Search

PKCe is highly expressed in mast cells and plays a fundamental role in the antigen-triggered activation of the allergic reaction. Although its regulation by diacylglycerols has been described, its regulation by acidic phospholipids and how this regulation leads to the control of downstream vesicle secretion is barely known. Here, we used structural and evolutionary studies to find the molecular mechanism that explains the selectivity of the C1B domain of PKCe by Phosphatidic Acid (PA). This resided in a collection of Arg residues that form a specific rim on the outer surface of the C1B domain, around the diacylglycerol binding cleft. In RBL-2H3 cells, this basic rim allowed the kinase to respond specifically to phosphatidic acid signals that induced its translocation to the plasma membrane and subsequent activation. Further experiments in cells that overexpress PKCe and a mutant of the PA binding site, showed that PA-dependent PKCe activation increased vesicle degranulation in RBL-2H3 cells, and this correlated with increased SNAP23 phosphorylation. Over-expression of PKCe in these cells also induced an increase in the number of docked vesicles containing SNAP23, when stimulated with PA. This accumulation could be attributed to the stabilizing effect of phosphorylation on the formation of the SNARE complex, which ultimately led to increased release of content in the presence of Ca2+ during the fusion process. Therefore, these findings reinforce the importance of PA signaling in the activation of PKCe, which could be an important target to inhibit the exacerbated responses of these cells in the allergic reaction.

Vitamin K1 and vitamin K2 play very important biological roles as members of chains of electron transport as antioxidants in membranes and as cofactors for the posttranslational modification of proteins that participate in a number of physiological functions such as coagulation. The interaction of these vitamins with dimyristoylphosphatidylcholine (DMPC) model membranes has been studied by using a biophysical approach. It was observed by using differential scanning calorimetry that both vitamins have a very limited miscibility with DMPC and they form domains rich in the vitamins at high concentrations. Experiments using X-ray diffraction also showed the formation of different phases as a consequence of the inclusion of either vitamin K at temperatures below the phase transition. However, in the fluid state, a homogeneous phase was detected, and a decrease in the thickness of the membrane was accompanied by an increase in the water layer thickness. 2H NMR spectroscopy showed that both vitamins K induced a decrease in the onset of the phase transition, which was bigger for vitamin K1, and both vitamins decreased the order of the membrane as seen through the first moment (M1). 1H NOESY MAS-NMR showed that protons located at the rings or at the beginning of the lateral chain of both vitamins K interacted with a clear preference with protons located in the polar part of DMPC. On the other hand, protons located on the lateral chain have a nearer proximity with the methyl end of the myristoyl chains of DMPC. In agreement with the 2H NMR, ATR-FTIR (attenuated total reflectance Fourier transform infrared spectroscopy) indicated that both vitamins decreased the order parameters of DMPC. It was additionally deduced that the lateral chains of both vitamins were oriented almost in parallel to the myristoyl chains of the phospholipid.

Aim: Collagen-enriched transfersomes, glycerosomes and glytransfersomes were specifically tailored for skin delivery of oleuropein. Methods: Vesicles were prepared by direct sonication and their main physicochemical and technological properties were measured. Biocompatibility, protective effect and promotion of the healing of a wounded cell monolayer were tested in vitro using fibroblasts. Results: Vesicles were mainly multicompartment, small (∼108 nm), slightly polydispersed (approximately 0.27) and negatively charged (~-49 mV). Oleuropein was incorporated in high amounts (approximately 87%) and vesicles were stable during four months of storage. In vitro studies confirmed the low toxicity of formulations (viability ≥95%), their effectiveness in counteracting nitric oxide generation and damages caused by free oxygen radicals, especially when collagen glytransfersomes were used (viability ~100%). These vesicles also promoted the regeneration of a wounded area by promoting the proliferation and migration of fibroblasts. Conclusion: Collagen-enriched vesicles are promising formulations capable of speeding up the healing of the wounded skin.

The C2 domain of novel protein kinases C (nPKC) binds to membranes in a Ca2+-independent way contributing to the activation of these enzymes. We have studied the C2 domain of one of these nPKCs, namely PKCe, and confirmed that it establishes a strong interaction with POPA, which is clearly visible through changes in chemical shifts detected through 31P-MAS-NMR and the protection that it exerts on the domain against thermal denaturation seen through DSC and FT-IR. In this study, using two-dimensional correlation analysis (2D-COS) applied to infrared spectra, we determined the sequence of events that occur during the thermal unfolding of the domain and highlighted some differences when phosphatidic acid or cardiolipin are present. Finally, by means of FRET and DLS experiments, we wanted to determine the effect of membrane curvature on the domain/membrane interaction by using lysophosphatidylcholine to introduce positive curvature as a control and we observed that the effect of these phospholipids on the protein binding is not exerted through the change of membrane curvature.

This commentary provides a short description of IUPAB’s past and present activities from the IUPAB’s Secretary General, Dr. Juan C. Gómez-Fernández.

A comparative study of three synthetic peptides, namely neutral Cecropin D-like G. mellonella (WT) and two cationic peptides derived from its sequence, ΔM1 (+5) and ΔM2 (+9) is reported in this work. The influence of charge on the interactions between peptides and membranes and its effect on phase were studied by calorimetric assays. Differential scanning calorimetry (DSC) showed that ΔM2 peptide showed the strongest effect when the membrane contained phosphatidylcholine (PC) and phosphatidylglycerol (PG), increasing membrane fluidization. Fourier transform infrared spectroscopy (FTIR) was used to determine lipid segregation in the presence of peptides. When WT and ΔM1 bound to model membrane containing PG and PC (1:1 molar ratio) a separation of both lipids was observed. Meanwhile, ΔM2 peptide also induced a demixing of PG-peptide rich domains separated from PC. FTIR experiments also suggested that the presence of ΔM1 and ΔM2 peptides increased lipid carbonyl group hydration in DMPG membrane fluid phase, However, hydration at the interface level in fluid phase was notably increased in the presence of WT and ΔM1 peptides in DMPC/DMPG. Overall the increase in positively charged residues favors the interaction of the peptides with the negatively charged membrane and its perturbation.

