Recombinant cannabinoid type 2 receptor in liposome model activates g protein in response to anionic lipid constituents.
ABSTRACT: Human cannabinoid type 2 (CB(2)) receptor expressed in Escherichia coli was purified and successfully reconstituted in the functional form into lipid bilayers composed of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and cholesteryl hemisuccinate (CHS). Reconstitution was performed by detergent removal from the protein/lipid/detergent mixed micelles either on an adsorbent column, or by rapid dilution to below the critical micelle concentration of detergent followed by removal of detergent monomers on a concentrator. Proteoliposomes prepared at a protein/phospholipid/CHS molar ratio of 1/620-650/210-220 are free of detergent as shown by (1)H NMR, have a homogeneous protein/lipid ratio shown by isopycnic gradient ultracentrifugation, and are small in size with a mean diameter of 150-200 nm as measured by dynamic light scattering. Functional integrity of the reconstituted receptor was confirmed by quantitative binding of (2)H-labeled agonist CP-55,940-d(6) measured by (2)H magic angle spinning NMR, as well as by activation of G protein. The efficiency of G protein activation by agonist-bound CB(2) receptor was affected by negative electric surface potentials of proteoliposomes controlled by the content of anionic CHS or POPS. The activation was highest at an anionic lipid content of about 50 mol %. There was no correlation between the efficiency of G protein activation and an increase of hydrocarbon chain order induced by CHS or cholesterol. The results suggest the importance of anionic lipids in regulating signal transduction by CB(2) receptor and other class A GPCR. The successful reconstitution of milligram quantities of pure, functional CB(2) receptor enables a wide variety of structural studies.
Project description:Prion diseases are neurodegenerative disorders of the central nervous system that are associated with the misfolding of the prion protein (PrP). PrP is glycosylphosphatidylinositol-anchored, and therefore the hydrophobic membrane environment may influence the process of prion conversion. This study investigates how the morphology and mechanism of growth of prion aggregates on membranes are influenced by lipid composition. Atomic force microscopy is used to image the aggregation of prions on supported lipid bilayers composed of mixtures of the zwitterionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS). Circular dichroism shows that PrP interactions with POPS membranes result in an increase in beta-sheet structure, whereas interactions with POPC do not influence PrP structure. Prion aggregation is observed on both zwitterionic and anionic membranes, and the morphology of the aggregates formed is dependent on the anionic phospholipid content of the membrane. The aggregates that form on POPC membranes have uniform dimensions and do not disrupt the lipid bilayer. The presence of POPS results in larger aggregates with a distinctive sponge-like morphology that are disruptive to membranes. These data provide detailed information on the aggregation mechanism of PrP on membranes, which can be described by classic models of growth.
Project description:Amyloid formation by islet amyloid polypeptide (IAPP) contributes to ?-cell dysfunction in type 2 diabetes. Perturbation of the ?-cell membrane may contribute to IAPP-induced toxicity. We examine the effects of lipid composition, salt, and buffer on IAPP amyloid formation and on the ability of IAPP to induce leakage of model membranes. Even low levels of anionic lipids promote amyloid formation and membrane permeabilization. Increasing the percentage of the anionic lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) or 1,2-dioleoyl-sn-glycero-3-phospho(1'-rac-glycerol), enhances the rate of amyloid formation and increases the level of membrane permeabilization. The choice of zwitterionic lipid has no noticeable effect on membrane-catalyzed amyloid formation but in most cases affects leakage, which tends to decrease in the following order: 1,2-dioleoyl-sn-glycero-3-phosphocholine > 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine > sphingomyelin. Uncharged lipids that increase the level of membrane order weaken the ability of IAPP to induce leakage. Leakage is due predominately to pore formation rather than complete disruption of the vesicles under the conditions used in these studies. Cholesterol at or below physiological levels significantly reduces the rate of vesicle-catalyzed IAPP amyloid formation and decreases the susceptibility to IAPP-induced leakage. The effects of cholesterol on amyloid formation are masked by 25 mol % POPS. Overall, there is a strong inverse correlation between the time to form amyloid and the extent of vesicle leakage. NaCl reduces the rate of membrane-catalyzed amyloid formation by anionic vesicles, but accelerates amyloid formation in solution. The implications for IAPP membrane interactions are discussed, as is the possibility that the loss of phosphatidylserine asymmetry enhances IAPP amyloid formation and membrane damage in vivo via a positive feedback loop.
