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The C2 domain of PKCα (C2α) induces fluorescence self-quenching of NBD-PS in the presence of Ca2+, which is interpreted as the demixing of phosphatidylserine from a mixture of this phospholipid with phosphatidylcholine. Self-quenching of NBD-PS was considerably increased when phosphatidylinositol-4,5-bisphosphate (PIP2) was present in the membrane. When PIP2 was the labeled phospholipid, in the form of TopFluor-PIP2, fluorescence self-quenching induced by the C2 domain was also observed, but this was dependent on the presence of phosphatidylserine. An independent indication of the phospholipid demixing effect given by the C2α domain was obtained by using 2H-NMR, since a shift of the transition temperature of deuterated phosphatidylcholine was observed as a consequence of the addition of the C2α domain, but only in the presence of PIP2. The demixing induced by the C2α domain may have a physiological significance since it means that the binding of PKCα to membranes is accompanied by the formation of domains enriched in activating lipids, like phosphatidylserine and PIP2. The formation of these domains may enhance the activation of the enzyme when it binds to membranes containing phosphatidylserine and PIP2.

The mKO is the monomeric version of Kusabira Orange, a GFP-like protein emitting bright orange fluorescence at 559 nm. This protein shows the characteristic beta-barrel motif typical of the fluorescent protein family which it belongs to, similar spectral properties to the tetrameric form and an exceptional photo-stability to pH changes. Here, we demonstrate that mKO in solution at physiological pH exhibits a secondary structure analogue to that of the crystal. Moreover, we describe the thermal unfolding, revealing an outstanding structural stability with a denaturation temperature close to 90 degrees C and identifying the existence of a thermodynamic intermediate. The denaturation process of mKO results to be absolutely irreversible because of the complete lost of the native structure and the consequent aggregation, while the presence of the intermediate state is most likely due to coexistence of two different species of mKO, with protonated and deprotonated chromophore respectively, that affects the fluorescence properties and the structural stability of the protein. (C) 2014 Elsevier B.V. All rights reserved.

Coenzyme Q (CoQ) is a component of the mitochondrial respiratory chain which carries out additional membrane functions, such as acting as an antioxidant. The location of CoQ in the membrane and the interaction with the phospholipid bilayer is still a subject of debate. The interaction of CoQ in the oxidized (ubiquinone-10) and reduced (ubiquinol-10) state with membrane model systems of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (Ela2Gro-P-Etn) has been studied by means of differential scanning calorimetry (DSC), 31P-nuclear magnetic resonance (31P-NMR) and small angle X-ray diffraction (SAXD). Ubiquinone-10 did not visibly affect the lamellar gel to lamellar liquid-crystalline phase transition of Ela2Gro-P-Etn, but it clearly perturbed the multicomponent lamellar liquid-crystalline to lamellar gel phase transition of the phospholipid. The perturbation of both transitions was more effective in the presence of ubiquinol-10. A location of CoQ forming head to head aggregates in the center of the Ela2Gro-P-Etn bilayer with the polar rings protruding toward the phospholipid acyl chains is suggested. The formation of such aggregates are compatible with the strong hexagonal HII phase promotion ability found for CoQ. This ability was evidenced by the shifting of the lamellar to hexagonal HII phase transition to lower temperatures and by the appearance of the characteristic hexagonal HII 31P-NMR resonance and SAXD pattern at temperatures at which the pure Ela2Gro-P-Etn is still organized in extended bilayer structures. The influence of CoQ on the thermotropic properties and phase behavior of Ela2Gro-P-Etn is discussed in relation to the role of CoQ in the membrane.

The antineoplastic ether phospholipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phophocholine (ET-18-OCH3) was incorporated into dimyristoylglycerophosphocholine (Myr2Gro-PCho)/dimyristoylglycerophosphoserine (Myr2Gro-PSer) (4 : 1 molar ratio) mixtures. Electron microscopy showed that the addition of ET-18-OCH3 reduced the size of the vesicles. Small vesicles could be detected even at 60 mol% ET-18-OCH3. Sedimentation studies showed the increasing presence of phospholipids in the supernatant, while turbidity measurements indicated a decrease in absorbance as the ET-18-OCH3 concentration was increased. These findings may be explained by the formation of small vesicles and/or mixed micelles. Infrared spectroscopy showed that at 60 mol% the fluidity of the membrane was considerably increased at temperatures below the phase transition, with only a small increase in the proportion of gauche isomers after the gel-to-fluid phase transition of this sample. On the other hand, protein kinase Cα (PKCα) activity progressively decreased when ET-18-OCH3 was incorporated into multilamellar vesicles, reaching a minimum value at 20 mol%, this inhibition being attributed to the modification of the membrane produced by a cone-shaped molecule. At higher concentrations, however, ET-18-OCH3 activated the enzyme with a maximum being attained at 50 mol%. This activation being attributed to the formation of small vesicles and/or micelles. At still higher concentrations of ET-18-OCH3 the enzyme was once again inhibited, inhibition being almost complete at 80 mol%. When PKC was assayed using large unilamellar vesicles a slight activation was observed at very low ET-18-OCH3 concentrations.

Lipid activation of protein kinase C α (PKC α) was studied using a model mixture containing POPC/POPS (molar ratio 4:1) and different proportions of either DPG or POG. The lipid mixtures containing DPG were physically characterized by using different physical techniques, and a phase diagram was constructed by keeping a constant POPC/POPS molar ratio of 4:1 and changing the concentration of 1,2-DPG. The phase diagram displayed three regions delimited by two compounds: compound 1 (CO1) with 35 mol % of 1,2-DPG and compound 2 (CO2) with 65 mol % of 1,2-DPG. PKC α activity was assayed at increasing concentrations of 1,2-DPG, maximum activity being reached at 30 mol % 1,2-DPG, which decreased at higher concentrations. Maximum activity occurred, then, at concentrations of 1,2-DPG which corresponded to the transition from region 1 to region 2 of the phase diagram. It was interesting that this protein was maximally bound to the membrane at all DPG concentrations. Similar results were observed when the enzyme was...

The C2 domain of classical PKCs binds to membranes through Ca(2+) bridging to phosphatidylserine as recently observed through X-ray diffraction of the isolated domain. Additionally, it has been proposed that N189, T251, R216, and R249A interact directly with phosphatidylserine [Verdaguer, N., et al. (1999) EMBO J. 18, 6329-6338]. When these four residues were mutated to Ala to determine their role in PKC binding to phospholipid membranes, PKC activation, and in its in vivo localization, the results revealed that they were very important for the activation of full-length PKCalpha. N189, in particular, was involved in the activation of the enzyme after its interaction with PS, since its mutation to Ala did not decrease the level of membrane binding but did prevent full enzyme activation. On the other hand, mutations R216A, R249A, and T251A affected both membrane binding and enzyme activation, although T251A had the most drastic effect, suggesting that the protein interactions with the carbonyl groups of the phospholipid are also a key event in the activation process. Taken together, these results show that the four residues located near the calcium binding site are critical in phosphatidylserine-dependent PKCalpha activation, in which N189 plays an important role, triggering the enzyme activation probably by interacting with neighboring residues of the protein when lipid binding occurs. Furthermore, these results provide strong evidence for better defining one of the two phosphatidylserine isomer models proposed in the previous crystallographic report.

The C-terminal domain of the pro-apoptotic protein Bax is a hydrophobic stretch which, it has been predicted, anchors this protein to the outer mitochondrial membrane when apoptosis is induced in the cell. A 21mer peptide imitating this domain has been synthesized together with two mutants, one with a S184 substituted by K and the other with the S184 deleted. When their structures were studied by infrared spectroscopy, it was seen that the three peptides formed aggregates both in solution and within lipid membranes, and that the peptide changed its secondary structure as a consequence of these two mutations. It was also observed that the wild-type peptide and the two mutants became membrane-integral molecules and changed their conformation when they were incorporated into model membranes with the same composition as the outer mitochondrial membrane. With the peptides incorporated in the membranes the location of W188 was studied by fluorescence quenching using the water soluble quencher acrylamide and different doxyl-PC located in the membrane, this residue being found at different membrane depths in each of the three peptides. The fact that the three peptides were able to perturb the motion of the fluorescent probe diphenylhexatriene confirmed their insertion in the membrane. However, whereas the wild type and the DeltaS184 mutant peptides were very efficient in releasing encapsulated carboxyfluorescein from liposomes, the mutant S184K was less efficient. Taken together, these results showed that the mutation tested changed the conformation of the C-terminal domain of Bax and the positions that they adopted when inserted in membranes, confirming the importance of S184 determining the conformation of this domain. At the same time, these results confirmed that the C-terminal domain of Bax participates in disrupting the barrier properties of biomembranes.