New three-dimensionally-structured hybrid phospholipid vesicles, able to load clotrimazole in a high amount (10 mg/mL), were obtained for the first time in this work by significantly reducing the amount of water (&le, 10%), which was replaced with a mixture of glycerol and ethanol (&asymp, 90%). A pre-formulation study was carried out to evaluate the effect of both the composition of the hydrating medium and the concentration of the phospholipid on the physico-chemical properties of hybrid vesicles. Four different three-dimensionally-structured hybrid vesicles were selected as ideal systems for the topical application of clotrimazole. An extensive physico-chemical characterization performed using transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM), 31P-NMR, and small-angle X-ray scattering (SAXS) displayed the formation of small, multi-, and unilamellar vesicles very close to each other, and was capable of forming a three-dimensional network, which stabilized the dispersion. Additionally, the dilution of the dispersion with water reduced the interactions between vesicles, leading to the formation of single unilamellar vesicles. The evaluation of the in vitro percutaneous delivery of clotrimazole showed an improved drug deposition in the skin strata provided by the three-dimensionally-structured vesicles with respect to the commercial cream (Canesten&reg, ) used as a reference. Hybrid vesicles were highly biocompatible and showed a significant antifungal activity in vitro, greater than the commercial cream Canesten&reg, The antimycotic efficacy of formulations was confirmed by the reduced proliferation of the yeast cells at the site of infection in vivo. In light of these results, clotrimazole-loaded, three-dimensionally-structured hybrid vesicles appear to be one of the most innovative and promising formulations for the treatment of candidiasis infections.

Curcumin and two bivalent compounds, namely 17MD and 21MO, both obtained by conjugation of curcumin with a steroid molecule that acts as a membrane anchor, were comparatively studied. When incorporated into 1,2-dipalmitoyl-sn-glycero-3-phosphocholine the compounds showed a very limited solubility in the model membranes. Curcumin and the two bivalent compounds were also incorporated in membranes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and quenching the fluorescence of pure curcumin or of the curcumin moiety in the bivalent compounds by acrylamide it was seen that curcumin was accessible to this water soluble quencher but the molecule was somehow located in a hydrophobic environment. This was confirmed by quenching with doxyl-phosphatidylcholines, indicating that the curcumin moieties of 17MD and 21MO were in a more polar environment than pure curcumin itself. NOESY-MAS-(1)HNMR analysis supports this notion by showing that the orientation of curcumin was parallel to the plane of the membrane surface close to C2 and C3 of the fatty acyl chains, while the curcumin moiety of 17MD and 21MO positioned close to the polar part of the membrane with the steroid moiety in the centre of the membrane. Molecular dynamics studies were in close agreement with the experimental results with respect to the likely proximity of the protons studied by NMR and show that 17MD and 21MO have a clear tendency to aggregate in a fluid membrane. The anchorage of the bivalent compounds to the membrane leaving the curcumin moiety near the polar part may be very important to facilitate the bioactivity of the curcumin moiety when used as anti-Alzheimer drugs.

Idebenone is a synthetic analog of coenzyme Q; both share a quinone moiety but idebenone has a shorter lipophilic tail ending with a hydroxyl group. Differential scanning calorimetry experiments showed that both idebenone and idebenol widened and shifted the phase transition of 1,2-dipalmitoylphosphatidylcholine (DPPC) to a lower temperature and a phase separation with different concentrations of these molecules was observed. Also small angle X-ray diffraction and wide angle X-ray diffraction revealed that both, idebenone and idebenol, induced laterally separated phases in fluid membranes when included in DPPC membranes. Electronic profiles showed that both forms, idebenone and idebenol, reduced the thickness of the fluid membrane. (2)H NMR measurements showed that the order of the membrane decreased at all temperatures in the presence of idebenone or idebenol, the greatest disorder being observed in the segments of the acyl chains close to the lipid-water interface. (1)H NOESY MAS NMR spectra were obtained using 1-palmitoyl-2-oleoyl-phosphatidylcholine membranes and results pointed to a similar location in the membrane for both forms, with the benzoquinone or benzoquinol rings and their terminal hydroxyl group of the hydrophobic chain located near the lipid/water interface of the phospholipid bilayer and the terminal hydroxyl group of the hydrophobic chain of both compounds located at the lipid/water interface. Taken together, all these different locations might explain the different physiological behavior shown by the idebenone/idebenol compared with the ubiquinone-10/ubiquinol-10 pair in which both compounds are differently localized in the membrane.

α-Tocopherol is considered to carry on a very important role as antioxidant of membranes and lipoproteins and other biological roles as membrane stabilizers and bioactive lipids. Given its essential role, it is very important to fully understand its location in the membrane. In this work, the vertical location of vitamin E in saturated membranes has been studied using biophysical techniques. Small- and wide-angle X-ray diffraction experiments show that α-tocopherol alters the water layer between bilayers in both DMPC and DPPC, indicating its proximity to this surface. The quenching of the intrinsic fluorescence of α-tocopherol indicates a low quenching efficiency by acrylamide and a higher quenching by 5-doxyl-PC than by 9- and 16-doxyl-PC. These results suggest that in both DMPC and DPPC membranes the chromanol ring is not far away from the surface of the membrane but within the bilayer. 1H-NMR-NOESY MAS-NMR studies showed that α-tocopherol is localized similarly in DMPC and DPPC membranes, with the chro...

