Molecular dynamics simulation of cation-phospholipid clustering in phospholipid bilayers: possible role in stalk formation during membrane fusion

In this study, we performed all-atom long-timescale molecular dynamics simulations of phospholipid bilayers incorporating three different proportions of negatively charged lipids in the presence of K(+), Mg(2+), and Ca(2+) ions to systemically determine how membrane properties are affected by cations and lipid compositions. Our simulations revealed that the binding affinity of Ca(2+) ions with lipids is significantly stronger than that of K(+) and Mg(2+) ions, regardless of the composition of the lipid bilayer. The binding of Ca(2+) ions to the lipids resulted in bilayers having smaller lateral areas, greater thicknesses, greater order, and slower rotation of their lipid head groups, relative to those of corresponding K(+)- and Mg(2+)-containing systems. The Ca(2+) ions bind preferentially to the phosphate groups of the lipids. The complexes formed between the cations and the lipids further assembled to form various multiple-cation-centered clusters in the presence of anionic lipids and at higher ionic strength-most notably for Ca(2+). The formation of cation-lipid complexes and clusters dehydrated and neutralized the anionic lipids, creating a more-hydrophobic environment suitable for membrane aggregation. We propose that the formation of Ca(2+)-phospholipid clusters across apposed lipid bilayers can work as a "cation glue" to adhere apposed membranes together, providing an adequate configuration for stalk formation during membrane fusion.     

Activating and deactivating roles of lipid bilayers on the Ca(2+)-ATPase/phospholamban complex

The physicochemical properties of the lipid bilayer shape the structure and topology of membrane proteins and regulate their biological function. Here, we investigated the functional effects of various lipid bilayer compositions on the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) in the presence and absence of its endogenous regulator, phospholamban (PLN). In the cardiac muscle, SERCA hydrolyzes one ATP molecule to translocate two Ca(2+) ions into the SR membrane per enzymatic cycle. Unphosphorylated PLN reduces SERCA's affinity for Ca(2+) and affects the enzymatic turnover. We varied bilayer thickness, headgroup, and fluidity and found that both the maximal velocity (V(max)) of the enzyme and its apparent affinity for Ca(2+) (K(Ca)) are strongly affected. Our results show that (a) SERCA's V(max) has a biphasic dependence on bilayer thickness, reaching maximum activity with 22-carbon lipid chain length, (b) phosphatidylethanolamine (PE) and phosphatidylserine (PS) increase Ca(2+) affinity, and (c) monounsaturated lipids afford higher SERCA V(max) and Ca(2+) affinity than diunsaturated lipids. The presence of PLN removes the activating effect of PE and shifts SERCA's activity profile, with a maximal activity reached in bilayers with 20-carbon lipid chain length. Our results in synthetic lipid systems compare well with those carried out in native SR lipids. Importantly, we found that specific membrane compositions closely reproduce PLN effects (V(max) and K(Ca)) found in living cells, reconciling an ongoing controversy regarding the regulatory role of PLN on SERCA function. Taken with the physiological changes occurring in the SR membrane composition, these studies underscore a possible allosteric role of the lipid bilayers on the SERCA/PLN complex.     

Synaptotagmin 1 modulates lipid acyl chain order in lipid bilayers by demixing phosphatidylserine

Synaptotagmin 1 (syt1) functions as the Ca(2+) sensor in neuronal exocytosis, and it has been proposed to act by modulating lipid bilayer curvature. Here we examine the effect of the two C2 domains (C2A and C2B) of syt1 on membrane lipid order and lateral organization. In mixtures of phosphatidylcholine and phosphatidylserine (PS), attenuated total internal reflection Fourier transform infrared spectroscopy indicates that a fragment containing both domains (C2AB) or C2B alone disorders the lipid acyl chains, whereas the C2A domain has little effect upon chain order. Two observations suggest that these changes reflect a demixing of PS. First, the changes in acyl chain order are reversed at higher protein concentration; second, selective lipid deuteration demonstrates that the changes in lipid order are associated only with the PS component of the bilayer. Independent evidence for lipid demixing is obtained from fluorescence self-quenching of labeled lipid and from natural abundance (13)C NMR, where heteronuclear single quantum correlation spectra reveal Ca(2+)-dependent chemical shift changes for PS, but not for phosphatidylcholine, in the presence of the syt1 C2 domains. The ability of syt1 to demix PS is observed in a range of lipid mixtures that includes cholesterol, phosphatidylethanolamine, and varied PS content. These data suggest that syt1 might facilitate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors)-mediated membrane fusion by phase separating PS, a process that is expected to locally buckle bilayers and disorder lipids due to the curvature tendencies of PS.     

