Magnetically orientable phospholipid bilayers containing small amounts of a bile salt analogue, CHAPSO.

Buffered mixtures of the detergent 3-(cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO) and dimyristoylphosphatidylcholine (DMPC) orient in the presence of a strong magnetic field over a wide range of water contents (at least 65-85%) and CHAPSO:DMPC molar ratios (typically 1:10-1:3). 31P NMR studies show that the phospholipid in such mixtures is oriented with its director axis perpendicular to the magnetic field. 31P and 2H NMR results also suggest that the structure and dynamics of the DMPC molecules are similar to that of pure phospholipids existing in the liquid crystalline (L alpha) bilayer phase. The ability of 1:5 CHAPSO:DMPC samples to orient is highly tolerant of large changes in temperature, pH, and ionic strength, as well as to the addition of substantial amounts of charged amphiphiles or soluble protein. However, 2H NMR studies of deuterated beta-dodecyl melibiose (DD-MB) solubilized in the system indicate the head group conformation and/or dynamics of this glycolipid analogue is dependent upon the CHAPSO concentration. Despite the latter results, the orientational versatility of the system, together with the nondenaturing properties of CHAPSO, makes this system useful in spectroscopic studies of membrane-associated phenomena.     

Membrane topology of a 14-mer model amphipathic peptide: a solid-state NMR spectroscopy study

We have investigated the interaction between a synthetic amphipathic 14-mer peptide and model membranes by solid-state NMR. The 14-mer peptide is composed of leucines and phenylalanines modified by the addition of crown ethers and forms a helical amphipathic structure in solution and bound to lipid membranes. To shed light on its membrane topology, 31P, 2H, 15N solid-state NMR experiments have been performed on the 14-mer peptide in interaction with mechanically oriented bilayers of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC). The 31P, 2H, and 15N NMR results indicate that the 14-mer peptide remains at the surface of the DLPC, DMPC, and DPPC bilayers stacked between glass plates and perturbs the lipid orientation relative to the magnetic field direction. Its membrane topology is similar in DLPC and DMPC bilayers, whereas the peptide seems to be more deeply inserted in DPPC bilayers, as revealed by the greater orientational and motional disorder of the DPPC lipid headgroup and acyl chains. 15N{31P} rotational echo double resonance experiments have also been used to measure the intermolecular dipole-dipole interaction between the 14-mer peptide and the phospholipid headgroup of DMPC multilamellar vesicles, and the results indicate that the 14-mer peptide is in contact with the polar region of the DMPC lipids. On the basis of these studies, the mechanism of membrane perturbation of the 14-mer peptide is associated to the induction of a positive curvature strain induced by the peptide lying on the bilayer surface and seems to be independent of the bilayer hydrophobic thickness.     

An NMR study of pyridine associated with DMPC liposomes and magnetically ordered DMPC-surfactant mixed micelles.

With molecular dynamics simulations of phospholipid membranes becoming a reality, there is a growing need for experiments that provide the molecular details necessary to test these computational results. Pyridine is used here to explore the interaction of planar aromatic groups with the water-lipid interface of membranes. It is shown by magic angle spinning 13C nuclear magnetic resonance (NMR) to bind between the glycerol and choline groups of dimyristoylphosphatidylcholine (DMPC) liposomes. The axial pattern for the 31P NMR spectrum of DMPC liposomes is preserved even with more than half of the interfacial sites occupied, indicating that pyridine does not disrupt the lamellar phase of this lipid. 2H NMR experiments of liposomes in deuterium oxide demonstrate that pyridine might promote greater penetration of water into restricted regions in the interface. Magnetically oriented DMPC/surfactant micelles were investigated as a means for improving resolution and sensitivity in NMR studies of species bound to bilayers. The quadrupolar splittings in the 2H NMR spectra of d5-pyridine in DMPC liposomes and magnetically oriented DMPC/Trixon X-100 micelles indicate a common bound state for the two bilayer systems. The well resolved quadrupolar splittings of d5-pyridine in oriented micelles were used to establish the tilt of the pyridine ring relative to the bilayer plane.     

