the mechanism of antitumour action of BS-RNase is based on the ability of the protein to resist the neutralizing action of the cytosolic RNase inhibitor, a resistance due to the dimeric structure of the enzyme. Monomeric RNases, and monomeric derivatives of BS-RNase, are strongly bound by the inhibitor and inactive as antitumor agents. Here we report on monomeric derivatives of BS-RNase that, although strongly inhibited by the cytosolic RNase inhibitor, are cytotoxic towards malignant cells. These monomers are produced by reductive cleavage of the intersubunit disulfides of the native, dimeric protein followed by linking the exposed sulfhydryls to small thiols through formation of mixed disulfides. We found that sulfhydryls from cell monolayers and cell membranes can attack these mixed disulfides in the monomeric derivatives, and reconstitute, through sulfhydryl-disulfide interchange reactions, the native dimeric protein, which is internalized as such, and displays its antitumour action.     

Membrane binding of the colicin E1 channel: activity requires an electrostatic interaction of intermediate magnitude.

In vitro channel activity of the C-terminal colicin E1 channel polypeptide under conditions of variable electrostatic interaction with synthetic lipid membranes showed distinct maxima with respect to pH and membrane surface potential. The membrane binding energy was determined from fluorescence quenching of the intrinsic tryptophans of the channel polypeptide by liposomes containing N-trinitrophenyl-phosphatidylethanolamine. Maximum in vitro colicin channel activity correlated with an intermediate magnitude of the electrostatic interaction. For conditions associated with maximum activity (40% anionic lipid, I = 0.12 M, pH 4.0), the free energy of binding was delta G approximately -9 kcal/mol, with nonelectrostatic and electrostatic components, delta Gnel approximately -5 kcal/mol and delta Gel approximately -4 kcal/mol, and an effective binding charge of +7 at pH 4.0. Binding of the channel polypeptide to negative membranes at pH 8 is minimal, whereas initial binding at pH 4 followed by a shift to pH 8 causes only 3-10% reversal of binding, implying that it is kinetically trapped, probably by a hydrophobic interaction. It was inferred that membrane binding and insertion involves an initial electrostatic interaction responsible for concentration and binding to the membrane surface. This is followed by insertion into the bilayer driven by hydrophobic forces, which are countered in the case of excessive electrostatic binding.     

Lipid demixing and protein-protein interactions in the adsorption of charged proteins on mixed membranes

The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic.     

Engineering the refolding pathway and the quaternary structure of seminal ribonuclease by newly introduced disulfide bridges

Seminal RNase (BS-RNase), a ribonuclease from bovine seminal vesicles, is a homodimeric enzyme with a strong cytotoxic activity selective for tumor cells. It displays the unusual structural feature of existing in solution as an equilibrium mixture of two quaternary isoforms. The major one is characterized by the swap between subunits of their N-terminal ends, whereas the minor isoform shows no swap. The tendency of the two isolated isoforms to interconvert into each other has so far made it difficult to attribute the functional properties of BS-RNase to either isoform. Herein, molecular modeling and site-directed mutagenesis were used to engineer the refolding pathway of BS-RNase and obtain a stable variant of its non-swapping isoform. The protein was engineered with two extra disulfide bridges linking the N-terminal helix of each subunit to the main body of the same subunit. Purified as an active enzyme, the BS-RNase variant was found to be very resistant to thermal denaturation. Its functional characterization revealed that the lack of swapping has a negative effect on the cytotoxic activity of BS-RNase.     

The Gbetagamma dimer drives the interaction of heterotrimeric Gi proteins with nonlamellar membrane structures