Effect of calcium and magnesium on phosphatidylserine membranes: experiments and all-atomic simulations

It is known that phosphatidylserine (PS(-)) lipids have a very similar affinity for Ca(2+) and Mg(2+) cations, as revealed by electrokinetic and stability experiments. However, despite this similar affinity, experimental evidence shows that the presence of Ca(2+) or Mg(2+) induces very different aggregation behavior for PS(-) liposomes as characterized by their fractal dimensions. Also, turbidity measurements confirm substantial differences in aggregation behavior depending on the presence of Ca(2+) or Mg(2+) cations. These puzzling results suggest that although these two cations have a similar affinity for PS(-) lipids, they induce substantial structural differences in lipid bilayers containing each of these cations. In other words, these cations have strong ion-specific effects on the structure of PS(-) membranes. This interpretation is supported by all-atomic molecular-dynamics simulations showing that Ca(2+) and Mg(2+) cations have different binding sites and induce different membrane hydration. We show that although both ions are incorporated deep into the hydrophilic region of the membrane, they have different positions and configurations at the membrane. Absorbed Ca(2+) cations present a peak at a distance ~2 nm from the center of the lipid bilayer, and their most probable binding configuration involves two oxygen atoms from each of the charged moieties of the PS molecule (phosphate and carboxyl groups). In contrast, the distribution of absorbed Mg(2+) cations has two different peaks, located a few angstroms before and after the Ca(2+) peak. The most probable configurations (corresponding to these two peaks) involve binding to two oxygen atoms from carboxyl groups (the most superficial binding peak) or two oxygen atoms from phosphate groups (the most internal peak). Moreover, simulations also show differences in the hydration structure of the membrane: we obtained a hydration of 7.5 and 9 water molecules per lipid in simulations with Ca(2+) and Mg(2+), respectively.     

Structural effects of neutral lipids on divalent cation-induced interactions of phosphatidylserine-containing bilayers.

Ca2+ is known to induce the adhesion and collapse of phosphatidylserine (PS) bilayers into dehydrated multilamellar structures. The aim of this study was to examine how that interaction and the resultant structures might be modified by neutral lipid species. A combination of rapid mixing, x-ray diffraction, thin-layer chromatography, density gradient centrifugation, and freeze-fracture electron microscopy was used in conjunction with osmotic stress techniques to characterize the structures formed by the Ca(2+)-induced interaction of multilamellar liposomes and of large unilamellar vesicles. The results showed that dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine at concentrations of up to approximately 30 mol % are accommodated in a single dehydrated multilamellar structure. Similar results were obtained using mixed PS species isolated from bovine brain. Principally, the data indicate that neutral lipid is both dehydrated during the rapid collapse process of Ca(PS)2 formation and accommodated within this dehydrated structure. The large energies available on formation of the Ca(PS)2 bilayers contribute to the dehydration of neighboring neutral lipids that likely form continuous bilayers with them. Higher concentrations of these neutral lipids modify Ca(2+)-induced bilayer interactions, leading to progressively weaker interactions, larger bilayer separations, and in some cases separation into two structures; phosphatidylethanolamine species favoring nonbilayer structures tended to promote such separation at lower concentrations than bilayer lipids.     