Membrane fusion is essential in a myriad of eukaryotic cell biological processes, including the synaptic transmission. Rabphilin-3A is a membrane trafficking protein involved in the calcium-dependent regulation of secretory vesicle exocytosis in neurons and neuroendocrine cells, but the underlying mechanism remains poorly understood. Here, we report the crystal structures and biochemical analyses of Rabphilin-3A C2B–SNAP25 and C2B–phosphatidylinositol 4,5-bisphosphate (PIP2) complexes, revealing how Rabphilin-3A C2 domains operate in cooperation with PIP2/Ca2+ and SNAP25 to bind the plasma membrane, adopting a conformation compatible to interact with the complete SNARE complex. Comparisons with the synaptotagmin1–SNARE show that both proteins contact the same SNAP25 surface, but Rabphilin-3A uses a unique structural element. Data obtained here suggest a model to explain the Ca2+-dependent fusion process by membrane bending with a myriad of variations depending on the properties of the C2 domain-bearing protein, shedding light to understand the fine-tuning control of the different vesicle fusion events., Work in Barcelona was supported by Grants BIO2014-54588-P [Ministry of Economy and Competitiveness (MINECO), Spain–European Fund for Economic and Regional Development (FEDER)] and Maria de Maeztu Unit of Excellence MDM-2014-0435. Work in Murcia was supported by Grants BFU2014-52269-P (MINECO, Spain–FEDER) and Fundación Séneca Region de Murcia 19409/PI/14. X-ray data were collected at ALBA-CELLS (beamline XALOC) (Cerdanyola del Valles, Barcelona, Spain) with the collaboration of ALBA staff and at ESRF beamline ID23.2 (Grenoble, France). Financial support was also provided by ALBA and ESRF.

α-Tocopherol is a natural preservative that prevents free radical chain oxidations in biomembranes. We have studied the location of α-tocopherol in model membranes formed by different unsaturated phosphatidylcholines, namely 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC). Small angle X-ray diffraction revealed that α-tocopherol was well mixed with all the phospholipids. In all the cases only one lamellar phase was detected. Very modest changes occasioned by α-tocopherol were observed in the electron density profiles. The results obtained from quenching of α-tocopherol intrinsic fluorescence by acrylamide showed that this vitamin was inefficiently quenched in the four types of membranes, indicating that the fluorescent chromanol ring was poorly accessible for this hydrophilic quencher. Compatible with that, quenching by doxyl derivatives of phosphatidylcholines indicated that the chromanol ring was close in the four membranes to the nitroxide probe located at position 5. Quenching by doxyl-phosphatidylcholines also indicated that the efficiency of quenching was higher in POPC than in the other unsaturated phospholipids. 1H-MAS-NMR showed that α-tocopherol induced chemical shifts of protons from the phospholipids, especially of those bonded to carbons 2 and 3 of the acyl chains of the four phospholipids studied. The 1H-MAS-NMR NOESY results suggested that the lower part of the chromanol ring was located between the C3 of the fatty acyl chains and the centre of the hydrophobic monolayer for the four phospholipid membranes studied. Taken together, these results suggest that α-tocopherol is located, in all the membranes studied, with the chromanol ring within the hydrophobic palisade but not far away from the lipid–water interface.

Sphingosine [(2S, 3R, 4E)-2-amino-4-octadecen-1, 3-diol] is the most common sphingoid long chain base in sphingolipids. It is the precursor of important cell signaling molecules, such as ceramides. In the last decade it has been shown to act itself as a potent metabolic signaling molecule, by activating a number of protein kinases. Moreover, sphingosine has been found to permeabilize phospholipid bilayers, giving rise to vesicle leakage. The present contribution intends to analyze the mechanism by which this bioactive lipid induces vesicle contents release, and the effect of negatively charged bilayers in the release process. Fluorescence lifetime measurements and confocal fluorescence microscopy have been applied to observe the mechanism of sphingosine efflux from large and giant unilamellar vesicles; a graded-release efflux has been detected. Additionally, stopped-flow measurements have shown that the rate of vesicle permeabilization increases with sphingosine concentration. Because at the physiological pH sphingosine has a net positive charge, its interaction with negatively charged phospholipids (e.g., bilayers containing phosphatidic acid together with sphingomyelins, phosphatidylethanolamine, and cholesterol) gives rise to a release of vesicular contents, faster than with electrically neutral bilayers. Furthermore, phosphorous 31-NMR and x-ray data show the capacity of sphingosine to facilitate the formation of nonbilayer (cubic phase) intermediates in negatively charged membranes. The data might explain the pathogenesis of Niemann-Pick type C1 disease.

Lipid lateral segregation into specific domains in cellular membranes is associated with cell signaling and metabolic regulation. This phenomenon partially arises as a consequence of the very distinct bilayer-associated lipid physico-chemical properties that give rise to defined phase states at a given temperature. Until now lamellar gel (Lβ) phases have been described in detail in single or two-lipid systems. Using x-ray scattering, differential scanning calorimetry, confocal fluorescence microscopy, and atomic force microscopy, we have characterized phases of ternary lipid compositions in the presence of saturated phospholipids, cholesterol, and palmitoyl ceramide mixtures. These phases stabilized by direct cholesterol-ceramide interaction can exist either with palmitoyl sphingomyelin or with dipalmitoyl phosphatidylcholine and present intermediate properties between raft-associated phospholipid-cholesterol liquid-ordered and phospholipid-ceramide Lβ phases. The present data provide novel, to our knowledge, evidence of a chemically defined, multicomponent lipid system that could cooperate in building heterogeneous segregated platforms in cell membranes.

The C2 domain of PKCε binds to negatively charged phospholipids but little is known so far about the docking orientation of this domain when it is bound. By using a FRET assay we have studied the binding of this domain to model membranes. We have also used ATR-Fourier transform infrared spectroscopy with polarized light (ATR-FTIR) to determine the docking mode by calculating the β-sandwich orientation when the domain is bound to different types of model membranes. The vesicle lipid compositions were: POPC/POPE/POPA (22:36:42) imitating the inner leaflet of a plasma membrane, POPC/POPA (50:50) in which POPE has been eliminated with respect to the former composition and POPC/POPE/CL (43:36:21) imitating the inner mitochondrial membrane. Results show that the β-sandwich of the PKCα-C2 domain is inclined at an angle α close to 45° to the membrane normal. Some differences were found with respect to the extent of binding as a function of phospholipid composition and small changes on secondary structure were only evident when the domain was bound to model membranes of POPC/POPA: in this case, the percentage of β-sheet of the C2 domain increases if compared with the secondary structure of the domain in the absence of vesicles. With respect to the β-sandwich orientation, when the domain is bound to POPC/POPE/CL membranes it forms an angle with the normal to the surface of the lipid bilayer (39°) smaller than that one observed when the domain interacts with vesicles of POPC/POPA (49°).

