Interaction of tea catechins with lipid bilayers investigated with liposome systems

Interaction of tea catechins with lipid bilayers was investigated with liposome systems, which enabled us to separate liposomes from the external medium by centrifugation. We found that epicatechin gallate had the highest affinity for lipid bilayers, followed by epigallocatechin gallate, epicatechin, and epigallocatechin. Epicatechin gallate and epigallocatechin gallate in the surface of lipid bilayer perturbed the membrane structure.     

Steric effects on interaction of tea catechins with lipid bilayers

Interaction of tea catechins with lipid bilayers has been investigated with liposome systems. Tea catechins are classified into cis-type and trans-type from the configuration of the two hydrogens at the 2 and 3 positions on the C-ring. The amount of trans-type catechins incorporated into liposomes was less than that of the respective cis-type catechins. Furthermore, the order of the partition coefficients of catechins in an n-octanol/PBS system is the same as that of the amount incorporated into liposomes. These results indicate that in addition to the number of hydroxyl groups on the B-ring and the presence of the galloyl moiety, the stereochemical structure of the C-ring also governs the hydrophobicity and the affinity for lipid bilayers. Trans-type catechins with the galloyl moiety were located on the surface of the lipid bilayer, as well as cis-type catechins with the galloyl moiety, and perturbed the membrane structure. These different stereochemical structures should influence the affinity for lipid bilayers, the alteration of membrane structures, and the difference in the order of the biological activities.     

Effects of external factors on the interaction of tea catechins with lipid bilayers

Green tea contains a high concentration of such catechins as (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Their biological activities have been evaluated by in vitro experiments using cultured cells or bacteria, but the order of activity of the various catechins differed with the study. We have been studying the interaction of tea catechins with lipid bilayers, and clarified that the number of hydroxyl groups on the B-ring, the presence of the galloyl moiety, and the stereochemical structure of each catechin govern their affinity for lipid bilayers. We investigated in this present study the effects of various external factors on the affinity of tea catechins for lipid bilayers by using liposomes as model membranes. The amount of tea catechins incorporated into the lipid bilayers increased with increasing salt concentration in an aqueous medium and decreased with increasing negative electric charge of the lipid bilayers. Furthermore, the amount of EGCg or ECg incorporated into the lipid bilayers increased with increasing EC concentration. These results reveal that the salt concentration in an aqueous medium, the electric charge of the membrane, and the presence of other catechins governed the affinity of tea catechins for the lipid bilayers.     

Interaction of local anesthetics with lipid bilayers investigated by 1H MAS NMR spectroscopy

The membrane location of the local anesthetics (LA) lidocaine, dibucaine, tetracaine, and procaine hydrochloride as well as their influence on phospholipid bilayers were studied by ³¹P and ¹H magic-angle spinning (MAS) NMR spectroscopy. The ³¹P NMR spectra of the LA/lipid preparations confirmed that the overall bilayer structure of the membrane remained preserved. The relation between the molecular structure of the LAs and their membrane localization and orientation was investigated quantitatively using induced chemical shifts, nuclear Overhauser enhancement spectroscopy, and paramagnetic relaxation rates. All three methods revealed an average location of the aromatic rings of all LAs in the lipid-water interface of the membrane, with small differences between the individual LAs depending on their molecular properties. While lidocaine is placed in the upper chain/glycerol region of the membrane, for dibucaine and procaine the maximum of the distribution are slightly shifted into the glycerol region. Finally for tetracaine the aromatic ring is placed closest to the aqueous phase in the glycerol/headgroup region of the membrane. The hydrophobic side chains of the LA molecules dibucaine and tetracaine were located deeper in the membrane and showed an orientation towards the hydrocarbon core. In contrast the side chains of lidocaine and procaine are oriented towards the aqueous phase.     