The effect of calcium on the properties of charged phospholipid bilayers

We have performed molecular dynamics simulations to investigate the structure and dynamics of charged bilayers as well as the distribution of counterions at the bilayer interface. For this, we have considered the negatively charged di-myristoyl-phosphatidyl-glycerol (DMPG) and di-myristoyl-phosphatidyl-serine (DMPS) bilayers as well as a protonated di-myristoyl-phosphatidyl-serine (DMPSH) bilayer. We were particularly interested in calcium ions due to their important role in biological systems. Simulations performed in the presence of calcium ions (DMPG, DMPS) or sodium ions (DMPS) were run for 45-60 ns. Simulation results for DMPG are compared with fluorescence measurements. The average areas per molecule were 47.4+/-0.5 A2 (DMPG with calcium), 47.3+/-0.5 A2 (DMPS with calcium), 51.3+/-1.0 A2 (DMPS with sodium) and 45.3+/-0.5 A2 (DMPSH). The structure of the negatively charged lipids is significantly affected by the counterions, where calcium ions have a more pronounced effect than sodium ions. Calcium ions were found to be tightly bound to the anionic groups of the lipid molecules and as such appear to constitute an integral part of the membrane interface on nanoseconds time scales. In contrast to sodium ions, calcium ions are localised in a narrow (approximately 10 A) band around the phosphate group. The interaction of calcium with the lipid molecules enhances the molecular packing of the PG and PS lipids. This observation is in good agreement with emission spectra of the membrane partitioning probe Laurdan in DMPG multilamellar vesicles that indicate an increase in the ordering of the DMPG bilayer due to the presence of calcium. Our results indicate that calcium ions, which often function as a second messengers in living cells have a pronounced effect on membrane structures, which may have implications during signal transduction events.     

Solid-state NMR investigation of the selective disruption of lipid membranes by protegrin-1

The interaction of a beta-hairpin antimicrobial peptide, protegrin-1 (PG-1), with various lipid membranes is investigated by (31)P, (2)H, and (13)C solid-state NMR. Mixed lipid bilayers containing anionic lipids and cholesterol are used to mimic the bacterial and mammalian cell membranes, respectively. (31)P and (2)H spectra of macroscopically oriented samples show that PG-1 induces the formation of an isotropic phase in anionic bilayers containing phosphatidylglycerol. Two-dimensional (31)P exchange experiments indicate that these isotropic lipids are significantly separate from the residual oriented lamellar bilayers, ruling out toroidal pores as the cause for the isotropic signal. (1)H spin diffusion experiments show that PG-1 is not exclusively bound to the isotropic phase but is also present in the residual oriented lamellar bilayers. This dynamic and morphological heterogeneity of the anionic membranes induced by PG-1 is supported by the fact that (13)C T(2) relaxation times measured under cross polarization and direct polarization conditions differ significantly. In contrast to the anionic membrane, the zwitterionic phosphatidylcholine (PC) membrane does not form an isotropic phase in the presence of PG-1 but shows significant orientational disorder. The addition of cholesterol to the PC bilayer significantly reduces this orientational disorder. The (13)C T(2) relaxation times of the PC lipids in the presence of both cholesterol and PG-1 suggest that the peptide may decrease the dynamic heterogeneity of the cholesterol-containing membrane. The observed selective interaction of PG-1 with different lipid membranes is consistent with its biological function and may be caused by its strong cationic and amphipathic structure.     

Synaptotagmin perturbs the structure of phospholipid bilayers

Synaptotagmin I (syt), an integral protein of the synaptic vesicle membrane, is believed to act as a Ca2+ sensor for neuronal exocytosis. Syt's cytoplasmic domain consists largely of two C2 domains, C2A and C2B. In response to Ca2+ binding, the C2 domains interact with membranes, becoming partially embedded in the lipid bilayer. We have imaged syt C2AB in association with lipid bilayers under fluid, using AFM. As expected, binding of C2AB to bilayers required both an anionic phospholipid [phosphatidylserine (PS)] and Ca2+. C2AB associated with bilayers in the form of aggregates of varying stoichiometries, and aggregate size increased with an increase in PS content. Repeated scanning of bilayers revealed that as C2AB dissociated it left behind residual indentations in the bilayer. The mean depth of these identations was 1.81 nm, indicating that they did not span the bilayer. Individual C2 domains (C2A and C2B) also formed aggregates and produced bilayer indentations. Binding of C2AB to bilayers and the formation of indentations were significantly compromised by mutations that interfere with binding of Ca2+ to syt or reduce the positive charge on the surface of C2B. We propose that bilayer perturbation by syt might be significant with respect to its ability to promote membrane fusion.     