Project description:PMP1, a regulatory subunit of the yeast plasma membrane H(+)-ATPase, is a single transmembrane helix protein. Its cytoplasmic C-terminus possesses several positively charged residues and interacts with phosphatidylserine lipids as shown through both (1)H- and (2)H-NMR experiments. We used all-atom molecular dynamics simulations to obtain atomic-scale data on the effects of membrane interface lipid composition on PMP1 structure and tilt. PMP1 was embedded in two hydrated bilayers, differing in the composition of the interfacial region. The neutral bilayer is composed of POPC (1-palmitoyl-2-oleoyl-3-glycero-phosphatidylcholine) lipids and the negatively charged bilayer is composed of POPC and anionic POPS (1-palmitoyl-2-oleoyl-3-glycero-phosphatidylserine) lipids. Our results were consistent with NMR data obtained previously, such as a lipid sn-2 chain lying on the W28 aromatic ring and in the groove formed on one side of the PMP1 helix. In pure POPC, the transmembrane helix is two residues longer than the initial structure and the helix tilt remains constant at 6 ± 3°. By contrast, in mixed POPC-POPS, the initial helical structure of PMP1 is stable throughout the simulation time even though the C-terminal residues interact strongly with POPS headgroups, leading to a significant increase of the helix tilt within the membrane to 20 ± 5°.
Project description:BACKGROUND: SoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1. METHODOLOGY/PRINCIPAL FINDING: We have characterized the protein structural stability after purification and after reconstitution into detergent micelles and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-?-D-glucopyranoside (OG) or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPhPC), or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE), 1-palmitoyl-2oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly ?-helical in accordance with crystallographic data. The protein has a high thermal structural stability in detergent solutions, with an irreversible thermal unfolding occurring at a melting temperature of 58°C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70°C. CONCLUSION/SIGNIFICANCE: The results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes for biotechnological applications.
Project description:Elucidation of the molecular mechanisms of activation of G protein-coupled receptors (GPCRs) is among the most challenging tasks for modern membrane biology. For studies by high resolution analytical methods, these integral membrane receptors have to be expressed in large quantities, solubilized from cell membranes and purified in detergent micelles, which may result in a severe destabilization and a loss of function. Here, we report insights into differential effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB(2), and provide guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. While we previously described the expression in Escherichia coli, purification and liposome-reconstitution of multi-milligram quantities of CB(2), here we report an efficient stabilization of the recombinant receptor in micelles - crucial for functional and structural characterization. The effects of detergents, lipids and specific ligands on structural stability of CB(2) were assessed by studying activation of G proteins by the purified receptor reconstituted into liposomes. Functional structure of the ligand binding pocket of the receptor was confirmed by binding of (2)H-labeled ligand measured by solid-state NMR. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS) and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB(2) in dodecyl maltoside (DDM)/CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS) exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB(2) reconstituted into lipid bilayers retained functionality for up to several weeks enabling high resolution structural studies of this GPCR at physiologically relevant conditions.
Project description:In this Data in Brief article we provide a data package of GROMACS input files for atomistic molecular dynamics simulations of multicomponent, asymmetric lipid bilayers using the OPLS-AA force field. These data include 14 model bilayers composed of 8 different lipid molecules. The lipids present in these models are: cholesterol (CHOL), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidyl-ethanolamine (SOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (SOPS), N-palmitoyl-D-erythro-sphingosyl-phosphatidylcholine (SM16), and N-lignoceroyl-D-erythro-sphingosyl-phosphatidylcholine (SM24). The bilayers? compositions are based on lipidomic studies of PC-3 prostate cancer cells and exosomes discussed in Llorente et al. (2013) , showing an increase in the section of long-tail lipid species (SOPS, SOPE, and SM24) in the exosomes. Former knowledge about lipid asymmetry in cell membranes was accounted for in the models, meaning that the model of the inner leaflet is composed of a mixture of PC, PS, PE, and cholesterol, while the extracellular leaflet is composed of SM, PC and cholesterol discussed in Van Meer et al. (2008) . The provided data include lipids? topologies, equilibrated structures of asymmetric bilayers, all force field parameters, and input files with parameters describing simulation conditions (md.mdp). The data is associated with the research article "Interdigitation of Long-Chain Sphingomyelin Induces Coupling of Membrane Leaflets in a Cholesterol Dependent Manner" (Róg et al., 2016) .