Interaction of antimicrobial peptides from Australian amphibians with lipid membranes

Solid-state NMR and CD spectroscopy were used to study the effect of antimicrobial peptides (aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1) from Australian tree frogs on phospholipid membranes. 31P NMR results revealed some effect on the phospholipid headgroups when the peptides interact with DMPC/DHPC (dimyristoylphosphatidylcholine/dihexanoylphosphatidylcholine) bicelles and aligned DMPC multilayers. 2H NMR showed a small effect of the peptides on the acyl chains of DMPC in bicelles or aligned multilayers, suggesting interaction with the membrane surface for the shorter peptides and partial insertion for the longer peptides. 15N NMR of selectively labelled peptides in aligned membranes and oriented CD spectra indicated an alpha-helical conformation with helix long axis approximately 50 degrees to the bilayer surface at high peptide concentrations. The peptides did not appear to insert deeply into PC membranes, which may explain why these positively charged peptides preferentially lyse bacterial rather than eucaryotic cells.     

Membrane interactions of peptides representing the polybasic regions of three Rho GTPases are sensitive to the distribution of arginine and lysine residues

Rho GTPases are a multifunctional family of proteins that are localized at cellular membranes via an isoprenyl group covalently linked to a C-terminal cysteine. Close to this primary site of membrane anchoring there is often found an additional polybasic region (PBR), which plays a secondary role in membrane binding and targeting of the complex. Here, peptides derived from the PBRs of the Rho family proteins Rac1 (K(183)KRKRK), TCL (K(198)KKKKR) and Cdc42 (P(182)KKSRR) were prepared with hexalysine (K(6)) and hexaarginine (R(6)) to study their interactions with multilamellar vesicles of phosphatidylglycerol (DOPG) and headgroup-deuterated dimyristoylphosphatidylcholine (DMPC-d(4)) using (2)H and (31)P NMR. The membranes retained their lamellar architecture after peptide binding, but the (2)H NMR line shapes for DMPC-d(4) indicated that the bound peptides altered the orientation of the choline headgroups, consistent with a change in membrane surface charge. Rac1 and TCL peptides appeared to affect the headgroup orientation similarly to K(6), although the perturbations were weaker and unlike those induced by the Cdc42 peptide and R(6). Magic-angle spinning (31)P NMR spectra of the membranes showed significant and selective broadening of the peak for DMPC after addition of the peptides, with R(6) and the Cdc42 peptide having the greatest effect. The selective broadening may be a consequence of the lipids separating into short-lived domains enriched in peptide-bound DOPG and peptide-free DMPC. These results illustrate that a complex relationship exists between the sequence of PBRs and their behaviour at membrane surfaces, which may have implications for the cellular functions and localization of Rho GTPases.     

Comparative effects of cholesterol and cholesterol sulfate on hydration and ordering of dimyristoylphosphatidylcholine membranes.

The comparative effect of cholesterol (CH) versus cholesterol sulfate (CS) on dimyristoylphosphatidylcholine (DMPC) membranes has been investigated by optical microscopy, freeze-fracture electron microscopy, x-ray diffraction, and solid state 2H and 31P nuclear magnetic resonance (NMR). The sulfate analogue extends the lamellar phase domain toward high water contents, and substitution of 30 mol % CH by CS in DMPC lamellae induces the trapping of 30 wt % additional water. The greater swelling of the CS-containing systems is evidenced by determination of lamellar repeat distances at maximal hydration: 147 +/- 4 A and 64 +/- 2 A in the presence of CS and CH, respectively. 2H-NMR of heavy water demonstrates that CS binds approximately 12 more water molecules at the interface than CH whereas NMR of deuterium-labeled DMPC chains reveals that 30 mol % CS orders the membrane as 15 mol % CH at high temperature and disorders much more than CH at low temperatures. The various effects of CS versus CH are discussed by taking into account attractive Van der Waals forces and repulsive steric/electrostatic interactions of the negatively charged sulfate group.     