Infrared spectroscopy (IR) and differential scanning calorimetry (DSC) were used to study the biophysical properties of the PKCepsilon-C2 domain, a C2 domain that possess special characteristics as it binds to acidic phospholipids in a Ca2+-independent manner and no structural information about it is available to date. When the secondary structure was determined by IR spectroscopy in H2O and D2O buffers, beta sheet was seen to be the major structural component. Spectroscopic studies of the thermal denaturation in D2O showed a broadening in the amide I' band starting at 45 degrees C. Curve fitting analysis of the spectra demonstrated that two components appear upon thermal denaturation, one at 1623 cm(-1) which was assigned to aggregation and a second one at 1645 cm(-1), which was assigned to unordered or open loop structures. A lipid binding assay has demonstrated that PKCepsilon-C2 domain has preferential affinity for PIP2 although it exhibits maximal binding activity for phosphatidic acid when 100 mol% of this negatively charged phospholipid was used. Thus, phosphatidic acid containing vesicles were used to characterize the effect of lipid binding on the secondary structure and thermal stability. These experiments showed that the secondary structure did not change upon lipid binding and the thermal stability was very high with no significant changes occurring in the secondary structure after heating. DSC experiments demonstrated that when the C2-protein was scanned alone, it showed a Tm of 49 degrees C and a calorimetric denaturation enthalpy of 144.318 kJ x mol(-1). However, when phoshatidic acid vesicles were included in the mixture, the transition disappeared and further IR experiments demonstrated that the protein structure was not modified under these conditions.

Resveratrol is a phytoalexin found in grapes and other foods that cancer chemopreventive and other biological activities have been attributed recently. We report that resveratrol is able to incorporate itself into model membranes in a location that is inaccessible to the fluorescence quencher, acrylamide. Differential scanning calorimetry revealed that resveratrol considerably affected the gel to liquid-crystalline phase transition of multilamellar vesicles made of phosphatidylcholine/phosphatidylserine and increased the temperature at which the fluid lamellar to H(II) inverted hexagonal transition took place in multilamellar vesicles made of 1,2-dielaidoyl-sn-phosphatidylethanolamine. Such a transition totally disappeared at 2.5 mM of resveratrol (resveratrol/lipid molar ratio of 2:1). This effect on 1, 2-dielaidoyl-sn-phosphatidylethanolamine polymorphism was confirmed through (31)P-NMR, which showed that an isotropic peak appeared at high temperature instead of the H(II)-characteristic peak of 42 mM of resveratrol (resveratrol/lipid molar ratio of 1.5:1). Finally, resveratrol inhibited PKCalpha when activated by phosphatidylcholine/phosphatidylserine vesicles with an IC(50) of 30 microM, whereas when the enzyme was activated by Triton X-100 micelles the IC(50) was 300 microM. These results indicate that the inhibition of PKCalpha by resveratrol can be mediated, at least partially, by membrane effects exerted near the lipid-water interface.

The secondary structure of amyloid betaAP(25-35) peptide was studied in pure form and in the presence of different phospholipid vesicles, by using Fourier transform infrared spectroscopy (FT-IR). Pure peptide aggregated with time, forming fibrils with beta-structure. Phospholipid vesicles formed by negatively charged phospholipids such as 1,2-dimyristoyl-sn-glycerol-3-phospho-L-serine (Myr2PtdSer), 1,2-dimyristoyl-sn-glycerol-3-phospho-rac-1-glycerol (Myr2PtdGro) and 1,2-dimyristoyl-sn-glycerol 3-phosphate (Myr2PtdH), greatly accelerated the aggregation of the peptide. However, the presence of vesicles formed by the zwitterionic phospholipid, 1, 2-dimyristoyl-sn-glycerol-3-phosphocholine (Myr2PtdCho), slowed down the aggregation process. Differential scanning calorimetry (DSC) measurements showed that the effect of betaAP(25-35) on the gel to crystal liquid phase transition was small at neutral pH for negatively charged phospholipids and practically nil for Myr2PtdCho. In the case of Myr2PtdSer the effect was also zero at pH 9 but the effect was large at pH 3. The effect on Myr2PtdH was not, however, very dependent on pH. These results were fully confirmed by the observation through FT-IR of the change with temperature of the CH2 antisymmetric stretching vibration. The case of Myr2PtdGro was special as this phospholipid presents polymorphism giving solid quasicrystalline phases when it is not sufficiently hydrated, and it is remarkable that betaAP(25-35) was able to induce the formation of crystalline phases in samples prepared through a method which ensure a good hydration of phospholipid. These results show that the interaction of amyloid betaAP(25-35) peptide with phospholipids is based on electrostatic interactions, that these interactions favour the aggregation of the peptides, and that the presence of the aggregates may disturb the lipid-water interphase of the membrane.

Fourier transform infrared (FTIR) spectroscopy was used to investigate the structural and thermal denaturation of the C2 domain of PKC alpha (PKC-C2) and its complexes with Ca(2+) and phosphatidic acid vesicles. The amide I regions in the original spectra of PKC-C2 in the Ca(2+)-free and Ca(2+)-bound states are both consistent with a predominantly beta-sheet secondary structure below the denaturation temperatures. Spectroscopic studies of the thermal denaturation revealed that for the PKC-C2 domain alone the secondary structure abruptly changed at 50 degrees C. While in the presence of 2 and 12.5 mM Ca(2+), the thermal stability of the protein increased to 60 and 70 degrees C, respectively. Further studies using a mutant lacking two important amino acids involved in Ca(2+) binding (PKC-C2D246/248N) demonstrated that these mutations were inherently more stable to thermal denaturation than the wild-type protein. Phosphatidic acid binding to the PKC-C2 domain was characterized, and the lipid-protein binding became Ca(2+)-independent when 100 mol% phosphatidic acid vesicles were used. The mutant lacking two Ca(2+) binding sites was also able to bind to phosphatidic acid vesicles. The effect of lipid binding on secondary structure and thermal stability was also studied. Beta-sheet was the predominant structure observed in the lipid-bound state, although the percentage represented by this structure in the total area of the amide I band significantly decreased from 60% in the lipid-free state to 47% in the lipid-bound state. This decrease in the beta-sheet component of the lipid-bound complex correlates well with the significant increase observed in the 1644 cm(-1) band which can be assigned to loops and disordered structure. Thermal stability after lipid binding was very high, and no sign of thermal denaturation was observed in the presence of lipids under the conditions that were studied.

The capacity of the antineoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (ET-18-OCH3) to modulate the polymorphic properties of dielaidoylphosphatidylethanolamine has been studied using biophysical techniques. Differential scanning calorimetry showed that ET-18-OCH3 depresses the onset of the Lbeta to Lalpha phase transition, decreasing also DeltaH of the transition. At the same time, the onset of the transition from Lalpha to inverted hexagonal HII phase was gradually increased as the ether lipid concentration was increased, totally disappearing at concentrations higher than 5 mol%. Small-angle X-ray diffraction and 31P-NMR confirmed that ET-18-OCH3 induced that the appearance of the inverted hexagonal HII phase was shifted towards higher temperatures completely disappearing at concentrations higher than 5 mol%. These results were used to elaborate a partial phase diagram and they were discussed as a function of the molecular action of ET-18-OCH3.