Heterotrimeric G proteins are peripheral membrane proteins that propagate signals from membrane receptors to regulatory proteins localized in distinct cellular compartments. To facilitate signal amplification, G proteins are in molar excess with respect to G protein-coupled receptors. Because G proteins are capable of translocating from membrane to cytosol, protein-lipid interactions play a crucial role in signal transduction. Here, we studied the binding of heterotrimeric G proteins (Galphabetagamma) to model membranes (liposomes) and that of the entities formed upon receptor-mediated activation (Galpha and Gbetagamma). The model membranes used were composed of defined membrane lipids capable of organizing into either lamellar or nonlamellar (hexagonal H(II)) membrane structures. We demonstrated that although heterotrimeric G(i) proteins and Gbetagamma dimers can bind to lipid bilayers of phosphatidylcholine, their binding to membranes was markedly and significantly enhanced by the presence of nonlamellar phases of phosphatidylethanolamine. Conversely, activated G protein alpha subunits showed an opposite membrane binding behavior with a marked preference for lamellar membranes. These results have important consequences in cell signaling. First, the binding characteristics of the Gbetagamma dimer account for the lipid binding behavior and the cellular localization of heterotrimeric G proteins. Second, the distinct protein-lipid interactions of heterotrimeric G proteins, Gbetagamma dimers, and Galpha subunits with membrane lipids explain, in part, their different cellular mobilizations during signaling upon receptor activation. Finally, their differential interactions with lipids suggest an active role of the membrane lipid secondary structure in the propagation of signals through G protein-coupled receptors.     

Effects of proteins on thermotropic phase transitions of phospholipid membranes.

A variety of proteins have been studied for their ability to interact and alter the thermotropic properties of phospholipid bilayer membranes as detected by differential scanning calorimeter. The proteins studied included: basic myelin protein (A1 protein), cytochrome c, major apoprotein of myelin proteolipid (N-2 apoprotein), gramicidin A, polylysine, ribonuclease and hemoglobin. The lipids used for the interactions were dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol. The interactions were grouped in three catagories each having very different effects on the phospholipid phase transition from solid to liquid crystalline. The calorimetric studies were also correlated with data from vesicle permeability and monolayer expansion. Ribonuclease and polylysine which exemplify group 1 interactions, show strong dependence on electrostatic binding. Their effects on lipid bilayers include an increase in the enthalpy of transition (deltaH) accompanied by either an increase or no change in the temperature of transition (Tc). In addition, they show minimal effects on vesicle permeability and monolayer expansion. It was concluded that these interactions represent simple surface binding of the protein on the lipid bilayer without penetration into the hydrocarbon region. Cytochrome c and A1 protein, which exemplify group 2 interactions, also show a strong dependence on the presence of net negative charges on the lipid bilayers for their binding. In contrast to the first group, however, they induce a drastic decrease in both Tc and deltaH of the lipid phase transition. Furthermore, they induce a large increase in the permeability of vesicles and a substantial expansion in area of closely packed monolayers at the air-water interface. It was concluded that group 2 interactions represent surface binding followed by partial penetration and/or deformation of the bilayer. Group 3 interactions, shown by proteolipid apoprotein and gramicidin A, were primarily non-polar in character, not requiring electrostatic charges and not inhibited by salt and pH changes. They had no appreciable effect on the Tc but did induce a linear decrease in the magnitude of the deltaH, proportional to the percentage of protein by weight. Membranes containing 50% proteolipid protein still exhibited a thermotropic transition with a deltaH one half that of the pure lipid, and only a small diminution of the size of the cooperative unit. It was concluded that in this case the protein was embedded within the bilayer, associating with a limited number of molecules via non-polar interactions, while the rest of the bilayer was largely unperturbed.     

Comparison of VILIP-1 and VILIP-3 Binding to Phospholipid Monolayers

The neuronal calcium sensor proteins Visinin-like Proteins 1 (VILIP-1) and 3 (VILIP-3) are effectors of guanylyl cyclase and acetyl choline receptors, and transduce calcium signals in the brain. The “calcium-myristoyl” switch, which involves a post-translationally added myristoyl moiety and calcium binding, is thought to regulate their membrane binding capacity and therefore, play a critical role in their mechanism of action. In the present study, we investigated the effect of membrane composition and solvent conditions on the membrane binding mechanisms of both VILIPs using lipid monolayers at the air/buffer interface. Results based on comparison of the adsorption kinetics of the myristoylated and non-myristoylated proteins confirm the pivotal role of calcium and the exposed myristol moiety for sustaining the membrane-bound state of both VILIPs. However, we also observed binding of both VILIP proteins in the absence of calcium and/or myristoyl conjugation. We propose a two-stage membrane binding mechanism for VILIP-1 and VILIP-3 whereby the proteins are initially attracted to the membrane surface by electrostatic interactions and possibly by specific interactions with highly negatively charged lipids head groups. The extrusion of the conjugated myristoyl group, and the subsequent anchoring in the membrane constitutes the second stage of the binding mechanism, and ensures the sustained membrane-bound form of these proteins.     