Response of the phosphatidylcholine headgroup to membrane surface charge in ternary mixtures of neutral, cationic, and anionic lipids: a deuterium NMR study.

Deuterium nuclear magnetic resonance (2H NMR) spectroscopy was used to investigate the response of the phosphatidylcholine headgroup of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) to changes in surface electrostatic charge in membranes consisting of ternary mixtures of lipids. DMPC was deuterated at the choline alpha- and beta-methylene segments. The membrane surface charge was manipulated by the simultaneous addition of cationic didodecyldimethylammonium bromide (DDAB) and anionic 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) to neutral DMPC. Addition of increasing amounts of DDAB caused a progressive decrease (increase) in the 2H NMR quadrupole splitting from DMPC-alpha-d2 (DMPC-beta-d2). Addition of increasing amounts of DMPG caused a progressive increase (decrease) in the quadrupole splitting from DMPC-alpha-d2 (DMPC-beta-d2). Qualitatively, the 2H NMR quadrupole splitting charge response exhibited the same main features for ternary mixtures of DDAB/DMPG/DMPC and binary mixtures of DDAB/DMPC or DMPG/DMPC. Quantitatively, however, the 2H NMR quadrupole splittings obtained from ternary mixtures did not coincide with those obtained from binary mixtures of nominally identical surface charge densities. Hence, the quadrupole splitting did not respond directly to the net membrane surface charge. Instead, the quadrupole splitting measured for a given ternary lipid composition could be reproduced by summing the individual effects of the charged lipids in binary mixtures, weighted according to their appropriate mole fractions.     

31P NMR and AFM studies on the destabilization of cell and model membranes by the major bovine seminal plasma protein, PDC-109

The effect of PDC-109 binding to dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylglycerol (DPPG) multilamellar vesicles (MLVs) and supported membranes was investigated by (31)P NMR spectroscopy and atomic force microscopy. Additionally, the effect of cholesterol on the binding of PDC-109 to phosphatidylcholine (PC) membranes was studied. Binding of PDC-109 to MLVs of DMPC and DPPG induced the formation of an isotropic signal in their (31)P NMR spectra, which increased with increasing protein/lipid ratio and temperature, consistent with protein-induced disruption of the MLVs and the formation of small unilamellar vesicles or micelles but not inverse hexagonal or cubic phases. Incorporation of cholesterol in the DMPC MLVs afforded a partial stabilization of the lamellar structure, consistent with previous reports of membrane stabilization by cholesterol. AFM results are consistent with the above findings and show that addition of PDC-109 leads to a complete breakdown of PC membranes. The fraction of isotropic signal in (31)P NMR spectra of DPPG in the presence of PDC-109 was less than that of DMPC under similar conditions, suggesting a significantly higher affinity of the protein for PC. Confocal microscopic studies showed that addition of PDC-109 to human erythrocytes results in a disruption of the plasma membrane and release of hemoglobin into the solution, which was dependent on the protein concentration and incubation time.     

Membrane thinning due to antimicrobial peptide binding: an atomic force microscopy study of MSI-78 in lipid bilayers

The interaction of an antimicrobial peptide, MSI-78, with phospholipid bilayers has been investigated using atomic force microscopy, circular dichroism, and nuclear magnetic resonance (NMR). Binding of amphipathic peptide helices with their helical axis parallel to the membrane surface leads to membrane thinning. Atomic force microscopy of supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers in the presence of MSI-78 provides images of the membrane thinning process at a high spatial resolution. This data reveals that the membrane thickness is not reduced uniformly over the entire bilayer area. Instead, peptide binding leads to the formation of distinct domains where the bilayer thickness is reduced by 1.1 +/- 0.2 nm. The data is interpreted using a previously published geometric model for the structure of the peptide-lipid domains. In this model, the peptides reside at the hydrophilic-hydrophobic boundary in the lipid headgroup region, which leads to an increased distance between lipid headgroups. This picture is consistent with concentration-dependent 31P and 2H NMR spectra of MSI-78 in mechanically aligned DMPC bilayers. Furthermore, 2H NMR experiments on DMPC-d54 multilamellar vesicles indicate that the acyl chains of DMPC are highly disordered in the presence of the peptide as is to be expected for the proposed structure of the peptide-lipid assembly.     