The C2 domain is a conserved protein module present in various signal-transducing proteins. To investigate the function of the C2 domain of protein kinase Calpha (PKCalpha), we have generated a recombinant glutathione S-transferase-fused C2 domain from rat PKCalpha, PKC-C2. We found that PKC-C2 binds with high affinity (half-maximal binding at 0.6 microM) to lipid vesicles containing the negatively charged phospholipid phosphatidylserine. When expressed into COS and HeLa cells, most of the PKC-C2 was found at the plasma membrane, whereas when the cells were depleted of Ca2+ by incubation with EGTA and ionophore, the C2 domain was localized preferentially in the cytosol. Ca2+ titration was performed in vivo and the critical Ca2+ concentration ranged from 0.1 to 0.32 microM. We also identified, by site-directed mutagenesis, three aspartic residues critical for that Ca2+ interaction, namely Asp-187, Asp-246 and Asp-248. Mutation of these residues to asparagine, to abolish their negative charge, resulted in a domain expressed as the same extension as wild-type protein that could interact in vitro with neither Ca2+ nor phosphatidylserine. Overexpression of these mutants into COS and HeLa cells also showed that they cannot localize at the plasma membrane, as demonstrated by immunofluorescence staining and subcellular fractionation. These results suggest that the Ca2+-binding site might be involved in promoting the interaction of the C2 domain of PKCalpha with the plasma membrane in vivo.

The lipid activation of protein kinase C alpha (PKC alpha) has been studied by comparing the activation capacity of different 1, 2-diacylglycerols and 1,3-diacylglycerols incorporated into mixed micelles or vesicles. Unsaturated 1,2-diacylglycerols were, in general, more potent activators than saturated ones when 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS)/Triton X-100 mixed micelles and pure POPS vesicles were used. In contrast, these differences were not observed when 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/POPS (4:1, molar ratio) vesicles were used. Diacylglycerols bearing short fatty acyl chains showed a very high activation capacity, however, the capacity was less in mixed micelles. Furthermore, 1, 2-diacylglycerols had a considerably higher activating capacity than 1,3-diacylglycerols in POPS/Triton X-100 mixed micelles and in POPC/POPS vesicles. However, the differences between the two types of diacylglycerols were smaller when pure POPS vesicles were used. Differential scanning calorimetry (DSC) showed that POPC/POPS membrane samples containing diacylglycerols had endothermic transitions in the presence of 200 microM Ca2+ and 5 mM Mg2+. Transitions were not detected when using pure POPS vesicles due to the formation of dehydrated phases as demonstrated by FTIR (Fourier-transform infrared) spectroscopy. PKC alpha binding studies, performed by differential centrifugation in the presence of 200 microM Ca2+ and 5 mM Mg2+, showed that 1,2-sn-dioleoylglycerol (1, 2-DOG) was more effective than 1,3-dioleoylglycerol (1,3-DOG) in promoting binding to POPC/POPS vesicles. However, when pure POPS vesicles were used, PKC alpha was able to bind to membranes containing either 1,2-DOG or 1,3-DOG to the same extent.

Fourier-transform infrared spectroscopy (FT-IR) has been applied to the quantitative study of the dehydration of the phosphatidylserine phosphate group in the presence of Ca2+ exerted by different molecules, such as diacylglycerol, sphingosine and stearylarnine, by using a partial least-squares statistical procedure. By using this method it was observed that diacylglycerol enhanced the dehydration of this PO2− group produced by Ca2+ whereas the amino-bases sphingosine and stearylamine protected the phosphate group from the dehydration produced by Ca2+ due to the very strong electrostatic interaction established. The apparent pKa of lipid carboxyl groups can also be estimated by using FTIR. The method consisted in quantifying the absorbance intensities due to the protonated and the unprotonated forms of the specific group being studied. The pKa of the carboxyl group of [1-13C]-palmitic acid included in dipalmitoylphosphatidylcholine membranes was found to be 8.7, a value much higher than that estimated from a molecular solution of the fatty acid. It was observed using the same method that the pKa of free fatty acids in model stratum corneum lipid mixtures was in the range 6.2–7.3 increasing with the preponderance of oleic acid over palmitic acid. Finally the pKa of the carboxyl group of phosphatidylserine was shifted from 4.6 in the pure phospholipid to 2.1 and 2.2 in the presence of equimolar sphingosine and stearylamine, respectively, as a consequence of electrostatic interactions.

Proteins containing C2 domains are the sensors for Ca2+ and PI (4,5)P2 in a myriad of secretory pathways. Here, the use of a freemounting system has enabled us to capture an intermediate state of Ca 2+ binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P2 and IP3 at resolutions of 1.75 and 1.9 A, respectively, unveiling that the polybasic cluster formed by strands β3-β4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P2/PI (3,4,5)P3, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI (4,5)P2. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca2+-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals., Work in Barcelona was supported by Grant BIO2011-24333 [Ministerio de Economía y Competitividad (MINECO), Spain-Fondo Europeo de Desarrollo Regional (FEDER)] and work in Murcia by Grants BFU2011-22828 (MINECO, Spain-FEDER) and 08700/PI/08 (Fundación Seneca, Region de Murcia).C.F.-O. is recipient of a Junta para la Amplificación de Estudios postdoctoral contract from Consejo Superior de Investigaciones Cientificas-Fondo Social Europeo. J.G. is recipient of a Juan de la Cierva postdoctoral contract from MINECO

Classical protein kinase C (PKC) enzymes are known to be important factors in cell physiology both in terms of health and disease. They are activated by triggering signals that induce their translocation to membranes. The consensus view is that several secondary messengers are involved in this activation, such as cytosolic Ca2+ and diacylglycerol. Cytosolic Ca2+ bridges the C2 domain to anionic phospholipids as phosphatidylserine in the membrane, and diacylglycerol binds to the C1 domain. Both diacylglycerol and the increase in Ca2+ concentration are assumed to arise from the extracellular signal that triggers the hydrolysis of phosphatidylinositol-4,5-bisphosphate. However, results obtained during the last decade indicate that this phosphoinositide itself is also responsible for modulating classical PKC activity and its localization in the plasma membrane.

The effect of alpha-tocopherol on the Ca(2+)-induced fusion of large unilamellar phosphatidylserine vesicles has been investigated. Mixing of aqueous vesicle contents was followed continually with the terbium-dipicolinic acid (Tb-DPA) assay, while the dissociation of preencapsulated Tb-DPA complex was taken as a measure of the release of vesicle contents. Vesicles consisting of pure phosphatidylserine and phosphatidylserine containing 2, 5, and 10 mol% of alpha-tocopherol were employed at different Ca2+ concentrations. The presence of low amounts of alpha-tocopherol decreased the initial rate of fusion without changing the Ca2+ threshold concentration. The reduction of the initial rate of fusion was proportional to the amount of alpha-tocopherol present in the bilayer. An alpha-tocopherol concentration-dependent decrease of both the initial rate and the final extent of release of vesicle contents was also observed. This effect was more evident as more alpha-tocopherol was incorporated in the bilayer, so that in the presence of 10 mol% of alpha-tocopherol no significant release was observed after 5 min. The stabilization of the vesicular structure exerted by alpha-tocopherol was responsible for the apparent increase of the fluorescence intensity of the Tb-DPA complex at later stages of the process. The results reflect a perturbation of the membrane by low concentrations of alpha-tocopherol which may account for a number of biological effects of this vitamin, not related to its antioxidant role.