Surface features of the lipid droplet mediate perilipin 2 localization

All eukaryotic organisms store excess lipid in intracellular lipid droplets. These dynamic structures are associated with and regulated by numerous proteins. Perilipin 2, an abundant protein on most lipid droplets, promotes neutral lipid accumulation in lipid droplets. However, the mechanism by which perilipin 2 binds to and remains anchored on the lipid droplet surface is unknown. Here we identify features of the lipid droplet surface that influence perilipin 2 localization. We show that perilipin 2 binding to the lipid droplet surface requires both hydrophobic and electrostatic interactions. Reagents that disrupt these interactions also decrease binding. Moreover, perilipin 2 binding does not depend on other lipid droplet-associated proteins but is influenced by the lipid composition of the surface. Perilipin 2 binds to synthetic vesicles composed of dioleoylphosphatidylcholine, a phospholipid with unsaturated acyl chains. Decreasing the temperature of the binding reaction, or introducing phospholipids with saturated acyl chains, decreases binding. We therefore demonstrate a role for surface lipids and acyl chain packing in perilipin 2 binding to lipid droplets. The ability of the lipid droplet phospholipid composition to impact protein binding may link changes in nutrient availability to lipid droplet homeostasis.     

The Bilayer Collective Properties Govern the Interaction of an HIV-1 Antibody with the Viral Membrane

Efficient engagement with the envelope glycoprotein membrane-proximal external region (MPER) results in robust blocking of viral infection by a class of broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV). Developing an accommodation surface that engages with the viral lipid envelope appears to correlate with the neutralizing potency displayed by these bnAbs. The nature of the interactions established between the antibody and the lipid is nonetheless a matter of debate, with some authors arguing that anti-MPER specificity arises only under pathological conditions in autoantibodies endowed with stereospecific binding sites for phospholipids. However, bnAb-lipid interactions are often studied in systems that do not fully preserve the biophysical properties of lipid bilayers, and therefore, questions on binding specificity and the effect of collective membrane properties on the interaction are still open. Here, to evaluate the specificity of lipid interactions of an anti-MPER bnAb (4E10) in an intact membrane context, we determine quantitatively its association with lipid bilayers by means of scanning fluorescence correlation spectroscopy and all-atom molecular dynamic simulations. Our data support that 4E10 establishes electrostatic and hydrophobic interactions with the viral membrane surface and that the collective physical properties of the lipid bilayer influence 4E10 dynamics therein. We conclude that establishment of peripheral, nonspecific electrostatic interactions with the viral membrane through accommodation surfaces may assist high-affinity binding of HIV-1 MPER epitope at membrane interfaces. These findings highlight the importance of considering antibody-lipid interactions in the design of antibody-based anti-HIV strategies.     

Ribonucleases and their antitumor activity

The antitumor effect of ribonucleases was studied with animal ribonucleolytic enzymes, bovine pancreatic RNase A, bovine seminal RNase (BS-RNase), onconase and angiogenin. While bovine pancreatic RNase A exerts a minor antitumor effect, BS-RNase and onconase exert significant effects. Angiogenin, as RNase, works in an opposite way, it initiates vascularization of tumors and subsequent tumor growth. Ribonunclease inhibitors are not able to inhibit the antitumor effectiveness of BS-RNase or onconase. However, they do so in the case of pancreatic RNases. Conjugation of BS-RNase with antibodies against tumor antigens (preparation of immunotoxins) like the conjugation of the enzyme with polymers enhances the antitumor activity of the ribonuclease. After conjugation with polymers, the half-life of BS-RNase in blood is extended and its immunogenicity reduced. Recombinant RNases have the same functional activity as the native enzymes. The synthetic genes have also been modified, some of them with gene sequences typical for the BS-RNase parts. Recent experimental efforts are directed to the preparation of ‘humanized antitumor ribonuclease’ that would be structurally similar to human enzyme with minimal immunogenicity and side effects. The angiogenesis of tumors is attempted to be minimized by specific antibodies or anti-angiogenic substances.     