Background Formulations incorporating nanoparticles (NPs) are widely used to prolong drug release. In this regard, poly(lactide-co-glicolide) (PLGA) is often used in their preparation due to its high degree of biocompatibility and biodegradability. In the present study, morphine HCl is incorporated in PLGA-NPs and different preparation alternatives are evaluated for their effects on the properties, stability and capacity of encapsulation. Methods NPs were prepared by a double emulsion solvent diffusion-ammonium loading (DESD-AL) or double emulsion solvent diffusion-traditional (DESD-T) technique. NP morphology, size, zeta potential and encapsulation efficiency were investigated. In vitro studies were performed in phosphate buffer pH 7.4 at 37 oC and deionized water at 4oC. Adult male Swiss mice were used to study the pharmacokinetic behavior in vivo. Results Our results show that DESD-AL provides a higher level of morphine entrapment and that increasing the sonication time reduces the size but does not appreciably reduce the entrapment percentage. It was also observed that NP stability was greater when Pluronic F68 was used rather than PVA, and that in vitro assays provided better results with low concentrations of both stabilizers. Lyophilized NPs, after rehydration showed properties that were only slightly different from those of the untreated ones, with no sign of precipitation or aggregation. Finally, the obtained NPs enhanced morphine bioavailability. Conclusions In conclusion, a useful method for encapsulating morphine in order to obtain an extended delivery period is described and its effects are compared with those of the free drug.

Protein kinase Cα (PKCα) is activated by its translocation to the membrane. Activity assays show the importance of PIP(2) in determining the specific activity of this enzyme. A FRET stopped flow fluorescence study was carried out to monitor the rapid kinetics of protein binding to model membranes containing POPC/POPS/DOG and eventually PIP(2). The results best fitted a binding mechanism in which protein bound to the membrane following a two-phase mechanism with a first bimolecular reaction followed by a slow unimolecular reaction. In the absence of PIP(2), the rapid protein binding rate was especially dependent on POPS concentration. Formation of the slow high affinity complex during the second phase seems to involve specific interactions with POPS and DOG since it is only sensitive to changes within relatively low concentration ranges of these lipids. Both the association and dissociation rate constants fell in the presence of PIP(2). We propose a model in which PKCα binds to the membranes via a two-step mechanism consisting of the rapid membrane initial recruitment of PKCα driven by interactions with POPS and/or PIP(2) although interactions with DOG are involved too. PKCα searches on the lipid bilayer in two dimensions to establish interactions with its specific ligands.

The structural and stability properties of a novel zinc-dependent alcohol dehydrogenase from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII) were investigated by Fourier transformed infrared spectroscopy (FTIR). This enzyme is a thermostable homo-tetramer belonging to the GroES-fold motif proteins characterized by an irregular β-barrel conformation. Our results revealed a protein with a secondary structure rich in β-sheet (32% of the total secondary elements) and it showed a three-step thermal unfolding pathway. The complete enzyme denaturation was preceded by the formation of a relaxed tertiary/quaternary structure and previously by an excited native-like conformation. Two-dimensional correlation spectroscopy analysis (2D-COS) and differential scanning calorimetry (DSC) experiments supported these data and allowed us to determine the protein melting temperature at 96.9 °C as well as to suggest the sequence of the events that occurred during the protein denaturation process.

QCM-D technique is based on the physical phenomenon that generates an acoustic shear wave with an oscillating resonance in quartz resulting in an evanescent wave that arises at the interface of the quartz and the solution. The amplitude of the acoustic wave is influenced by the deposition of material onto the quartz surface and from the subsequent decrease of the frequency the bound mass can be calculated. The dissipation shift which arises inform about viscoelasticity and flexibility of the adsorbed material. QCM-D can be applied for real-time studies of several biological systems since it is a simple, fast, low-cost and sensitive technique without having to label any sample. Common applications in biological field include measurements on adsorption of lipids, proteins, DNA and cells directly onto the surface of the sensor, which generally are chemically modified by self-assembled monolayer (SAM) technique or by spin-coated polymers. QCM-D can also be used to study molecular interactions between macromolecules and adsorbed materials. Three examples of the use of this technique are presented, namely the docking orientation of the C2 domain of PKCe on phospholipid membranes, the conformational changes of fibrinogen adsorbed to model acrylic polymers and the attachment of endothelial cells to carboxylated polymers of different configuration.

Curcumin modulates the activity of protein kinase Cα (PKCα) when assayed in the presence of vesicles including phosphatidylcholine, phosphatidylserine and diacylglycerol. Increasing concentrations of curcumin progressively increased PKCα activity at concentrations lower than 20 μM, but at higher concentrations of curcumin the activity decreased although, at concentrations of curcumin of up to 100 μM the activity was always higher than the basal one (in the absence of curcumin). The maximum activity was reached at 3 μM curcumin, at 20 and 30 mol% of phosphatidylserine, 10 μM Ca2+ and 2 mol% diacylglycerol. The same type of modulation was observed when changing the concentration of phosphatidylserine, diacylglycerol and Ca2+. No effect of curcumin was found when the activity was assayed in the presence of Triton X-100 mixed micelles which included phosphatidylserine and diacylglycerol, indicating that the effect of curcumin was membrane-dependent. The pattern of binding of PKCα to membrane vesicles as a function of curcumin concentration closely correlated with the pattern of activating effect. It was concluded that the effect of curcumin on PKCα activity was related to its effect on the membrane, which may modulate the binding of the enzyme to the membrane.

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.