Detergent-like properties of magainin antibiotic peptides: a 31P solid-state NMR spectroscopy study

(31)P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in (31)P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid (31)P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type (31)P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.     

Effect of staphylococcal delta-lysin on the thermotropic phase behavior and vesicle morphology of dimyristoylphosphatidylcholine lipid bilayer model membranes. Differential scanning calorimetric, 31P nuclear magnetic resonance and Fourier transform infrared spectroscopic, and X-ray diffraction studies

We investigated the effects of various concentrations of staphylococcal delta-lysin on the thermotropic phase behavior of large multilamellar dimyristoylphosphatidylcholine (DMPC) vesicles by differential scanning calorimetry (DSC), 31P nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction. The DSC studies revealed that at all concentrations, the addition of delta-lysin progressively decreases the enthalpy of the pretransition of DMPC bilayers without significantly affecting its temperature or cooperativity. Similarly, the addition of smaller quantities of peptide has little effect on the temperature of the main phase transition of DMPC bilayers but does reduce the cooperativity and enthalpy of this transition somewhat. However, at higher peptide concentrations, a second phase transition with a slightly increased temperature and a markedly reduced cooperativity and enthalpy is also induced, and this latter phase transition resolves itself into two components at the highest peptide concentrations that are tested. Moreover, our 31P NMR spectroscopic studies reveal that at relatively low delta-lysin concentrations, essentially all of the phospholipid molecules produce spectra characteristic of the lamellar phase, whereas at the higher peptide concentrations, an increasing proportion exhibit an isotropic signal. Also, at the highest delta-lysin concentrations that are studied, the isotropic component of the 31P NMR spectrum also resolves itself into two components. At the highest peptide concentration that was tested, we are also able to effect a macroscopic separation of our sample into two fractions by centrifugation, a pellet containing relatively smaller amounts of delta-lysin and a supernatant containing larger amounts of peptide relative to the amount of lipid present. We are also able to show that the more cooperative phase transition detected calorimetrically, and the lamellar phase 31P NMR signal, arise from the pelleted material, while the less cooperative phase transition and the isotropic 31P NMR signal arise from the supernatant. In addition, we demonstrate by X-ray diffraction that the pelleted material corresponds to delta-lysin-containing large multilamellar vesicles and the supernatant to a mixture of delta-lysin-containing small unilamellar vesicles and discoidal particles. We also show by FTIR spectroscopy that delta-lysin exists predominantly in the alpha-helical conformation in aqueous solution or when interacting with DMPC, and that a large fraction of the peptide bonds undergo H-D exchange in D(2)O. However, upon interaction with DMPC, the fraction of exchangeable amide protons decreases. We also demonstrate by this technique that both of the phase transitions detected by DSC correspond to phospholipid hydrocarbon chain-melting phase transitions. Finally, we show by several techniques that the absolute concentrations of delta-lysin and the thermal history, as well as the lipid:peptide ratio, can affect the thermotropic phase behavior and morphology of peptide-lipid aggregates.     