Finding a Needle in a Haystack: The Role of Electrostatics in Target Lipid Recognition by PH Domains

Interactions between protein domains and lipid molecules play key roles in controlling cell membrane signalling and trafficking. The pleckstrin homology (PH) domain is one of the most widespread, binding specifically to phosphatidylinositol phosphates (PIPs) in cell membranes. PH domains must locate specific PIPs in the presence of a background of approximately 20% anionic lipids within the cytoplasmic leaflet of the plasma membrane. We investigate the mechanism of such recognition via a multiscale procedure combining Brownian dynamics (BD) and molecular dynamics (MD) simulations of the GRP1 PH domain interacting with phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)). The interaction of GRP1-PH with PI(3,4,5)P(3) in a zwitterionic bilayer is compared with the interaction in bilayers containing different levels of anionic 'decoy' lipids. BD simulations reveal both translational and orientational electrostatic steering of the PH domain towards the PI(3,4,5)P(3)-containing anionic bilayer surface. There is a payoff between non-PIP anionic lipids attracting the PH domain to the bilayer surface in a favourable orientation and their role as 'decoys', disrupting the interaction of GRP1-PH with the PI(3,4,5)P(3) molecule. Significantly, approximately 20% anionic lipid in the cytoplasmic leaflet of the bilayer is nearly optimal to both enhance orientational steering and to localise GRP1-PH proximal to the surface of the membrane without sacrificing its ability to locate PI(3,4,5)P(3) within the bilayer plane. Subsequent MD simulations reveal binding to PI(3,4,5)P(3), forming protein-phosphate contacts comparable to those in X-ray structures. These studies demonstrate a computational framework which addresses lipid recognition within a cell membrane environment, offering a link between structural and cell biological characterisation.     

Effect of PIP2 binding on the membrane docking geometry of PKC alpha C2 domain: an EPR site-directed spin-labeling and relaxation study

Protein kinase C isoform alpha (PKCalpha) is a ubiquitous, conventional PKC enzyme that possesses a conserved C2 domain. Upon activation by cytoplasmic Ca (2+) ions, the C2 domain specifically binds to the plasma membrane inner leaflet where it recognizes the target lipids phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PIP 2). The membrane penetration depth and docking angle of the membrane-associated C2 domain is not well understood. The present study employs EPR site-directed spin labeling and relaxation methods to generate a medium-resolution model of the PKCalpha C2 domain docked to a membrane of lipid composition similar to the plasma membrane inner leaflet. The approach measures EPR depth parameters for 10 function-retaining spin labels coupled to the C2 domain, and for spin labels coupled to depth calibration molecules. The resulting depth parameters, together with the known structure of the free C2 domain, provide a sufficient number of constraints to define two membrane docking geometries for C2 domain bound to physiological membranes lacking or containing PIP 2, respectively. In both the absence and presence of PIP 2, the two bound Ca (2+) ions of the C2 domain lie near the anionic phosphate plane in the headgroup region, consistent with the known ability of the Ca (2+) and membrane-binding loops (CMBLs) to bind the headgroup of the PS target lipid. In the absence of PIP 2, the polybasic lipid binding site on the beta3-beta4 hairpin is occupied with PS, but in the presence of PIP 2 this larger, higher affinity target lipid competitively displaces PS and causes the long axis of the domain to tilt 40 +/- 10 degrees toward the bilayer normal. The ability of the beta3-beta4 hairpin site to bind PS as well as PIP 2 extends the lifetime of the membrane-docked state and is predicted to enhance the kinase turnover number of PKCalpha during a single membrane docking event. In principle, PIP 2-induced tilting of the C2 domain could modulate the activity of membrane-docked PKCalpha as it diffuses between membrane regions with different local PS and PIP 2 concentrations. Finally, the results demonstrate that EPR relaxation methods are sufficiently sensitive to detect signaling-induced changes in the membrane docking geometries of peripheral membrane proteins.     