1. Chemical modification of the Ca2+-ATPase with phenylglyoxal, as a modifier of arginine residues, leads to an almost total loss of the ATPase activity. The presence of nucleotides in the reaction medium protects against the binding of 18 nmol of phenylglyoxal/mg of protein and this reduction in the binding of phenylglyoxal is accompanied by a substantial retention of ATPase activity. The incorporation of phenylglyoxal to the protein alters neither calcium binding nor phosphorylation from inorganic phosphate. Nevertheless the binding of nucleotides is dramatically inhibited and, consequently, so is phosphorylation from ATP. Fluorescein 5´-isothiocyanate labelling of the phenylglyoxal-modified ATPase is not affected but, on the other hand, phenylglyoxal is not able to modify the fluorescein 5´-isothiocyanate-prelabelled ATPase. The way in which ATPase inhibition depends on the presence of phenylglyoxal indicates that this process occurs in a pseudo-first-order reaction. However, the dependence of the apparent first-order rate constant on phenylglyoxal concentration appears to be more complex and an inhibition mechanism of two steps, with phenylglyoxal binding, has to be taken into account. 2. We have found that phenylglyoxal labels both A and B tryptic fragments, but only B fragment labelling is prevented by ATP. The sequencing of peptides from mild acid hydrolysis of phenylglyoxal-labelled ATPase shows that phenylglyoxal is located in the Ala506–Gly595 peptide that is a part of the B fragment. 3. We conclude that phenylglyoxal inactivates the calcium pump in a two-step mechanism in which the second step is irreversible. Phenylglyoxal labels an arginyl residue in the Ala506–Gly595 peptide that can be protected by the binding of ATP to its site.

The interaction of alpha-tocopherol with dimyristoylphosphatidylserine (DMPS) has been studied in the presence and in the absence of Ca2+ by using differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR) and 45Ca2+-binding. In the absence of Ca2+, DSC showed that alpha-tocopherol decreases the temperature of the lamellar gel to lamellar liquid crystalline phase transition as well as it decreases delta H of this transition. Two different peaks were detected at 10 mol% of alpha-tocopherol and probably one of the peaks correspond to pure or nearly pure DMPS and the other to DMPS incorporating most of the alpha-tocopherol. The phase transition was totally abolished at 30 mol% of alpha-tocopherol. In the presence of Ca2+ this L(beta) to L(alpha) phase transition of DMPS was even more perturbed by alpha-tocopherol, so that it was totally abolished by only 7 mol% of alpha-tocopherol, at Ca2+ concentrations which were clearly non-saturating, like those giving DMPS/Ca2+ molar ratio of 4:1 and 10:1. Furthermore, the transition of the DMPS/Ca2+ complex observed at 91.6 degrees C was perturbed by the presence of alpha-tocopherol, indicating a change in the structure of the crystalline complex. The FT-IR analysis of the effect of alpha-tocopherol on DPMS phase transition confirmed the decrease in the phase transition temperature of the phospholipid, and also that alpha-tocopherol increases the number of gauche isomers in the gel state but has no effect in the liquid crystalline state. The binding of 45Ca2+ was also affected by the presence of alpha-tocopherol, so that the number of binding sites was decreased, and this may be interesting for situations in which phosphatidylserine and Ca2+ are simultaneously implicated in biological functions, such as membrane fusion and enzyme activation.

The metastability of dimiristoylphosphatidylethanolamine (DMPE) has been studied by means of Fourier transform infrared spectroscopy (FT-IR), both in the absence and in the presence of alpha-tocopherol. Two different methods of hydration were used to prepare the samples, poorly hydrated and well hydrated, and the results have been compared with anhydrous DMPE. Poorly hydrated DMPE gave place to a high-melting phase formed upon melting from gel to L alpha at approx. 49 degrees C, with a new transition to L alpha at approx. 55 degrees C. However, well hydrated DMPE incubated at 4 degrees C for 49 days gave place to a subgel phase which was transformed by heating into a L beta phase at about 40 degrees C and this into a L alpha phase after further heating at 52 degrees C. The subgel phase was more hydrated and less rigid than the high-melting phase. On the other hand, alpha-tocopherol, when included in poorly hydrated DMPE, stabilized a high-melting phase, which was transformed by heating, directly into a L alpha. However, when a sample of DMPE containing alpha-tocopherol was incubated for 49 days at 4 degrees C a dehydrated solid phase different from the subgel and the high-melting phases was formed.

Treatment of sarcoplasmic reticulum vesicles with diethylpyrocarbonate in the presence of a large excess of reagent, at pH 6.2 and at room temperature, reveals both a fast- and a slow-reacting population of protein residues. The loss of the Ca(2+)-ATPase activity is mainly associated with the fast-reacting population being partially sensitive to hydroxylamine. There is also an effect on the Ca(2+)-binding mechanism. Shorter derivatization times (5 min) produce a loss of the positive cooperativity of Ca2+ binding. When the treatment was prolonged for 30 min there was an additional decrease in the overall Ca2+ affinity. Curve-fitting procedures applied to the non-cooperative binding isotherms provide the equilibrium constants for the two Ca2+ sites, although they cannot discriminate between interacting and independent site mechanisms. Prestationary kinetics assays show 2 Ca2+:1 ATP ratios, at any extent of Ca2+ saturation, indicating that the Ca2+ sites are not independent. The Ca2+ dissociation profile after derivatization shows a decrease in the dissociation constant for the release of the second Ca2+, which is consistent with interacting sites. Isotopic exchange experiments show fast and slow components of equal amplitude even at subsaturating Ca2+ concentrations, which is incompatible with independent binding sites. The experimental data suggest a modification of the equilibrium binding constants making them more similar, but keeping the interacting character. The structural position of the external (cytoplasmic) and the internal (lumenal) Ca2+ sites remains unaltered in the absence of positive cooperativity.

Ritodrine inhibits the steady-state Ca 2+ -ATPase activity of isolated sarcoplasmic reticulum vesicles in a dose-dependent manner, The observed K 0.5 value for inhibition (∼3 mM) gives proof of a low-affinity interaction. Ritodrine also interferes with the steady-state Ca 2+ transport ability decreasing the maximal rate without modification of the Ca 2+ or ATP affinity for the enzyme, This is consistent with an absence of competition for the transport and the catalytic sites. Analysis of the catalytic and transport cycle shows that: (i) ritodrine inhibits the phosphorylation partial reaction, This is supported by pre-steady-state kinetic experiments of Ca 2+ transport and also by the temperature dependence of the phosphoenzyme level. (ii) At high ritodrine concentrations the dephosphorylation step becomes rate-limiting. This is suggested by the biphasic profile (V-shape) of phosphoenzyme accumulation at different ritodrine concentrations, This was also confirmed by chase experiments of radioactive phosphoenzyme decomposition at steady state. These data reveal a complex pattern of inhibition involving two sites for interaction with low and high ritodrine concentrations. It is envisaged that ritodrine does not exert any direct effect on the smooth muscle sarcoplasmic reticulum Ca 2+ -ATPase when used in the treatment of preterm birth.

Capsaicin is a natural compound with pharmacological and toxicological effects, which given its hydrophobicity, can influence the structure of membranes. The interaction of capsaicin with model membranes of dipalmitoylphosphatidylcholine and dielaidoylphosphatidylethanolamine has been studied by using differential scanning calorimetry, fluorescent probe spectroscopy and 31 P-nuclear magnetic resonance. Capsaicin remarkably affects the phase transition of dipalmitoylphosphatidylcholine, shifting the transition temperature to lower values, and giving rise, at relatively high capsaicin concentrations, to the appearance of two peaks in the thermogram. These peaks may correspond to separated phases as indicated by the partial phase diagram. Whereas capsaicin did not affect the fluorescence polarization of the probes diphenylhexatriene and trimethylammonium-diphenylhexatriene, it clearly affected that of the probe 2-anthroyloxystearic acid, indicating that the perturbation produced by capsaicin on the membrane would be mainly at the position where this fluorophore is located. On the other hand, capsaicin, at relatively low concentrations, gives rise to immiscible phases in the presence of dielaidoylphoshatidylethanolamine and decrease the temperature of the lamellar to hexagonal H II phase transition. At concentrations of capsaicin higher than 0.3 mol fraction, isotropic phases were detected. The possible implications of the effects of capsaicin on biological membranes are discussed.