PKCα is a key mediator of the neuronal differentiation controlled by NGF and ATP. However, its downstream signaling pathways remain to be elucidated. To identify the signaling partners of PKCα, we analyzed proteins coimmunoprecipitated with this enzyme in PC12 cells differentiated with NGF and ATP and compared them with those obtained with NGF alone or growing media. Mass spectrometry analysis (LC-MS/MS) identified plectin, peripherin, filamin A, fascin, and β-actin as potential interacting proteins. The colocalization of PKCα and its interacting proteins increased when PC12 cells were differentiated with NGF and ATP. Peripherin and plectin organization and the cortical remodeling of β-actin were dramatically affected when PKCα was down-regulated, suggesting that all three proteins might be functional targets of ATP-dependent PKCα signaling. Taken together, these data demonstrate that PKCα is essential for controlling the neuronal development induced by NGF and ATP and interacts with the cytoskeletal components at two levels: assembly of the intermediate filament peripherin and organization of cortical actin.

The C-terminal domain of the pro-apoptotic protein Bax (Bax-C) is supposed to act as a membrane anchor motif when Bax is activated leading to programmed cell death. A synthetic peptide which imitates this domain has been used to study the mechanism of peptide-phospholipid interaction. We have used static and MAS-NMR techniques to show that the interaction of Bax-C with membranes is modulated by the presence of a negatively charged phospholipid like phosphatidylglycerol. Bax-C slightly shifted upfield the (31)P resonances coming from phosphatidylglycerol and phosphatidylcholine. However the width of the resonance peaks was considerably higher when phosphatidylglycerol was present. Bax-C substantially decreased the T(1) relaxation times of phosphatidylglycerol and those of phosphatidylcholine when mixtured with phosphatidylglycerol, but T(1) values were not decreased when phosphatidylcholine was the only phospholipid present in the membrane. (13)C-MAS-NMR showed that T(1) values were decreased when Bax-C was incorporated into the lipid vesicles and this reduction affected similarly to carbons located in different regions of the membrane when the only phospholipid present was phosphatidylcholine. However, when phosphatidylglycerol was also present, the decrease in T(1) affected considerably more to some carbons in the polar region. These results indicate that Bax-C interacts differently with the polar part of the membrane depending on whether phosphatidylglycerol is present or not, suggesting that an electrostatic interaction of Bax-C with the membrane determines the location of this domain. Fluorescence spectroscopy showed that the Trp residues of Bax-C were placed in a microenvironment more hydrophobic and less accessible to quenching by acrylamide when phosphatidylglycerol was present.

Fourier transform infrared spectroscopy (FTIR) was used to study the secondary structure of peptides which imitate the amino acid sequences of the C-terminal domain of the pro-apoptotic protein Bax (Bax-C) when incorporated into different lipid vesicles with or without negatively charged phospholipids. The infrared spectroscopy results showed that while the beta-sheet components are predominant in the membrane-free Bax-C secondary structure as well as in the presence of phosphatidylcholine vesicles, the peptide changes its secondary structure in the presence of negatively charged membranes, including phospholipids such as phosphatidylglycerol or phosphatidylinositol, depending on both the lipid composition and their molar ratio. The negative charges in the model membrane surface caused a marked change from beta-sheet to alpha-helix structure. Moreover, using attenuated total reflection infrared spectroscopy (ATR-FTIR), we investigated the orientation of Bax-C alpha-helical structures with respect to the normal to the internal reflection element. The orientation of Bax-C in membranes was also affected by negatively charged lipids, the presence of phosphatidylglycerol reduced the angle it forms with the normal to the germanium plate from 45 degrees in phosphatidylcholine to 27 degrees in phosphatidylglycerol vesicles. These results highlight the importance of lipid-protein interaction for the correct folding of membrane proteins and they suggest that the C-terminal domain of Bax will only span membranes with a net negative charge in their surface.

The interaction between oxidized (ubiquinone-10) and reduced (ubiquinol-10) coenzyme Q 10 with dimyristoylphosphatidylcholine has been examined by differential scanning microcalorimetry, X-ray diffraction, infrared spectroscopy, and (2)H NMR. Microcalorimetry experiments showed that ubiquinol-10 perturbed considerably more the phase transition of the phospholipids than ubiquinone-10, both forms giving rise to a shoulder of the main transition peak at lower temperatures. Small angle X-ray diffraction showed an increase in d-spacing suggesting a thicker membrane in the presence of both ubiquinone-10 and ubiquinol-10, below the phase transition and a remarkable broadening of the peaks indicating a loss of the repetitive pattern of the lipid multilamellar vesicles. Infrared spectroscopy showed an increase in wavenumbers of the maximum of the CH 2 stretching vibration at temperatures below the phase transition, in the presence of ubiquinol-10, indicating an increase in the proportion of gauche isomers in the gel phase, whereas this effect was smaller for ubiquinone-10. A very small effect was observed at temperatures above the phase transition. (2)H NMR spectroscopy of perdeuterated DMPC showed only modest changes in the spectra of the phospholipids occasioned by the presence of coenzyme Q 10. These small changes were reflected, in the presence of ubiquinol-10, by a decrease in resolution indicating that the interaction between coenzyme Q and phospholipids changed the motion of the lipids. The change was also visible in the first spectral moment (M1), which is related with membrane order, which was slightly decreased at temperatures below the phase transition especially with ubiquinol-10. A slight decrease in M 1 values was also observed above the phase transition but only for ubiquinol-10. These results can be interpreted to indicate that most ubiquinone-10 molecules are localized in the center of the bilayer, but a considerable proportion of ubiquinol-10 molecules may span the bilayer interacting more extensively with the phospholipid acyl chains.