Trans interactions between galactosylceramide and cerebroside sulfate across apposed bilayers

The two glycosphingolipids galactosylceramide (GalC) and its sulfated form, cerebroside sulfate (CBS), are present at high concentrations in the multilayered myelin sheath and are involved in carbohydrate-carbohydrate interactions between the lipid headgroups. In order to study the structure of the complex of these two glycolipids by Fourier transform infrared (FTIR) spectroscopy, GalC dispersions were combined with CBS dispersions in the presence and absence of Ca(2+). The FTIR spectra indicated that a strong interaction occurred between these glycolipids even in the absence of Ca(2+). The interaction resulted in dehydration of the sulfate, changes in the intermolecular hydrogen bonding interactions of the sugar and other oxygens, decreased intermolecular hydrogen bonding of the amide C==O of GalC and dehydration of the amide region of one or both of the lipids in the mixture, and disordering of the hydrocarbon chains of both lipids. The spectra also show that Ca(2+) interacts with the sulfate of CBS. Although they do not reveal which other groups of CBS and GalC interact with Ca(2+) or which groups participate in the interaction between the two lipids, they do show that the sulfate is not directly involved in interaction with GalC, since it can still bind to Ca(2+) in the mixture. The interaction between these two lipids could be either a lateral cis interaction in the same bilayer or a trans interaction between apposed bilayers. The type of interaction between the lipids, cis or trans, was investigated using fluorescent and spin-label probes and anti-glycolipid antibodies. The results confirmed a strong interaction between the GalC and the CBS microstructures. They suggested further that this interaction caused the CBS microstructures to be disrupted so that CBS formed a single bilayer around the GalC multilayered microstructures, thus sequestering GalC from the external aqueous phase. Thus the CBS and GalC interacted via a trans interaction across apposed bilayers, which resulted in dehydration of the headgroup and interface region of both lipid bilayers. The strong interaction between these lipids may be involved in stabilization of the myelin sheath.     

Experimental and theoretical studies of emodin interacting with a lipid bilayer of DMPC

Emodin is one of the most abundant anthraquinone derivatives found in nature. It is the active principle of some traditional herbal medicines with known biological activities. In this work, we combined experimental and theoretical studies to reveal information about location, orientation, interaction and perturbing effects of Emodin on lipid bilayers, where we have taken into account the neutral form of the Emodin (EMH) and its anionic/deprotonated form (EM^?). Using both UV/Visible spectrophotometric techniques and molecular dynamics (MD) simulations, we showed that both EMH and EM^? are located in a lipid membrane. Additionally, using MD simulations, we revealed that both forms of Emodin are very close to glycerol groups of the lipid molecules, with the EMH inserted more deeply into the bilayer and more disoriented relative to the normal of the membrane when compared with the EM^?, which is more exposed to interfacial water. Analysis of several structural properties of acyl chains of the lipids in a hydrated pure DMPC bilayer and in the presence of Emodin revealed that both EMH and EM^? affect the lipid bilayer, resulting in a remarkable disorder of the bilayer in the vicinity of the Emodin. However, the disorder caused by EMH is weaker than that caused by EM^?. Our results suggest that these disorders caused by Emodin might lead to distinct effects on lipid bilayers including its disruption which are reported in the literature.     

Distinct structural and adhesive roles of Ca2+ in membrane binding of blood coagulation factors

The GLA domain, a common membrane-anchoring domain of several serine protease coagulation factors, is a key element in membrane association and activation of these factors in a highly Ca2+-dependent manner. However, the critical role of Ca2+ ions in binding is only poorly understood. Here, we present the atomic model of a membrane-bound GLA domain by using MD simulations of the GLA domain of human factor VIIa and an anionic lipid bilayer. The binding is furnished through a complete insertion of the omega-loop into the membrane and through direct interactions of structurally bound Ca2+ ions and protein side chains with negative lipids. The model suggests that Ca2+ ions play two distinct roles in the process: the four inner Ca2+ ions are primarily responsible for optimal folding of the GLA domain for membrane insertion, whereas the outer Ca2+ ions anchor the protein to the membrane through direct contacts with lipids.     