We have studied the effect of physiological concentrations of different diacylglycerols on Ca(2+)-induced fusion between phosphatidylserine vesicles. We monitored vesicle fusion as mixing of membrane lipids under conditions where the limiting factor was the aggregation and also in conditions where this aggregation was not the limiting factor. We found that diacylglycerols have a different modulating effect on the Ca(2+)-induced fusion: i) depending on their interfacial conformation, so that 1,2-isomers of diacylglycerols containing unsaturated or short saturated acyl chains stimulated fusion and their 1,3-isomers did not, and ii) depending on their specific type of bilayer interior perturbation, so that diacylglycerols containing unsaturated or short chain saturated acyl chains stimulated fusion but those containing long-chain saturated acyl chains did not. These requirements resembled those required for the diacylglycerol activation of protein kinase C, suggesting that diacylglycerol acts in both the specific activation of this enzyme and the induction of membrane fusion through the same perturbation of lipid structure. We found that polylysine affected the stimulatory role of 1,2-dioleoylglycerol differently, depending on whether aggregation was the limiting factor of fusion. When we studied the effect of very low concentrations of diacylglycerols on the bulk structural properties of phosphatidylserine, we found that they neither significantly perturbed the thermotropic transitions of phosphatidylserine nor affected the interaction of Ca2+ with the phosphate group of phosphatidylserine. The underlying mechanism of fusion between phosphatidylserine vesicles is discussed.

The role of phosphatidylserine and diacylglycerol in the fusion of chromaffin granules with target membranes was investigated in vitro, monitoring the mixing of membrane lipids with the octadecyl rhodamine B fluorescence dequenching assay. Polylysine was able to induce fusion of chromaffin granule ghosts with plasma membrane vesicles in the absence of Ca2+. This fusion was maximal at 1.6 μM polylysine and the kinetics were dependent on the amount of plasma membrane added. Polylysine lowered the Ca2+ threshold concentration for inducing fusion, increased the extent of fusion at a given Ca2+ concentration and acted synergistically with Ca2+ when added prior to the cation. Removal of membrane proteins by trypsinization of chromaffin granule ghost and/or plasma membranes increased the extent of polylysine induced fusion. Incorporation of diacylglycerol into the plasma membrane promoted Ca2+-induced fusion. Chromaffin granule ghosts were induced to fuse with model membranes of different complexities from one resembling the inner monolayer of the erythrocyte membrane to one composed solely of pure phosphatidylserine. The characteristics of the fusion with model membranes were qualitatively similar to that observed with target plasma membrane, although differences in kinetics and stoichiometries were found. Interestingly, considerably low (μM) Ca2+ concentrations were able to induce fusion of chromaffin granule ghosts with diacylglycerol containing phosphatidylserine vesicles in the presence of polylysine. Our results indicate that acidic phospholipid-like phosphatidylserine may have an important role in the process of fusion and suggest that diacylglycerol, as a destabilizing lipid, may have a role by itself in promoting exocytosis in chromaffin cells.

The interactions of sphingosine (SPH) with dipalmitoylphosphatidylcholine (DPPC) and dielaidoylphosphatidylethanolamine (DEPE) have been studied by means of differential scanning calorimetry (DSC) and 31P-nuclear magnetic resonance (31P-NMR). Experiments were carried out with the fully protonated form of SPH, at pH 6.0. DSC studies showed that the main Tc transition temperature of DPPC was perturbed by the presence of SPH so that Tc of the mixture was higher than those of pure components at concentrations of SPH up to 50 mol%, with an azeotropic point at 30 mol% of SPH. At higher concentrations solid phase separations were observed from 70 to 95 mol% of SPH with an eutectic point at 90 mol% of SPH. 31P-NMR showed lamellar phases in DPPC/SPH mixtures, at all the range of concentrations. The behavior of DEPE/SPH mixtures was somewhat different since no azeotropic point was detected, the gel to liquid-crystalline transition being depressed by the presence of SPH, and an eutectic point was detected at 60 mol%. Solid phase immiscibilities were present between 50 mol% and 85 mol% of SPH. It is also remarkable that the liquid-crystalline to hexagonal HII phase transition of DEPE was only slightly shifted to lower temperatures at concentrations of SPH lower than 33 mol% of SPH but, this transition disappeared at concentrations of SPH higher than 33 mol% of SPH, so that isotropic phases were formed instead, as seen through 31P-NMR. The present results show the importance of taking into account the effects appearing in mixtures of SPH with zwitterionic phospholipids when considering their influence on the organization of biomembranes.

The purified (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum was subjected to extensive proteolysis by using trypsin and proteinase K. This digestion led to the elimination of a considerable portion of the protein, so that the lipid to protein weight ratio was increased from 0.44 in the purified ATPase to 1.20 after extensive proteolysis. After the digestion, the residue was found to be considerably enriched in hydrophobic amino acids. FT-IR spectroscopic studies indicated that the secondary structure of the proteolytic residue was enriched in alpha-helix with 75%, compared with 48% in the intact purified ATPase. FT-IR studies using ATR polarization showed that the alpha-helical part of the residue of proteolytic digestion was considerably more polarized than the purified ATPase, indicating that, on average, the alpha-helices of the residual protein should lie with an orientation closer to the normal to the plane of the membrane. Thermal denaturation studies showed that the residue of proteolysis was considerably more stable than the intact purified ATPase. This would be compatible with the residue being protected from denaturation by its hydrophobic location within the membrane. This study is experimental evidence of the alpha-helical structure of the membrane part of this protein, as suggested by predictions made from its known primary structure (Brandl et al., 1986).

The interaction of 1,2-dipalmitoylglycerol (1,2-DPG) with dipalmitoylphosphatidylserine (DPPS) has been studied in the presence and in the absence of Ca2+ by using differential scanning calorimetry (DSC) and 31P-nuclear magnetic resonance (31P-NMR). In the absence of Ca2+, DSC showed that 1,2-DPG increased the phase transition of DPPS, effect already noticed at very low 1,2-DPG concentrations, whereas lipid immiscibilities were detected at concentrations of 1,2-DPG higher than about 30 mol%. 31P-NMR indicated that lamellar phases were always present at concentration of 1,2-DPG lower than about 35 mol%, but at higher concentrations non-lamellar phases may be present in the fluid phase. As observed by DSC, the apparent pKa of the carboxyl group of DPPS was increased slightly in the presence of 1,2-DPG. In the presence of Ca2+, the effect of 1,2-DPG was to further increase the temperature of the onset of the phase transition, indicating an stabilization of the most rigid phase in the DPPS/1,2-DPG/Ca2+ samples. Even concentrations of 1,2-DPG as low as 1 mol% of the total lipid already produced a noticeable effect. Moreover, lipid immiscibilities were apparent at concentrations of 1,2-DPG higher than 20 mol%. Furthermore, the transition of the DPPS/Ca2+ complex observed by DSC at 155 degrees C was perturbed by the presence of 1,2-DPG, indicating a change in the structure of the crystalline complex. Interestingly, the effect of non-saturating Ca2+ concentrations on the DPPS phase transition was enhanced by the presence of 1,2-DPG. The effect reported here may be significant for a number of situations where Ca2+, phosphatidylserine and diacylglycerols are involved, such as fusion of membranes, where diacylglycerol may facilitate Ca(2+)-induced fusion, or the activation of enzymes such as protein kinase C and phospholipases.

A fluorescence study of a diacylglycerol derivatized with the n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) chromophore (NBD) was carried out. Fluorescence self-quenching was observed for this probe in lecithin model membranes due to collisional interaction rather than to an aggregational behaviour of the probe. The efficient energy migration (Ro = 28 A) of the NBD fluorophore was studied via the fluorescence depolarization upon increase of probe concentration in membranes, and the results are compared with a model where a random distribution of the probes is assumed. A surface location of the chromophore was concluded for the NBD derivative of diacylglycerol, both from the fluorescence parameters and from the study of its fluorescence quenching by spin label probes. Very high lateral diffusion coefficients were obtained for these probes, both from the self-quenching (D = 2-6 x 10(-6) cm2 s-1) and from the spin probe quenching (D = 3.5 x 10(-6) cm2 s-1) studies. A concomitant fluorescence study of the related probe NBD-phosphatidylcholine revealed that its photophysical behaviour is similar to the derivatized diacylglycerol.