Rapamycin-triggered heterodimerization strategy is becoming an excellent tool for rapidly modifying phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P2] levels at the plasma membrane and for studying their influence in different processes. In this work, we studied the effect of modulation of the PtdIns(4,5)P2 concentration on protein kinase C (PKC) alpha membrane localization in intact living cells. We showed that an increase in the PtdIns(4,5)P2 concentration enlarges the permanence of PKCalpha in the plasma membrane when PC12 cells are stimulated with ATP, independently of the diacylglycerol generated. The depletion of this phosphoinositide decreases both the percentage of protein able to translocate to the plasma membrane and its permanence there. Our results demonstrate that the polybasic cluster located in the C2 domain of PKCalpha is responsible for this phosphoinositide-protein interaction. Furthermore, the C2 domain acts as a dominant interfering module in the neural differentiation process of PC12 cells, a fact that was also supported by the inhibitory effect obtained by knocking down PKCalpha with small interfering RNA duplexes. Taken together, these data demonstrate that PtdIns(4,5)P2 itself targets PKCalpha to the plasma membrane through the polybasic cluster located in the C2 domain, with this interaction being critical in the signaling network involved in neural differentiation.

Bcl-2 family proteins are involved in the cell homeostasis by regulating programmed cell death. Some of these proteins promote apoptosis, while others inhibit the same process. The C-terminal hydrophobic domain of some of these proteins is predicted to be involved in anchoring them to a variety of cell membranes, such as mitochondrial, endoplasmic reticulum and nuclear membranes. We have used five synthetic peptides imitating the C-terminal domain from both anti-apoptotic (Bcl-2) and pro-apoptotic members (Bak, Bax, and two mutants of this last protein) of this family to study their interaction with model membranes. Some differences were detected in the interaction with these peptides. The addition of all the peptides to large unilamellar vesicles destabilized them and released encapsulated carboxyfluorescein to different degrees, so that fluidity and the increase in negative curvature favoured the extent in the release of carboxyfluorescein. Bcl-2-C and Bax-C peptides produced the highest release levels in most cases, while BaxS184K-C was the least efficient in this respect. These results indicate that these C-terminal domains are able to insert themselves in the membranes, each in a different way that is probably related with their different way which can be related to their differing locations within the cell and their different roles in regulating apoptosis.

C2 domains are conserved protein modules in many eukaryotic signaling proteins, including the protein kinase (PKCs). The C2 domains of classical PKCs bind to membranes in a Ca(2+)-dependent manner and thereby act as cellular Ca(2+) effectors. Recent findings suggest that the C2 domain of PKCalpha interacts specifically with phosphatidylinositols 4,5-bisphosphate (PtdIns(4,5)P(2)) through its lysine rich cluster, for which it shows higher affinity than for POPS. In this work, we compared the three C2 domains of classical PKCs. Isothermal titration calorimetry revealed that the C2 domains of PKCalpha and beta display a greater capacity to bind to PtdIns(4,5)P(2)-containing vesicles than the C2 domain of PKCgamma. Comparative studies using lipid vesicles containing both POPS and PtdIns(4,5)P(2) as ligands revealed that the domains behave as PtdIns(4,5)P(2)-binding modules rather than as POPS-binding modules, suggesting that the presence of the phosphoinositide in membranes increases the affinity of each domain. When the magnitude of PtdIns(4,5)P(2) binding was compared with that of other polyphosphate phosphatidylinositols, it was seen to be greater in both PKCbeta- and PKCgamma-C2 domains. The concentration of Ca(2+) required to bind to membranes was seen to be lower in the presence of PtdIns(4,5)P(2) for all C2 domains, especially PKCalpha. In vivo experiments using differentiated PC12 cells transfected with each C2 domain fused to ECFP and stimulated with ATP demonstrated that, at limiting intracellular concentration of Ca(2+), the three C2 domains translocate to the plasma membrane at very similar rates. However, the plasma membrane dissociation event differed in each case, PKCalpha persisting for the longest time in the plasma membrane, followed by PKCgamma and, finally, PKCbeta, which probably reflects the different levels of Ca(2+) needed by each domain and their different affinities for PtdIns(4,5)P(2).

The C2 domain from protein kinase Cepsilon (PKCepsilon) binds to membranes but does not require Ca2+ to do so. This work examines the mode in which the conformation and organization of the phospholipids present in model membranes are altered by the presence of the C2 domain from PKCepsilon (C2-PKCepsilon). It is concluded from the results of differential scanning calorimetry that the protein shifted the temperature of the gel to the fluid phase transition of pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), widening the transition and increasing it to a higher temperature. When POPA was mixed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), the changes in the transition were smaller and no phase separation was observed. Experiments performed using magic angle spinning NMR showed that this C2 domain specifically affected POPA when the phospholipid was mixed with POPC, as indicated by the downfield shift in the isotropic resonance of POPA, the widening of the resonance peak, the decrease in T2, and the decrease in T1 observed at all temperatures. All these effects were quite marked compared with the very small effect observed with POPC, indicating the specificity of the effect. The presence of the C2-PKCepsilon protein changed the conformation of the polar head group of POPA, as shown by infrared spectroscopy. All these results clearly illustrate the electrostatic interaction that takes place between this C2 domain and membranes which contain POPA in the absence of Ca2+.

One of the most powerful techniques used to gain structural information of membrane proteins is the Attenuated Total Reflectance Fourier Transformed Infrared spectroscopy (ATR-FTIR). Secondary structure, conformational changes, interactions with lipids and spatial positioning of membranes, proteins and peptides are commonly analyzed by this method that allows having a simultaneous vision of the membrane and the protein that is bound. In particular, ATR-FTIR is especially advantageous to evaluate the membrane lipids order and the effective position of a protein relative to the membrane. This technique has been successfully applied to study alpha-helical peptides and proteins, and also beta-sheet, beta-barrel and beta-sandwich proteins, but potentially it could be applicable for any protein structure suitable for a geometric approximation. The present review wants to examine and summarize the different models developed to calculate the orientation of these proteins giving some practical examples, analyzing lipid alignment, protein secondary structure and orientation.