Project description:KCNE1 (minK), found in the human heart and cochlea, is a transmembrane protein that modulates the voltage-gated potassium KCNQ1 channel. While KCNE1 has previously been the subject of extensive structural studies in lyso-phospholipid detergent micelles, key observations have yet to be confirmed and refined in lipid bilayers. In this study, a reliable method for reconstituting KCNE1 into lipid bilayer vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (POPG) was developed. Microinjection of the proteoliposomes into Xenopus oocytes expressing the human KCNQ1 (K(V)7.1) voltage-gated potassium channel led to nativelike modulation of the channel. Circular dichroism spectroscopy demonstrated that the percent helicity of KCNE1 is significantly higher for the protein reconstituted in lipid vesicles than for the previously described structure in 1.0% 1-myristoyl-2-hydroxy-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (LMPG) micelles. SDSL electron paramagnetic resonance spectroscopic techniques were used to probe the local structure and environment of Ser28, Phe54, Phe57, Leu59, and Ser64 of KCNE1 in both POPC/POPG vesicles and LMPG micelles. Spin-labeled KCNE1 cysteine mutants at Phe54, Phe57, Leu59, and Ser64 were found to be located inside POPC/POPG vesicles, whereas Ser28 was found to be located outside the membrane. Ser64 was shown to be water inaccessible in vesicles but found to be water accessible in LMPG micelle solutions. These results suggest that key components of the micelle-derived structure of KCNE1 extend to the structure of this protein in lipid bilayers but also demonstrate the need to refine this structure using data derived from the bilayer-reconstituted protein to more accurately define its native structure. This work establishes the basis for such future studies.
Project description: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.
Project description:The histidine-rich designer peptide LAH4-L1 exhibits antimicrobial and potent cell-penetrating activities for a wide variety of cargo including nucleic acids, polypeptides, adeno-associated viruses, and nanodots. The non-covalent complexes formed between the peptide and cargo enter the cell via an endosomal pathway where the pH changes from neutral to acidic. Here, we investigated the membrane interactions of the peptide with phospholipid bilayers and its membrane topology using static solid-state NMR spectroscopy. Oriented 15N solid-state NMR indicates that in membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) 3:1 mol/mole and at neutral pH, the peptide adopts transmembrane topologies. Furthermore, 31P and 2H solid-state NMR spectra show that liquid crystalline 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and POPC/POPS 3:1 liposomes retain a bilayer macroscopic phase even at the highest peptide concentrations investigated, with an oblate orientational distribution of the phospholipids at a peptide/lipid ratio of 1:5. At pH 5, as it occurs in the endosome, the alignment of LAH4-L1 at a peptide/lipid ratio of 1:25 is predominantly parallel to POPC/POPS 3:1 bilayers (prolate deformation) when at the same time it induces a considerable decrease of the deuterium order parameter of POPC/2H31-POPS 3:1. In addition, when studied in mechanically supported lipid membranes, a pronounced disordering of the phospholipid alignment is observed. In the presence of even higher peptide concentrations, lipid spectra are observed that suggest the formation of magnetically oriented or isotropic bicelles. This membrane-disruptive effect is enhanced for gel phase DMPC membranes. By protonation of the four histidines in acidic environments, the overall charge and hydrophobic moment of LAH4-L1 considerably change, and much of the peptide is released from the cargo. Thus, the amphipathic peptide sequences become available to disrupt the endosomal membrane and to assure highly efficient release from this organelle.
Project description:The interaction of Saposin C (Sap C) with negatively charged phospholipids such as phosphatidylserine (PS) is essential for its biological function. In this study, Sap C (initially protonated in a weak acid) was inserted into multilamellar vesicles (MLVs) consisting of either 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (negatively charged, POPS) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (neutrally charged, POPC). The MLVs were then investigated using solid-state NMR spectroscopy under neutral pH (7.0) conditions. The (2)H and (31)P solid-state NMR spectroscopic data of Sap C-POPS and Sap C-POPC MLVs (prepared under the same conditions) were compared using the (2)H order parameter profiles of the POPC-d(31) or POPS-d(31) acyl chains as well as the (31)P chemical shift anisotropy width and (31)P T(1) relaxation times of the phospholipids headgroups. All those solid-state NMR spectroscopic approaches indicate that protonated Sap C disturbs the POPS bilayers and not the POPC lipid bilayers. These observations suggest for the first time that protonated Sap C inserts into PS bilayers and forms a stable complex with the lipids even after resuspension under neutral buffer conditions. Additionally, (31)P solid-state NMR spectroscopic studies of mechanically oriented phospholipids on glass plates were conducted and perturbation effect of Sap C on both POPS and POPC bilayers was compared. Unlike POPC bilayers, the data indicates that protonated Sap C (initially protonated in a weak acid) was unable to produce well-oriented POPS bilayers on glass plates at neutral pH. Conversely, unprotonated Sap C (initially dissolved in a neutral buffer) did not interact significantly with POPS phospholipids allowing them to produce well-oriented bilayers at neutral pH.