C2-domains are independently folded modules, of about 130 residues, found in a large and diverse set of eukaryotic proteins. Many of the best-characterized C2-domains are found in proteins involved in membrane trafficking and fusion, such as synaptotagmins or rabphilin, and in signal transduction, such as phospholipases A2 (cPLA2) and C (PLC) or the protein kinase C isoforms (PKCs). All C2-domains share a common overall fold: a single compact Greek-key motif organized as an eight-stranded antiparallel β-sandwich consisting of a pair of four-stranded β-sheets – the A and B sheets – with a long axis of about 50 A. Connections between β-strands emerge from the top and bottom of the domain according to the two distinct topologies of C2-domains. In topology I (the S family), the first β-strand occupies the same structural position as the eighth β-strand of topology II (the P family), which shifts in a circular permutation the order of homologous strands in the primary structure. The structural information defines adjacent calcium binding sites within a broad acidic cleft that contains highly conserved acidic residues. These provide most of the oxygen atoms that coordinate with the calcium ions. The present work will focus on the wealth of functional and structural information, which is being obtained at an exponential pace, on the membrane-binding C2-domains that require calcium. 3D Structure Keywords: C2-Domain; calcium; protein kinase C; phospholipases; membrane binding; synaptotagmin

A methodology for the kinetic study of the self-inactivation of an unstable enzyme has been developed by using the transient-phase approach when the enzymatic activity is measured through a coupled enzyme system. An experimental design has been developed and applied to the inactivation of the Ca(2+)-ATPase from sarcoplasmic reticulum solubilized in the monomeric state. The catalytic activity of the ATP hydrolysis is determined in the presence of pyruvate kinase and lactate dehydrogenase as auxiliary enzymes, and the oxidation of the last substrate, NADH, is continuously monitored. The experimental results show that both substrates, ATP and calcium, protect against enzyme inactivation. This enzyme, the monomeric ATPase, fulfills the catalytic cycle of the native ATPase, and free enzyme and first-calcium bound enzyme are proposed as the intermediates which are being inactivated.

Sarcoplasmic reticulum membrane derived from the terminal cisternae region reacts with the carboxyl reagent N,N'-dicyclohexylcarbodiimide. The extension of this reaction is dependent on the reagent/protein ratio. By using a low ratio (10 microM reagent and 1 mg of protein/mL), we can selectively prevent the closure of the 450-kDa Ca2+ channel. Rapid filtration experiments indicate no alteration in the activating mechanism of Ca2+ release induced by Ca2+ or Sr2+ whereas the Ca2+ efflux inhibition by Ca2+, Mg2+, or ruthenium red disappears after the chemical treatment. The activating/inhibitory effect of ryanodine on the Ca2+ channel does not appear to be perturbed by N,N'-dicyclohexylcarbodiimide. The negligible incorporation of the 14C radioactive reagent to the 450-kDa band (the Ca2+ channel subunit) indicates the possibility of protein cross-linking in addition to simple derivatization. The functional alterations produced by this reagent suggest the presence of critical acidic residue(s) in a hydrophobic environment which are involved in the low-affinity cationic binding site. They can be tentatively associated with hydrophobic domains of the channel subunits contributing to the lining of the pore for Ca2+ release. The data also indicate that the channel activation by micromolar Ca2+ occurs in a different protein domain which is carbodiimide-insensitive under the experimental conditions tested.

Lipid composition and specially their electrostatic properties, were found to greatly influence the stability of liposomes in human blood serum. The amount and type of serum proteins bound to the liposomes were also clearly influenced by lipid composition and charge of liposomes. a good correlation was found between the amount of serum proteins adsorbed to a given type of liposome and its instability as measured by the release of an encapsulated fluorescent probe. Liposomes that bind the highest amount of protein were the least stable, except for the case of liposomes containing gangliosides, which were fairly stable even at a high amount of bound protein. Liposomes with neutral charge containing phosphatidylcholine were the most stable and bound the lowest amount of protein. Liposomes with positive charge behaved similarly to those with neutral charge. However, the stability of negatively charged liposomes was very dependent on their composition. Those liposomes containing only one class of negatively charged phospholipids bound a great amount of protein and were very unstable. However, those liposomes containing also phosphatidylcholine bound less protein and were more stable. The examination of the electrophoresis patterns of serum proteins bound to the different types of liposomes indicated the presence of specific proteins which correlated with liposome instability. (Mol Cell Biochem120: 119–126, 1993)

Synthetic diacylglycerol-lactones (DAG-lactones) are effective modulators of critical cellular signaling pathways, downstream of the lipophilic second messenger diacylglycerol, that activate a host of protein kinase C (PKC) isozymes and other nonkinase proteins that share similar C1 membrane-targeting domains with PKC. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study examines the biological properties of charged DAG-lactones exhibiting different alkyl groups attached to the heterocyclic nitrogen of an α-pyridylalkylidene chain, and particularly the relationship between membrane interactions of the substituted DAG-lactones and their respective biological activities. Our results suggest that bilayer interface localization of the N-alkyl chain in the R(2) position of the DAG-lactones inhibits translocation of PKC isoenzymes onto the cellular membrane. However, the orientation of a branched alkyl chain at the bilayer surface facilitates PKC binding and translocation. This investigation emphasizes that bilayer localization of the aromatic side residues of positively charged DAG-lactone derivatives play a central role in determining biological activity, and that this factor contributes to the diversity of biological actions of these synthetic biomimetic ligands.

We have used attenuated total internal reflection infrared spectroscopy (ATR-IR) spectroscopy to study the association of the C2 domain from protein kinase Cα (PKCα) with different phospholipid membranes, so as to characterise the mode of membrane docking and its modulation by the second-messenger lipid PIP₂. In parallel, we have also examined the membrane interaction of the C2 domain from cytosolic phospholipase A₂. PIP₂ did not induce significant changes in secondary structure of the membrane-bound PKCα-C2 domain, nor did binding of the PKCα-C2 domain change the dichroic ratios of the lipid chains, whereas the C2 domain from phospholipase A₂ did perturb the lipid chain orientation. Measurements of the dichroic ratios for the amide I and amide II protein bands were combined so as to distinguish the tilt of the β-sheets from that of the β-strands within the sheet. When associated with POPC/POPS membranes, the β-sandwich of the PKCα-C2 domain is inclined at an angle α=35° to the membrane normal, i.e., is oriented more nearly perpendicular than parallel to the membrane. In the process of membrane docking, the tilt angle increases to α=44° in the presence of PIP₂, indicating that the β-sandwich comes closer to the membrane surface, so confirming the importance of this lipid in determining docking of the C2 domain and consequent activation of PKCα.

The C2 domains of classical and novel protein kinases C play a very important role in decoding signals, which trigger the translocation of these enzymes to the plasma membrane and/or other membrane subcellular compartments. The C2 domain of classical PKCs has a long reputation as a paradigm of protein responding to intracytosolic Ca2+ elevations through a calcium-binding region, where this cation acts as a bridge with the phosphatidylserine located in the inner leaflet of the plasma membrane. However, more recently, it has been discovered that a second site on the C2 domain interacts specifically with the phosphoinositide, PtdIns(4,5)P(2). Furthermore, several in vivo studies have shown that both calcium and PtdIns(4,5)P(2)-interacting regions are essential for the translocation of classical PKCs to the membrane. Other molecules like arachidonic and retinoic acid have also been observed to bind to these domains, modulating the activity of classical PKCs. The C2 domains of novel PKCs, on the other hand, were supposed to play only a secondary role with respect to the C1 domain in the activation process of these enzymes. New insights reveal that these C2 domains may also receive regulatory inputs and play an important role in the localization and activation of these enzymes. In this way, the C2 domain of PKCepsilon has been observed to respond to phosphatidic acid and to act together with the C1 domain in the membrane anchorage and activation of the protein. These domains are also regulated by lipid-independent events like protein-protein interactions and phosphorylation. In this review we will focus in describing the recent findings on structural and functional properties of the C2 domains of PKCs, mainly as lipid-interacting modules able to integrate a wide variety of signals in the cell.