Membrane interactions and dynamics of a 21-mer cytotoxic peptide: A solid-state NMR study

We have investigated the membrane interactions and dynamics of a 21-mer cytotoxic model peptide that acts as an ion channel by solid-state NMR spectroscopy. To shed light on its mechanism of membrane perturbation, ³¹P and ²H NMR experiments were performed on 21-mer peptide-containing bicelles. ³¹P NMR results indicate that the 21-mer peptide stabilizes the bicelle structure and orientation in the magnetic field and perturbs the lipid polar head group conformation. On the other hand, ²H NMR spectra reveal that the 21-mer peptide orders the lipid acyl chains upon binding. ¹⁵N NMR experiments performed in DMPC bilayers stacked between glass plates also reveal that the 21-mer peptide remains at the bilayer surface. ¹⁵N NMR experiments in perpendicular DMPC bicelles indicate that the 21-mer peptide does not show a circular orientational distribution in the bicelle planar region. Finally, ¹³C NMR experiments were used to study the 21-mer peptide dynamics in DMPC multilamellar vesicles. By analyzing the ¹³CO spinning sidebands, the results show that the 21-mer peptide is immobilized upon membrane binding. In light of these results, we propose a model of membrane interaction for the 21-mer peptide where it lies at the bilayer surface and perturbs the lipid head group conformation.     

Interaction of the neuropeptide met-enkephalin with zwitterionic and negatively charged bicelles as viewed by 31P and 2H solid-state NMR

The interaction of the neuropeptide methionine-enkephalin (Menk) with bicelles was investigated by solid-state NMR. Bicelles composed of dimyristoylphosphatidylcholine (DMPC) and dicaproylphosphatidylcholine (DCPC) were modified to investigate the effect of the lipid headgroup and electrostatic charges on the association with Menk. A total of 10 mol % of DMPC was replaced by zwitterionic phosphatidylethanolamine (DMPE), anionic phosphatidylglycerol (DMPG), or phosphatidylserine (DMPS). The preparation of DMPE-doped bicelles (Bic/PE) is reported for the first time. The (31)P and (2)H NMR results revealed changes in the lipid dynamics when Menk interacts with the bicellar systems. (2)H NMR experiments showed a disordering effect of Menk on the lipid chains in all the bicelles except Bic/PG, whereas the study of the choline headgroups indicated a decreased order of the lipids only in Bic/PE and Bic/PG. Our results suggest that the insertion depth of Menk into bicelles is modulated by their composition, more specifically by the balance between hydrophobic and electrostatic interactions. Menk would be buried at the lipid polar/apolar interface, the depth of penetration into the hydrophobic membrane core following the scaling Bic > Bic/PE > Bic/PS at the slightly acidic pH used in this study. The peptide would not insert into the bilayer core of Bic/PG and would rather remain at the surface.     

The orientation effect of gramicidin A on bicelles and Eu3+ -doped bicelles as studied by solid-state NMR and FT-IR spectroscopy

We have explored the effect of gramicidin A (gA) on bicelle (Bic) orientation in the absence and presence of Eu(3+) by (31)P and (2)H NMR at different DMPC/gA ratios. FT-IR spectroscopy was used to assess the lipid chain ordering and verify the transmembrane peptide conformation. Our results show a time-dependent flipping of the bilayer normal alignment at high temperatures and high proportion of gA. The results are explained by both the diamagnetic susceptibility anisotropy of the beta(6.3) helical peptides and viscosity of the lipid mixture. The concentration effect of gramicidin on Bic/Eu(3+) is compared to that on Eu(3+)-doped DMPC liposomes. The Bic/Eu(3+) system is no longer oriented in the presence of gA and adopts a vesicular morphology while the peptide incorporation induces the formation of ellipsoidal DMPC/Eu(3+) assemblies aligned with their normal parallel to the magnetic field. The difference is explained in terms of lipid chain disorder and size of the bilayers.     

Investigation of the interaction of myelin basic protein with phospholipid bilayers using solid-state NMR spectroscopy

Interaction of bovine myelin basic protein and its constituent charge isomers (C1-C3) with phospholipid bilayers was studied using solid-state NMR experiments on model membranes. 31P NMR experiments on multilamellar vesicles and mechanically aligned bilayers were used to measure the degree of protein-induced disorder in the lipid headgroup region while 2H NMR data provided the disorder caused by the protein in the hydrophobic core of the bilayers. Our results suggest that MBP and its charge isomers neither fragment nor significantly disrupt DMPC, POPC, POPC:POPG, and POPE bilayers. These results demonstrate that the MBP-induced fragmentation of POPC bilayers is due to the freeze-thaw cycles used in the preparation of multilamellar vesicles and not due to intrinsic protein-lipid interactions.     