Solid-state NMR investigation of the depth of insertion of protegrin-1 in lipid bilayers using paramagnetic Mn2+

The depth of insertion of an antimicrobial peptide, protegrin-1 (PG-1), in lipid bilayers is investigated using solid-state NMR. Paramagnetic Mn(2+) ions bind to the surface of lipid bilayers and induce distance-dependent dipolar relaxation of nuclear spins. By comparing the signal dephasing of the peptide with that of the lipids, whose segmental depths of insertion are known, we determined the depths of several residues of PG-1 in 1,2 dilauryl-sn-glycero-3-phosphotidylcholine (DLPC) bilayers. We found that residues G2 at the N-terminus and F12 at the beta-turn of the peptide reside near the membrane surface, whereas L5 and V16 are embedded in the acyl chain region. The depths increase in the order of G2 < F12 < L5 < V16. These intensity-dephasing results are confirmed by direct measurement of the paramagnetically enhanced (13)C transverse relaxation rates. The relative depths indicate that PG-1 is tilted from the bilayer normal, which is consistent with independent solid-state NMR measurements of PG-1 orientation in the same lipids (Yamaguchi et al., 2001). They also indicate that PG-1 is fully immersed in the lipid bilayer. However, a quantitative mismatch between the bilayer thickness and PG-1 length suggests a local thinning of the DLPC bilayer by 8-10 A. The depth sensitivity of this Mn(2+) dephasing technique is tunable with the Mn(2+) concentration to focus on different regions of the lipid bilayer.     

Solid-state NMR investigation of the selective perturbation of lipid bilayers by the cyclic antimicrobial peptide RTD-1

RTD-1 is a cyclic beta-hairpin antimicrobial peptide isolated from rhesus macaque leukocytes. Using (31)P, (2)H, (13)C, and (15)N solid-state NMR, we investigated the interaction of RTD-1 with lipid bilayers of different compositions. (31)P and (2)H NMR of uniaxially oriented membranes provided valuable information about how RTD-1 affects the static and dynamic disorder of the bilayer. Toward phosphatidylcholine (PC) bilayers, RTD-1 causes moderate orientational disorder independent of the bilayer thickness, suggesting that RTD-1 binds to the surface of PC bilayers without perturbing its hydrophobic core. Addition of cholesterol to the POPC membrane does not affect the orientational disorder. In contrast, binding of RTD-1 to anionic bilayers containing PC and phosphatidylglycerol lipids induces much greater orientational disorder without affecting the dynamic disorder of the membrane. These correlate with the selectivity of RTD-1 for anionic bacterial membranes as opposed to cholesterol-rich zwitterionic mammalian membranes. Line shape simulations indicate that RTD-1 induces the formation of micrometer-diameter lipid cylinders in anionic membranes. The curvature stress induced by RTD-1 may underlie the antimicrobial activity of RTD-1. (13)C and (15)N anisotropic chemical shifts of RTD-1 in oriented PC bilayers indicate that the peptide adopts a distribution of orientations relative to the magnetic field. This is most likely due to a small fraction of lipid cylinders that change the RTD-1 orientation with respect to the magnetic field. Membrane-bound RTD-1 exhibits narrow line widths in magic-angle spinning spectra, but the sideband intensities indicate rigid-limit anisotropies. These suggest that RTD-1 has a well-defined secondary structure and is likely aggregated in the membrane. These structural and dynamical features of RTD-1 differ significantly from those of PG-1, a related beta-hairpin antimicrobial peptide.     