Curcumin is a polyphenol present in turmeric, widely used in Asian traditional medicine and cooking, which has many and diverse biological effects and is found incorporated in membranes. We have studied the mode in which curcumin modulates the physical properties of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) multilamellar membranes and 1, 2-dielaidoyl-sn-glycero-3-phosphoetnanolamine (DEPE). Curcumin disordered DPPC membranes at temperatures below Tc as seen through DSC, FT-IR, 2H-NMR, WAXD and SAXD. The decrease induced in Tc, suggests that curcumin is oriented in the bilayer with its main axis parallel to the acyl chains. Above Tc curcumin also introduced disordering as seen by FT-IR. FT-IR also showed that curcumin alters the conformation of the polar group of DPPC, increasing the percentage of unhydrated C=O groups, but however it does not form hydrogen bonds with neither the C=O group nor the phosphate group of DPPC. SAXD showed a remarkable increase in the repeating spacings by the presence of curcumin probably indicating the formation of a ripple phase. A partial phase diagram was built, which suggest the formation of a phospholipid/curcumin complex given place to immiscibilities in both the fluid and the rigid states, between curcumin and DPPC. Additionally DEPE was used to test the effect of curcumin on its polymorphism, and it was found that the temperature at which HII phase is formed was decreased, indicating that curcumin favours negative curvature of the membrane, which may be important to explain its effect on membrane dynamics and on membrane proteins or on proteins which may be activated through membrane insertion.

Fourier transform infrared spectroscopy has been used to study [1- 13 C]palmitic acid and l -α-[ dipalmitoyl -D31 2 ]phosphatidylcholine mixtures. The specific isotope labelling of these molecules has made possible the individualized study of the most characteristic bands originating from both lipids in the mixture. It has been possible, thanks to the labelling of palmitic acid, to directly determine its apparent p K in dDPPC, which was found to be ∼8.7 at 25°C by measuring the intensities of the unionized and ionized carboxylic bands of [1- 13 C]palmitic acid in the infrared spectra. The phase transitions of both l -α-[ dipalmitoyl -D31 2 ]phosphatidylcholine and [1- 13 C]palmitic acid in aqueous mixtures at different molar ratios were observed to occur at very similar temperatures at bulk pH 7.4, i.e., when the fatty acid was protonated by more than 95%, providing evidence of a nearly ideal mixing behavior. However, they occurred at well differentiated temperatures at bulk pH 11.0, i.e., when the fatty acid was ionized by more than 99%, showing that a phase separation between the two types of molecules occurred. Hence, the protonation state of the fatty acid affects its miscibility with the phospholipid. On the other hand, the fatty acid did not appear to establish hydrogen bonds with the phospholipid as monitored through the absorption bands due to the ester carbonyl and phosphate groups of the phospholipid. A significant effect that was noticed on the phospholipid was the increase in the proportion of the ester carbonyl groups in the dehydrated state as a consequence of the presence of palmitic acid. The dehydration effect was more considerable at neutral than at alkaline pH.

Diethylstilbestrol is a potent inhibitory agent of the Ca(2+)-ATPase activity of sarcoplasmic reticulum membranes. Other structurally related molecules, such as dienestrol or hexestrol having hydroxyl groups at para positions of the two benzene rings produce similar effects. The absence or derivatization of the hydroxyl groups as occurs with trans-stilbene or diethylstilbestrol dipropionate converts the structure in an activating agent of the enzyme. The Ca2+ transport profiles in the presence of the referred drugs reproduces the same behavior observed for the hydrolytic activity. There is also a clear indication of a membrane-mediated mechanism of these drugs. Ligand binding experiments at equilibrium indicate that diethylstilbestrol decreases the affinity for Ca2+ of the high affinity Ca2+ sites. Functional studies reveal that the activation/inhibition induced by these drugs is correlated with decreased levels of phosphoenzyme at steady state, and these levels are sensitive to the Ca2+ concentration. Chase experiments of [32P]phosphoenzyme and 45Ca2+ indicate a slight activation effect of diethylstilbestrol dipropionate on Ca2+ dissociation during the enzyme turnover. The use of different anthroyloxy derivatives of stearic acid as a fluorescent probe suggest that diethylstilbestrol and other inhibitory agents could be located close to the polar region of the lipid bilayer, which interferes with the Ca(2+)-binding sites, whereas the activators trans-stilbene and diethylstilbestrol dipropionate may have a deeper position into the membrane, which accelerates the Ca2+ translocation process.

C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca2+-binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKCα-C2 domain in complex with Ca 2+, 1,2-dihexanoyl-sn-glycero-3-[phospho-L-serine] (PtdSer), and 1,2-diayl-sn-glycero-3-[phosphoinositol-4,5-bisphos-phate] [Ptdlns(4,5)P2] shows that PtdSer binds specifically to the calcium-binding region, whereas Ptdlns(4,5)P2 occupies the concave surface of strands β3 and β4. Strikingly, the structure reveals a Ptdlns(4,5)P2-C2 domain-binding mode in which the aromatic residues Tyr-195 and Trp-245 establish direct interactions with the phosphate moieties of the inositol ring. Mutations that abrogate Tyr-195 and Trp-245 recognition of Ptdlns(4,5)P2 severely impaired the ability of PKCα to localize to the plasma membrane. Notably, these residues are highly conserved among C2 domains of topology I, and a general mechanism of C2 domain-membrane docking mediated by Ptdlns(4,5)P 2 is presented., Work in Murcia was supported by the Fundación Médica Mutua Madrileña, Fundación Ramón Areces, and Fundación Séneca 08700/PI/08 (to S.C.-G.), and Ministerio de Ciencia e Innovación Grants BFU2005-02482 and BFU2008-01010 (to J.G.-F.). Work in Barcelona was supported by Ministerio de Ciencia e Innovación Grants BFU2005-02376/BMC (to N.V.) and BFU2005-08686-C02-01 (to I.F.).Financial support was provided by the European Synchrotron Radiation Facility

The effect of low concentrations of lysophosphatidylcholine (LPC), platelet-activating factor (PAF) and other surfactants (Triton X-100, C 12 E 8 , sodium dodecyl sulfate, sodium cholate and sodium deoxycholate) on membrane permeability of native sarcoplasmic reticulum vesicles and sarcoplasmic reticulum lipid vesicles, has been studied. Triton X-100, C 12 E 8 , sodium dodecyl sulfate, sodium cholate and sodium deoxycholate were all able to permeabilize membranes at concentrations of surfactants below their critical micellar concentration (CMC) in both lipid and native vesicles, being the K 0.5 of calcium release from native vesicles lower than that from lipid vesicles. The values of these K 0.5 were well correlated with the corresponding CMC values for each type of membrane. However, both LPC and PAF behaved in a different way since, although they induced permeabilization of the native vesicles at values of K 0.5 close to their CMC, their K 0.5 values for permeabilizing vesicles, prepared by using lipids extracted from sarcoplasmic reticulum, were much higher than their corresponding CMC

The effect of edelfosine (1- O-octadecyl-2- O-methyl-rac-glycero-3-phosphocholine or ET-18-OCH3) on model membranes containing 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine/sphingomyelin/cholesterol (POPC/SM/cholesterol) was studied by several physical techniques. The sample POPC/SM (1:1 molar ratio) showed a broad phase transition as seen by DSC, X-ray diffraction, and 2H NMR. The addition of edelfosine to this sample produced isotropic structures at temperatures above the phase transition, as seen by 2H NMR and by 31P NMR. When cholesterol was added to give a POPC/SM/cholesterol (at a molar ratio 1:1:1), no transition was observed by DSC nor X-ray diffraction, and 2H NMR indicated the presence of a liquid ordered phase. The addition of 10 mol % edelfosine increased the thickness of the membrane as seen by X-ray diffraction and led to bigger differences in the values of the molecular order of the membrane detected at high and low temperatures, as detected through the M 1 first spectral moment from 2H NMR. These differences were even greater when 20 mol % edelfosine was added, and a transition was now clearly visible by DSC. In addition, a gel phase was clearly indicated by X-ray diffraction at low temperatures. The same technique pointed to greater membrane thickness in this mixture and to the appearance of a second membrane structure, indicating the formation of two separated phases in the presence of edelfosine. All of these data strongly suggest that edelfosine associating with cholesterol alter the phase status present in a POPC/SM/cholesterol (1:1:1 molar ratio) mixture, which is reputed to be a model of a raft structure. However, cell experiments showed that edelfosine colocalizes in vivo with rafts and that it may reach concentrations higher than 20 mol % of total lipid, indicating that the concentrations used in the biophysical experiments were within what can be expected in a cell membrane. The conclusion is that molecular ways of action of edelfosine in cells may involve the modification of the structure of rafts.