A deuterium and phosphorus-31 nuclear magnetic resonance study of the interaction of melittin with dimyristoylphosphatidylcholine bilayers and the effects of contaminating phospholipase A2

The interaction of bee venom melittin with dimyristolphosphatidylcholine (DMPC) selectively deuteriated in the choline head group has been studied by deuterium and phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. The action of residual phospholipase A2 in melittin samples resulted in mixtures of DMPC and its hydrolytic products that underwent reversible transitions at temperatures between 30 and 35 degrees C from extended bilayers to micellar particles which gave narrow single-line deuterium and phosphorus-31 NMR spectra. Similar transitions were observed in DMPC-myristoyllysophosphatidylcholine (lysoPC)-myristic acid mixtures containing melittin but not in melittin-free mixtures, indicating that melittin is able to stabilize extended bilayers containing DMPC and its hydrolytic products in the liquid-crystalline phase. Melittin, free of phospholipase A2 activity, and at 3-5 mol% relative to DMPC, induced reversible transitions between extended bilayers and micellar particles on passing through the liquid-crystalline to gel phase transition temperature of the lipid, effects similar to those observed in melittin-acyl chain deuterated dipalmitoylphosphatidylcholine (DPPC) mixtures [Dufourc, E. J., Smith, I. C. P., & Dufourcq, J. (1986) Biochemistry 25, 6448-6455]. LysoPC at concentrations of 20 mol% or greater relative to DMPC induced transitions between extended bilayers and micellar particles with characteristics similar to those induced by melittin. It is proposed that these melittin- and lysoPC-induced transitions share similar mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tetrahydromethanopterin, a carbon carrier in methanogenesis

Evidence obtained by 13C NMR spectroscopy indicates that tetrahydromethanopterin (H4MPT) serves as a carbon carrier for C1 units at the methine, methylene, and methyl levels of oxidation. All three derivatives of H4MPT served as substrates for methanogenesis by cell extracts under a hydrogen atmosphere; in each instance, methane evolved at a rate comparable to that obtained when 2-(methylthio)ethanesulfonic acid was used as the substrate. Each C1 derivative of H4MPT stimulated the reduction of CO2 as efficiently as 2-(methylthio)ethanesulfonic acid. High resolution fast atom bombardment mass spectrometry indicated that the product of the spontaneous reaction of formaldehyde with H4MPT was methylene-H4MPT, with the molecular formula C31H45N6O16P. 13C NMR spectroscopy of hexamethylenetetramine, a model compound, suggested that the methylene group in methylene-H4MPT was bound to two nitrogen atoms. Molecular formulas of C31H44N6O16P and C31H47N6O16P were assigned to methenyl-H4MPT+, and methyl-H4MPT, by high resolution fast atom bombardment mass spectrometry. 1H NMR spectroscopy of methyl-H4MPT indicated that the methyl group was bound to a nitrogen atom. Sensitivity of each derivative to oxygen was noted. Apparent extinction coefficients of H4MPT and its derivatives were recorded. Evidence for the enzymatic synthesis of methylene-H4MPT from methenyl-H4MPT+ is presented.     