The role of electrostatic interactions in the regulation of the membrane association of G protein beta gamma heterodimers

In this paper we report calculations of electrostatic interactions between the transducin (G(t)) betagamma heterodimer (G(t)betagamma) and phospholipid membranes. Although membrane association of G(t)betagamma is due primarily to the hydrophobic penetration into the membrane interior of a farnesyl chain attached to the gamma subunit, structural studies have revealed that there is a prominent patch of basic residues on the surface of the beta subunit surrounding the site of farnesylation that is exposed upon dissociation from the G(t)alpha subunit. Moreover, phosducin, which produces dissociation of G(t)betagamma from membranes, interacts directly with G(t)betagamma and introduces a cluster of acidic residues into this region. The calculations, which are based on the finite difference Poisson-Boltzmann method, account for a number of experimental observations and suggest that charged residues play a role in mediating protein-membrane interactions. Specifically, the calculations predict the following. 1) Favorable electrostatic interactions enhance the membrane partitioning due to the farnesyl group by an order of magnitude although G(t)betagamma has a large net negative charge (-12). 2) This electrostatic attraction positions G(t)betagamma so that residues implicated in mediating the interaction of G(t)betagamma with its membrane-bound effectors are close to the membrane surface. 3) The binding of phosducin to G(t)betagamma diminishes the membrane partitioning of G(t)betagamma by an order of magnitude. 4) Lowering the ionic strength of the solution converts the electrostatic attraction into a repulsion. Sequence analysis and homology model building suggest that our conclusions may be generalized to other Gbetagamma and phosducin isoforms as well.     

Structural basis of the membrane-targeting and unmasking mechanisms of the radixin FERM domain

Radixin is a member of the ezrin/radixin/moesin (ERM) family of proteins, which play a role in the formation of the membrane-associated cytoskeleton by linking actin filaments and adhesion proteins. This cross-linking activity is regulated by phosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PIP2) in the downstream of the small G protein Rho. The X-ray crystal structures of the radixin FERM domain, which is responsible for membrane binding, and its complex with inositol-(1,4, 5)-trisphosphate (IP3) have been determined. The domain consists of three subdomains featuring a ubiquitin-like fold, a four-helix bundle and a phosphotyrosine-binding-like domain, respectively. These subdomains are organized by intimate interdomain interactions to form characteristic grooves and clefts. One such groove is negatively charged and so is thought to interact with basic juxta-membrane regions of adhesion proteins. IP3 binds a basic cleft that is distinct from those of pleckstrin homology domains and is located on a positively charged flat molecular surface, suggesting an electrostatic mechanism of plasma membrane targeting. Based on the structural changes associated with IP3 binding, a possible unmasking mechanism of ERM proteins by PIP2 is proposed.     

Interaction of charged and uncharged calcium channel antagonists with phospholipid membranes. Binding equilibrium, binding enthalpy, and membrane location.

The membrane location and the binding mechanism of two Ca2+ channel antagonists, amlodipine and nimodipine, in pure lipid membranes were investigated with deuterium and phosphorus-31 nuclear magnetic resonance, with thermodynamic methods such as high-sensitivity titration calorimetry, and by measuring the membrane surface charge via the zeta-potential. The two drugs exhibit quite different physical-chemical properties. The noncharged nimodipine is strongly hydrophobic, and selective deuteration of the lipid membrane reveals a homogeneous distribution of nimodipine across the whole hydrocarbon layer, but no interaction at the lipid headgroup level. The membrane behavior of the amiphiphilic amlodipine (electric charge z = +1) is distinctly more complex. Deuterium magnetic resonance demonstrates that amlodipine adopts a well-defined position in the bilayer membrane. In particular, the charged ethanolamine side group of amlodipine is located near the water-lipid interface, interacting with the dipoles of the headgroup region according to a nonspecific, electrostatic mechanism and inducing a reorientation of the phosphocholine dipoles toward the water phase. At the level of the hydrocarbon segment, the nonpolar ring system of amlodipine interacts specifically with the cis double bond of the membrane lipid, forming a weak association complex. With increasing amlodipine concentration the deuterium signal of the cis double bond gradually loses intensity, a phenomenon previously observed only in related studies on protein-lipid interactions. The binding equilibrium of amlodipine to phosphatidylcholine membranes was studied by measuring the electrophoretic mobility of lipid vesicles and with a centrifugation assay. Hydrophobic interactions of the nonpolar ring systems and electrostatic repulsions at the membrane surface contribute to the binding energy.(ABSTRACT TRUNCATED AT 250 WORDS)