The distribution of free fatty acids at equilibrium after incubation of small sonicated unilamellar vesicles (SUV) with large unilamellar vesicles (LUV) of different lipid composition has been determined. Stearic acid (SA) and oleic acid (OA) showed similar preferences for SUV and LUV of egg yolk phosphatidylcholine (EYPC). Both ionized and protonated forms of the free fatty acids (FFAs) behaved similarly with respect to the equilibrium distribution between EYPC of different size. The charge of the vesicles was found, however, to be important, since both FFAsin their ionized form preferentially associated to vesicles of phosphatidylcholine (PC) as compared with vesicles of phosphatidylglycerol (PC). While SA preferred membranes in the gel state, OA showed preference for the membrane in fluid state. The insertion of both OA and SA in phosphatidylethanolamine (PE)/ phosphatidylcholine vesicles is less favourable than in vesicles of pure PC. All these data suggest that membrane lipid content may play a role in determining the distribution of free fatty acids among the membranes of a cell.

The C2 domains of cPKCs [classical/conventional PKCs (protein kinase Cs)] bind to membranes in a Ca 2+ -dependent manner and thereby act as cellular Ca 2+ effectors. Recent findings have demonstrated that the C2 domain of cPKCs interacts specifically with PtdIns(4,5) P 2 through its polybasic cluster located in the β3–β4-strands, this interaction being critical for the membrane localization of these enzymes in living cells. In addition, these C2 domains exhibit higher affinity to bind PtdIns(4,5) P 2 than any other polyphosphate phosphatidylinositols. It has also been shown that the presence of PtdIns(4,5) P 2 in model membranes decreases the Ca 2+ concentration required for classical C2 domains to bind them. Overall, the studies reviewed here suggest a new mechanism of membrane docking by the C2 domains of cPKCs in which the local densities of phosphatidylserine and PtdIns(4,5) P 2 on the inner leaflet of the plasma membrane are sufficient to drive Ca 2+ -activated membrane docking during a physiological Ca 2+ signal.

Diacylglycerols are second messengers confined to biomembranes and, although relatively simple molecules from the structural point of view, they are able of triggering a surprisingly wide range of biological responses. Diacylglycerols are recognized by a well conserved protein motif, such as the C1 domain. This domain was observed for the first time in protein kinases C but is now known to be present in many other proteins. The effect of diacylglycerols is not limited to binding to C1 domains and they are able to alter the biophysical properties of biomembranes and hence modulate the activity of membrane associated proteins and also facilitate some processes like membrane fusion.

The C2 domain is a targeting domain that responds to intracellular Ca 2+ signals in classical protein kinases (PKCs) and mediates the translocation of its host protein to membranes. Recent studies have revealed a new motif in the C2 domain, named the lysine-rich cluster, that interacts with acidic phospholipids. The purpose of this work was to characterize the molecular mechanism by which PtdIns(4,5)P 2 specifically interacts with this motif. Using a combination of isothermal titration calorimetry, fluorescence resonance energy transfer and time-lapse confocal microscopy, we show here that Ca 2+ specifically binds to the Ca 2+ -binding region, facilitating PtdIns(4,5)P 2 access to the lysine-rich cluster. The magnitude of PtdIns(4,5)P 2 binding is greater than in the case of other polyphosphate phosphatidylinositols. Very importantly, the residues involved in PtdIns(4,5)P 2 binding are essential for the plasma membrane localization of PKCα when RBL-2H3 cells are stimulated through their IgE receptors. Additionally, CFP-PH and CFP-C1 domains were used as bioprobes to demonstrate the co-existence of PtdIns(4,5)P 2 and diacylglycerol in the plasma membrane, and it was shown that although a fraction of PtdIns(4,5)P 2 is hydrolyzed to generate diacylglycerol and IP 3 , an important amount still remains in the membrane where it is available to activate PKCα. These findings entail revision of the currently accepted model of PKCα recruitment to the membrane and its activation.

The effect of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3, edelfosine), and six other analog asymmetric phosholipids on the physical properties of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) model membranes was studied using differential scanning calorimetry (DSC), 31P-nuclear magnetic resonance (31P NMR) and X-ray diffraction. DSC data revealed that, at concentrations of 40 mol% and higher, a new type of mixtures with higher Tc and narrower transitions appeared with all the asymmetric lipids studied. At very high concentrations of these lipids (50-80 mol%), destabilization was observed in the systems probably because of the formation of micelles or small vesicles. In all cases, the asymmetric lipids at concentrations of 40 mol% induced the formation of interdigitated structures in the lamellar gel phase, as deduced from X-ray diffraction. The asymmetric phospholipids were also added to 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) model membranes and DSC data revealed that the lipids primarily affected transition from the lamellar gel (Lβ) to the lamellar liquid crystalline (Lα) phase in two aspects: the transition temperature was reduced, and the transition itself became broader and smaller. The lamellar liquid crystalline (Lα) to inverted hexagonal phase (HII) transition was also affected, as detected by DSC and 31P NMR data. Increasing concentrations of the asymmetric lipids reduced the formation of inverted hexagonal phases, which were completely inhibited in the case of ET-18-OCH3. Since these compounds have been shown to have important biological actions through the plasma membrane, these results may help to understand the mechanism of action of these compounds. In addition these asymmetric lipids were tested for their capacity to induce cell apoptosis, and only ET-18-OCH3 was found to have a clear effect, thus suggesting that the apoptotic effect is not exerted through changes in the biophysical properties of model membranes. © 2006 Elsevier B.V. All rights reserved., This paper was supported by Grant 1FD97-0622 from the European Commission and Comisión Interministerial de Ciencia y Tecnología, Grant BFU2005-02482/BMC (to J.C.G.F.) from Dirección General de Investigación (Spain), Grant 00591/PI/04 (to J.C.G.F.) from Fundación Séneca (Comunidad Autónoma de Murcia, Spain), and Grant FIS04-0843 from Fondo de Investigación Sanitaria (to F.M.). C.G. and S.C.-G were supported by the Ramón y Cajal Program from the Ministerio de Educación y Ciencia of Spain.

All-trans-retinoic acid (atRA) is a derivative of vitamin A and possesses antitumor activity. We demonstrate that atRA is able to modulate the activity of protein kinase C alpha (PKCalpha), which is related to tumor development. In vitro, it was found that atRA activated PKCalpha in the presence of Ca(2+) and in the absence of phosphatidylserine, although such activity is considerably inhibited in mutations affecting residues D246 and D248 and also residue N189, all of which are known to be essential for the interaction with Ca(2+) and phosphatidylserine in the C2 domain. It was concluded that atRA substitutes phosphatidylserine although with lower specific activities. However, atRA had a biphasic effect on PKCalpha activity in the presence of activating phospholipids, such as phosphatidylserine and phosphatidylinositol 4,5-bisphosphate, yielding activation at low concentrations but inactivation at higher ones. This second inhibitory characteristic was not shown with K209 and K211 mutations (residues located in the Lys-rich cluster in the C2 domain) in PKCalpha. This interesting effect revealed the importance of phospholipid binding at this site to ensure maximum activity for the wild-type PKCalpha. The C1 domain was not related with the atRA effect on PKCalpha. It was concluded that whereas atRA may activate PKCalpha through the Ca(2+)-phosphatidylserine-binding site of the C2 domain, it may also inhibit the activity of this enzyme when displacing the phospholipid from the Lys-rich cluster also located in the C2 domain.