Membrane-bound orientation and position of the synaptotagmin C2B domain determined by site-directed spin labeling

Site-directed spin labeling is used to determine the orientation and depth of insertion of the second C2 domain from synaptotagmin I (C2B) into membrane vesicles composed of phosphatidylcholine (PC) and phosphatidylserine (PS). EPR line shapes of spin-labeled mutants located with the Ca(2+)-binding loops of C2B broaden in the presence of Ca(2+) and PC/PS vesicles, indicating that these loops undergo a Ca(2+)-dependent insertion into the membrane interface. Power saturation of the EPR spectra provides a position for each spin-labeled site along the bilayer normal, and these EPR-derived distance constraints, along with a high-resolution structure of the C2B domain, are used to generate a model for the domain orientation and position at the membrane interface. Our data show that the isolated C2B domain from synaptotagmin I penetrates PC/PS membranes, and that the backbone of Ca(2+)-binding loops 1 and 3 is inserted below the level of a plane defined by the lipid phosphates. The side chains of several loop residues are within the bilayer interior, and both Ca(2+)-binding sites are positioned near a plane defined by the lipid phosphates. A Tb(3+)-based fluorescence assay is used to compare the membrane affinity of the C2B domain to that of the first synaptotagmin C2 domain (C2A). Both C2A and C2B bind PC/PS (75:25) membrane vesicles with a micromolar lipid affinity in the presence of metal ion. These results indicate that C2A and C2B have a similar membrane affinity and position when bound to PC/PS (75:25) membrane vesicles. EPR spectroscopy indicates that the C2B domain has different interactions with PC/PS membranes containing 1 mol % phosphatidylinositol 4,5-bisphosphate.     

Solid-state NMR investigations of peptide-lipid interaction and orientation of a beta-sheet antimicrobial peptide,protegrin

Protegrin-1 (PG-1) is a broad-spectrum beta-sheet antimicrobial peptide found in porcine leukocytes. The mechanism of action and the orientation of PG-1 in lipid bilayers are here investigated using (2)H, (31)P, (13)C, and (15)N solid-state NMR spectroscopy. (2)H spectra of mechanically aligned and chain-perdeuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers indicate that PG-1 at high concentrations destroys the orientational order of the aligned lamellar bilayer. The conformation of the lipid headgroups in the unoriented region is significantly altered, as seen from the (31)P spectra of POPC and the (2)H spectra of headgroup-deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine. These observations indicate that PG-1 disrupts microbial membranes by breaking the extended bilayer into smaller disks, where a significant fraction of lipids is located in the edges of the disks with a distribution of orientations. These edges allow the lipid bilayer to bend back on itself as in toroidal pores. Interestingly, this loss of bilayer orientation occurs only in long-chain lipids such as POPC and not in shorter chain lipids such as 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC). To understand the mode of binding of PG-1 to the lipid bilayer, we determined the orientation of PG-1 in DLPC bilayers. The (13)CO and (15)N chemical shifts of Val-16 labeled PG-1 indicate that the beta-strand axis is tilted by 55 degrees +/- 5 degrees from the bilayer normal while the normal of the beta-sheet plane is 48 degrees +/- 5 degrees from the bilayer normal. This orientation favors interaction of the hydrophobic backbone of the peptide with the hydrophobic core of the bilayer and positions the cationic Arg side chains to interact with the anionic phosphate groups. This is the first time that the orientation of a disulfide-stabilized beta-sheet membrane peptide has been determined by solid-state NMR.     

A solid-state NMR study of the phospholamban transmembrane domain: local structure and interactions with Ca(2+)-ATPase

The structure and dynamics of a double (13)C-labelled 24-residue synthetic peptide ([(13)C(2)]CAPLB(29-52)), corresponding to the membrane-spanning sequence of phospholamban (PLB), were examined using (13)C cross-polarisation magic-angle spinning (CP-MAS) NMR spectroscopy. CP-MAS spectra of [(13)C(2)]CAPLB(29-52) reconstituted into unsaturated lipid membranes indicated that the peptide was mobile at temperatures down to -50 degrees C. The NMR spectra showed that peptide motion became constrained in the presence of the SERCA1 isoform of Ca(2+)-ATPase, and chemical cross-linking experiments indicated that [(13)C(2)]CAPLB(29-52) and Ca(2+)-ATPase came into close contact with one another. These results together suggested that the peptide and the 110-kDa calcium pump were interacting in the membrane. Rotational resonance CP-MAS (13)C-(13)C distance measurements on [(13)C(2)]CAPLB(29-52) reconstituted into lipid bilayers confirmed that the sequence spanning Phe-32 and Ala-36 was alpha-helical, and that this structure was not disrupted by interaction with Ca(2+)-ATPase. These results support the finding that the transmembrane domain of PLB is partially responsible for regulation of Ca(2+) transport through interactions with cardiac muscle Ca(2+)-ATPase in the lipid bilayer, and also demonstrate the feasibility of performing structural measurements on PLB peptides when bound to their physiological target.     