Hibernation, reversible cell growth inhibition by epigallocatechin-3-O-gallate

Epigallocatechin-3-O-gallate (EGCg) and related polyphenolic compounds found in tea are known to have antioxidative activities. However, they also have pro-oxidative activities such as generation of hydrogen peroxide. In this report, we investigated the effect on cells and showed the potential usage of EGCg in cell preservation. H(2)O(2) was generated from EGCg at concentrations of more than 300 microg/mL for 6 h at 37 degrees C, and high cytotoxicity for L929 cells were shown. In contrast, in the presence of 1 microg/mL catalase, the amount of generated H(2)O(2) was significantly low and cytotoxicity decreased markedly. This indicates that catalase eliminated H(2)O(2) generated by degradation of EGCg. Although H(2)O(2) generation was prevented, L929 cell proliferation was slightly inhibited in proportion to the concentrations of EGCg. L929 was exposed able to be 300 microg/mL to EGCg and 1 microg/mL catalase for maximum 18 days. EGCg inhibited the growth of L929 cells, and cell proliferation was restarted immediately after medium change for removing EGCg. We concluded that EGCg had a reversible growth inhibition when H(2)O(2) was eliminated from cell cultures.     

Conformational changes of phospholipid headgroups induced by a cationic integral membrane peptide as seen by deuterium magnetic resonance

Deuterium nuclear magnetic resonance (2H NMR) was used to study the interaction of a cationic amphiphilic peptide with pure DMPC membranes and with mixed bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS). The choline and serine headgroups were selectively deuteriated at the alpha and beta positions. The amphiphilic peptide, with 20 leucine residues in the hydrophobic core and two cationic hydrophilic lysine residues at each end, spanned the lipid bilayer. Although 2H NMR experiments using DMPC with perdeuteriated fatty acyl chains showed that the average order parameter of the hydrophobic region was not significantly modified by the incorporation of the amphiphilic peptide, for either DMPC or DMPC/DMPS (5:1) bilayers, large perturbations of the quadrupolar splittings of the choline and serine headgroups were observed. The results obtained with the DMPC headgroup suggest that the incorporation of the cationic peptide in both DMPC and DMPC/DMPS (5:1) bilayers leads to a structural perturbation directly related to the net charge on the membrane surface. The magnitude of the observed effect seems to be similar to those observed previously with other cationic molecules [Seelig, J., MacDonald, P.M., & Scherer, P.G. (1987) Biochemistry 26, 7535-7541]. Two of the three quadrupolar splittings of the PS headgroup exhibited large variations in the presence of the amphiphilic peptide, while the third one remained unchanged. Our data have led us to propose a model describing the influence of membrane surface charges on headgroup conformation. In this model, the surface charge is represented as a uniform charge distribution. The electric field due to the charges produces a torque which rotates the polar headgroups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane interactions of peptides representing the polybasic regions of three Rho GTPases are sensitive to the distribution of arginine and lysine residues

Rho GTPases are a multifunctional family of proteins that are localized at cellular membranes via an isoprenyl group covalently linked to a C-terminal cysteine. Close to this primary site of membrane anchoring there is often found an additional polybasic region (PBR), which plays a secondary role in membrane binding and targeting of the complex. Here, peptides derived from the PBRs of the Rho family proteins Rac1 (K¹⁸³KRKRK), TCL (K¹⁹⁸KKKKR) and Cdc42 (P¹⁸²KKSRR) were prepared with hexalysine (K₆) and hexaarginine (R₆) to study their interactions with multilamellar vesicles of phosphatidylglycerol (DOPG) and headgroup-deuterated dimyristoylphosphatidylcholine (DMPC-d₄) using ²H and ³¹P NMR. The membranes retained their lamellar architecture after peptide binding, but the ²H NMR line shapes for DMPC-d₄ indicated that the bound peptides altered the orientation of the choline headgroups, consistent with a change in membrane surface charge. Rac1 and TCL peptides appeared to affect the headgroup orientation similarly to K₆, although the perturbations were weaker and unlike those induced by the Cdc42 peptide and R₆. Magic-angle spinning ³¹P NMR spectra of the membranes showed significant and selective broadening of the peak for DMPC after addition of the peptides, with R₆ and the Cdc42 peptide having the greatest effect. The selective broadening may be a consequence of the lipids separating into short-lived domains enriched in peptide-bound DOPG and peptide-free DMPC. These results illustrate that a complex relationship exists between the sequence of PBRs and their behaviour at membrane surfaces, which may have implications for the cellular functions and localization of Rho GTPases.