Capsaicin is the chemical responsible for making some peppers spicy hot, but additionally it is used as a pharmaceutical to alleviate different pain conditions. Capsaicin binds to the vanilloid receptor TRPV1, which plays a role in coordinating chemical and physical painful stimuli. A number of reports have also shown that capsaicin inserts in membranes and its capacity to modify them may be part of its molecular mode of action, affecting the activity of other membrane proteins. We have used differential scanning calorimetry, X-ray diffraction, 31P NMR, and 2H NMR spectroscopy to show that capsaicin increases the fluidity and disorder of 1,2-palmitoyl-sn-glycero-3-phosphocholine membrane models. By using 1H NOESY MAS NMR based on proton-proton cross-peaks between capsaicin and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine resonances, we determined the location profile of this molecule in a fluid membrane concluding that it occupies the upper part of the phospholipid monolayer, between the lipid-water interface and the double bond of the acyl chain in position sn-2. This location explains the disorganization of the membrane of both the lipid-water interface and the hydrophobic palisade. © 2015 American Chemical Society.

Protein kinase C (PKC) is a member of a family of Ser/Thr phosphotransferases that are involved in many cellular signaling pathways. These enzymes possess two regulatory domains, C1 and C2, that are the targets of different second messengers. The purpose of this review is to describe in molecular terms the diverse mechanisms of activation of PKCs in the light of very significant advances made in this field over recent years. The role of some critical amino acid residues concerning activation of the enzymes and their location within known structures of isolated domains will be presented. For example, the recently deduced 3D structures of the C2 domains show that these domains can additionally act as PtdIns(4,5)P(2)-binding or phosphotyrosine-binding modules depending on the isoenzyme. All these capacities to play different roles in the cell wide web of signals underline the notion that we are dealing with a multifunctional family of enzymes which, after 30 years of investigation, we are just beginning to understand.

The secondary structure of the catalytic domain from protein kinase C zeta was studied using IR spectroscopy. In the presence of the substrate MgATP, there was a significant change in the secondary structure. After heating to 80 degrees C, a 14% decrease in the alpha-helix component was observed, accompanied by a 6% decrease in the beta-pleated sheet; no change was observed in the large loops or in 3(10)-helix plus associated loops. The maximum increase with heating was observed in the aggregated beta-sheet component, with an increase of 14%. In the presence of MgATP, and compared with the sample heated in its absence, there was a substantial decrease in the 3(10)-helix plus associated loops and an increase in alpha-helix. Synchronous 2D-IR correlation showed that the main changes occurred at 1617 cm(-1), which was assigned to changes in the intermolecular aggregated beta-sheet of the denaturated protein. This increase was mainly correlated with the change in alpha-helix. In the presence of MgATP, the main correlation was between aggregated beta-sheet and the large loops component. The asynchronous 2D-correlation spectrum indicated that a number of components are transformed in intermolecularly aggregated beta-sheet, especially the alpha-helix and beta-sheet components. It is interesting that changes in 3(10)-helix plus associated loops and in alpha-helix preceded changes in large loops, which suggests that the open loops structure exists as an intermediate state during denaturation. In summary, IR spectroscopy revealed an important effect of MgATP on the secondary structure and on the thermal unfolding process when this was induced, whereas 2D-IR correlation spectroscopy allowed us to show the establishment of the denaturation pathway of this protein.

Arachidonic acid, one of the major unsaturated fatty acids released during cell stimulation, participates in the signaling necessary for activation of different enzymes, including protein kinase C (PKC). Here, we demonstrate that arachidonic acid is a direct activator of PKCalpha, but needs the cooperation of Ca(2+) to exert its function. By using several mutants of the C2 and C1 domains, we were able to determine the molecular mechanism of this activation. More specifically, site-directed mutagenesis in key residues found in the C2 domain showed that the Ca(2+)-binding region was essential for the arachidonic acid-dependent localization and activation of PKCalpha. However, the lysine-rich cluster, also located in the C2 domain, played no relevant role in either the membrane localization or activation of the enzyme. Moreover, site-directed mutagenesis in key residues placed in the C1A and C1B subdomains, which are responsible for the diacylglycerol/phorbil ester interaction, demonstrated that the C1A subdomain was involved in the membrane localization and activation mechanism. Taken together, these data suggest a very precise mechanism for PKCalpha activation by arachidonic acid, involving a sequential model of activation in which an increase in intracytosolic Ca(2+) leads to the interaction of arachidonic acid with the Ca(2+)-binding region; only after this step, does the C1A subdomain interact with arachidonic acid, leading to full activation of the enzyme.

The effects of the anti-neoplastic ether lipid ET-18-OCH3 and some structural homologues on the activity of protein kinase C alpha (PKC alpha) were studied and compared with the effects the same had on the activity of PKC epsilon. ET-18-OCH3 progressively inhibited the activity of PKC alpha as the concentration was increased up to 30 mol% of the total lipid, above which the effect was one of activation. The experiments carried out with the homologues showed that the methoxy group bound at the sn-2 position of the glycerol of ET-18-OCH3 is essential for both the initial inhibitory effect and the subsequent activation effect. On the other hand, variations in the type of bond linking substitutions in the sn-1 position, ether or ester, do not seem to play an important role in determining the activity of the enzyme. The effects were different on PKC epsilon since ET-18-OCH3 had a triphasic effect, activating the enzyme at low concentrations, inhibiting it at slightly higher concentrations and then activating it again at higher concentrations. In this case, when the homologues were used, it was observed that the presence of the methoxy group linked to the sn-2 position of glycerol and the type of bond linking substitutions to the sn-1 position were important for activating the enzyme, so that only homologues with ester bonds as LPC and PAPC were able to induce the initial activation step in a way similar to ET-18-OCH3. Substitution of the phosphocholine group of ET-18-OCH3 by phosphoserine led to a greater activation of PKC alpha, an effect that comes from the Ca(2+)-phospholipid binding site probably because of the specific interaction of this site with the phosphoserine group. The action of ET-18-OCH3 and its homologues, as demonstrated in this paper, may permit the selective inhibition or activation of PKC alpha and PKC epsilon by using the most suitable range of concentrations.