Calorimetric study of the interaction of the C2 domains of classical protein kinase C isoenzymes with Ca2+ and phospholipids

The affinities of Ca(2+) and anionic lipid vesicles from the C2 domains of classical protein kinase C subfamily (alpha, betaII, and gamma) were studied using isothermal titration calorimetry (ITC). In addition, the thermal stability of these C2 domains in the presence of different ligand concentrations was analyzed using differential scanning calorimetry (DSC). These three closely related C2 domains bind Ca(2+) in a similar way, demonstrating the presence of two sets of sites. The first set of sites binds one Ca(2+) ion exothermically with similar high affinity for the three proteins (K(d) around 1 microM), while the second set of sites binds endothermically approximately two Ca(2+) ions with lower affinity, which varies for each C2 domain: 22.2 microM for the PKCalpha-C2 domain, 17.2 microM for the PKCbetaII-C2 domain, and 4.3 microM for the PKCgamma-C2 domain. In the absence of Ca(2+), the three C2 domains showed a weak interaction with vesicles containing anionic phospholipids. However, in the presence of a saturating Ca(2+) concentration, the C2 domains increased their affinities for the anionic lipid vesicles. In all cases, the C2 domains bound the vesicles exothermically and with similar affinities. A DSC thermal stability study of the C2 domains in the presence of Ca(2+) and anionic lipids provided further information about this protein-ligand interaction. The presence of increasing Ca(2+) concentrations was matched by an increase in the T(m) in all cases, which was even greater in the presence of anionic lipid vesicles. The extent of the change in T(m) differed for each C2 domain, reflecting the differing effect of the ligands bound during the protein stabilization. Denaturation of the C2 domains was irreversible both in the absence and in the presence of ligands, although the thermograms were not kinetically controlled. The dependence of the T(m) on the Ca(2+) concentration indicates that the protein stabilization observed by DSC primarily reflects the saturation by the cation of the low-affinity set of sites.     

The effect of calcium on the properties of charged phospholipid bilayers

We have performed molecular dynamics simulations to investigate the structure and dynamics of charged bilayers as well as the distribution of counterions at the bilayer interface. For this, we have considered the negatively charged di-myristoyl-phosphatidyl-glycerol (DMPG) and di-myristoyl-phosphatidyl-serine (DMPS) bilayers as well as a protonated di-myristoyl-phosphatidyl-serine (DMPSH) bilayer. We were particularly interested in calcium ions due to their important role in biological systems. Simulations performed in the presence of calcium ions (DMPG, DMPS) or sodium ions (DMPS) were run for 45-60 ns. Simulation results for DMPG are compared with fluorescence measurements. The average areas per molecule were 47.4 ± 0.5 Å² (DMPG with calcium), 47.3 ± 0.5 Å² (DMPS with calcium), 51.3 ± 1.0 Å² (DMPS with sodium) and 45.3 ± 0.5 Å² (DMPSH). The structure of the negatively charged lipids is significantly affected by the counterions, where calcium ions have a more pronounced effect than sodium ions. Calcium ions were found to be tightly bound to the anionic groups of the lipid molecules and as such appear to constitute an integral part of the membrane interface on nanoseconds time scales. In contrast to sodium ions, calcium ions are localised in a narrow (∼ 10 Å) band around the phosphate group. The interaction of calcium with the lipid molecules enhances the molecular packing of the PG and PS lipids. This observation is in good agreement with emission spectra of the membrane partitioning probe Laurdan in DMPG multilamellar vesicles that indicate an increase in the ordering of the DMPG bilayer due to the presence of calcium. Our results indicate that calcium ions, which often function as a second messengers in living cells have a pronounced effect on membrane structures, which may have implications during signal transduction events.