Diacylglycerols are generated in the membrane as the result of extracellular signals and are able to stimulate the activity of protein kinase C, acting as membrane second messengers. Diacylglycerols are recognized by protein kinases C through the C1 domain and established models propose that they will stabilize the translocation of the protein to the membrane. However, diacylglycerols also act by modulating the physical properties of the membrane, thus favouring the translocation of the enzyme. This is done through alteration of the membrane surface curvature, dehydration of the surface and the separation of phospholipid surface groups. Good correlations have been observed between the physical state of the membrane and protein kinase C activity.

The secondary structure of protein kinase C alpha (PKC alpha) has been studied using infrared spectroscopy in the presence of both H(2)O and D(2)O buffers. In the absence of ligands at 20 degrees C, it was shown that beta-sheet is the major component, representing about 44% of the total structure, whereas the alpha-helix amounts to 22%. The addition of Ca(2+) produced only small changes in the secondary structure at 20 degrees C with the beta-sheet increasing up to 48%. On the other hand, the other ligands, such as phorbol 12-myristate 13-acetate (PMA), ATP, and phospholipids, did not produce any significant change. When the thermal unfolding of PKC alpha was studied after heating to 75 degrees C, the presence of the ligands affected the unfolding process. PKC alpha was better preserved from thermal denaturation in the presence of Ca(2+), the aggregated beta-sheet at 1618 cm(-1) decreasing from 19% in the absence of this ligand to 13% in its presence. Protection was also afforded by the presence of PMA or phospholipids. A two-dimensional correlation study of the denaturation of PKC alpha in the presence of these different ligands also showed differences among them. Synchronous 2D-IR correlation showed that the main change occurred at 1616-1619 cm(-1), this component being assigned to the intermolecular aggregated beta-sheet of the denaturated protein. This increase was mainly correlated with the change in the alpha-helix component in all cases except in the presence of a mixture of ligands including Ca(2+), ATP, PMA, and phospholipids, when the intermolecular aggregation of beta-sheet was correlated with the change in the beta-sheet component. In addition, the asynchronous 2D-IR correlation study of PKC alpha showed that the aggregated beta-sheet increased after changes in other components. It was interesting that alpha-helix changed before the beta-sheet in the control experiment and in the presence of Ca(2+), while the order of change was reversed when PMA was added.

To evaluate the role of the C2 domain in protein kinase Cepsilon (PKCepsilon) localization and activation after stimulation of the IgE receptor in RBL-2H3 cells, we used a series of mutants located in the phospholipid binding region of the enzyme. The results obtained suggest that the interaction of the C2 domain with the phospholipids in the plasma membrane is essential for anchoring the enzyme in this cellular compartment. Furthermore, the use of specific inhibitors of the different pathways that generate both diacylglycerol and phosphatidic acid has shown that the phosphatidic acid generated via phospholipase D (PLD)-dependent pathway, in addition to the diacylglycerol generated via phosphoinosite-phospholipase C (PLC), are involved in the localization of PKCepsilon in the plasma membrane. Direct stimulation of RBL-2H3 cells with very low concentrations of permeable phosphatidic acid and diacylglycerol exerted a synergistic effect on the plasma membrane localization of PKCepsilon. Moreover, the in vitro kinase assays showed that both phosphatidic acid and diacylglycerol are essential for enzyme activation. Together, these results demonstrate that phosphatidic acid is an important and essential activator of PKCepsilon through the C2 domain and locate this isoenzyme in a new scenario where it acts as a downstream target of PLD.

The secondary structure of the C2 domains of the classical PKC isoenzymes, alpha, betaII, and gamma, has been studied using infrared spectroscopy. Ca(2+) and phospholipids were used as protein ligands to study their differential effects on the isoenzymes and their influence on thermal protein denaturation. Whereas the structures of the three isoenzymes were similar in the absence of Ca(2+) and phospholipids at 25 degrees C, some differences were found upon heating in their presence, the C2 domain of the gamma-isoenzyme being better preserved from thermal denaturation than the domain from the alpha-isoenzyme and this, in turn, being better than that from the beta-isoenzyme. A two-dimensional correlation study of the denaturation of the three domains also showed differences between them. Synchronous 2D-IR correlation showed changes (increased aggregation of denaturated protein) occurring at 1616-19 cm(-1), and this was found in the three isoenzymes. On the other hand, the asynchronous 2D-IR correlation study of the domains in the absence of Ca(2+) showed that, in all cases, the aggregation of denaturated protein increased after changes in other structural components, an increase perhaps related with the hard-core role of the beta-sandwich in these proteins. The differences observed between the three C2 domains may be related with their physiological specialization and occurrence in different cell compartments and in different cells.

In view of the interest shown in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) as a second messenger, we studied the activation of protein kinase Calpha by this phosphoinositide. By using two double mutants from two different sites located in the C2 domain of protein kinase Calpha, we have determined and characterized the PtdIns(4,5)P(2)-binding site in the protein, which was found to be important for its activation. Thus, there are two distinct sites in the C2 domain: the first, the lysine-rich cluster located in the beta3- and beta4-sheets and which activates the enzyme through direct binding of PtdIns(4,5)P(2); and the second, the already well described site formed by the Ca(2+)-binding region, which also binds phosphatidylserine and a result of which the enzyme is activated. The results obtained in this work point to a sequential activation model, in which protein kinase Calpha needs Ca(2+) before the PtdIns(4,5)P(2)-dependent activation of the enzyme can occur.

Bcl-2 family proteins are involved in cell homeostasis, where they regulate cell death. Some of these proteins are pro-apoptotic and others pro-survival. Moreover, many of them share a similar domain composition with several of the so-called BH domains, although some only have a BH3 domain. A C-terminal domain is present in all the multi-BH domain proteins and in some of the BH3-only ones. This C-terminal domain is hydrophobic or amphipathic, for which reason it was thought when they were discovered that they were membrane anchors. Although this is indeed one of their functions, it has since been observed that they may also serve as regulators of the function of some members of this family, such as Bax. They may also serve to recognize the target membrane of some of these proteins, which only after an apoptotic signal, are incorporated into a membrane. It has been shown that peptides that imitate the sequence of C-terminal domains can form pores and may serve as a model to design cytotoxic molecules.

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.