Interaction of local anesthetics with lipid bilayers investigated by (1)H MAS NMR spectroscopy

The membrane location of the local anesthetics (LA) lidocaine, dibucaine, tetracaine, and procaine hydrochloride as well as their influence on phospholipid bilayers were studied by (31)P and (1)H magic-angle spinning (MAS) NMR spectroscopy. The (31)P NMR spectra of the LA/lipid preparations confirmed that the overall bilayer structure of the membrane remained preserved. The relation between the molecular structure of the LAs and their membrane localization and orientation was investigated quantitatively using induced chemical shifts, nuclear Overhauser enhancement spectroscopy, and paramagnetic relaxation rates. All three methods revealed an average location of the aromatic rings of all LAs in the lipid-water interface of the membrane, with small differences between the individual LAs depending on their molecular properties. While lidocaine is placed in the upper chain/glycerol region of the membrane, for dibucaine and procaine the maximum of the distribution are slightly shifted into the glycerol region. Finally for tetracaine the aromatic ring is placed closest to the aqueous phase in the glycerol/headgroup region of the membrane. The hydrophobic side chains of the LA molecules dibucaine and tetracaine were located deeper in the membrane and showed an orientation towards the hydrocarbon core. In contrast the side chains of lidocaine and procaine are oriented towards the aqueous phase.     

Interaction of fatty acids with lipid bilayers

We present a method by which it is possible to describe the binding of fatty acids to phospholipid bilayers. Binding constants for oleic acid and a number of fatty acids used as spectroscopic probes are deduced from electrophoresis measurements. There is a large shift in $\text{p}\text{K}$ value for the fatty acids on binding to the phospholipid bilayers, consistent with stronger binding of the uncharged form of the fatty acid. For dansylundecanoic acid, fluorescence titrations are consistent with the binding constants derived from the electrophoresis experiments. For 12-(9-anthroyloxy)stearic acid, fluorescence and electrophoresis data are inconsistent, and we attribute this to quenching of fluorescence at high molar ratios of 12-anthroylstearic acid to phospholipid in the bilayer.     

Interaction of small peptides with lipid bilayers.

Molecular dynamics simulations of the tripeptide Ala-Phe-Ala-O-tert-butyl interacting with dimyristoylphosphatidylcholine lipid bilayers have been carried out. The lipid and aqueous environments of the peptide, the alkyl chain order, and the lipid and peptide dynamics have been investigated with use of density profiles, radial distribution functions, alkyl chain order parameter profiles, and time correlation functions. It appears that the alkyl chain region accommodates the peptides in the bilayer with minimal perturbation to this region. The peptide dynamics in the bilayer bound form has been compared with that of the free peptide in water. The peptide structure does not vary on the simulation time scale (of the order of hundreds of picoseconds) compared with the solution structure in which a random structure is observed.     

Interaction of synaptotagmin with lipid bilayers, analyzed by single-molecule force spectroscopy

Synaptotagmin I is the major Ca²⁺ sensor for membrane fusion during neurotransmitter release. The cytoplasmic domain of synaptotagmin consists of two C2 domains, C2A and C2B. On binding Ca²⁺, the tips of the two C2 domains rapidly and synchronously penetrate lipid bilayers. We investigated the forces of interaction between synaptotagmin and lipid bilayers using single-molecule force spectroscopy. Glutathione-S-transferase-tagged proteins were attached to an atomic force microscope cantilever via a glutathione-derivatized polyethylene glycol linker. With wild-type C2AB, the force profile for a bilayer containing phosphatidylserine had both Ca²⁺-dependent and Ca²⁺-independent components. No force was detected when the bilayer lacked phosphatidylserine, even in the presence of Ca²⁺. The binding characteristics of C2A and C2B indicated that the two C2 domains cooperate in binding synaptotagmin to the bilayer, and that the relatively weak Ca²⁺-independent force depends only on C2A. When the lysine residues K189-192 and K326, 327 were mutated to alanine, the strong Ca²⁺-dependent binding interaction was either absent or greatly reduced. We conclude that synaptotagmin binds to the bilayer via C2A even in absence of Ca²⁺, and also that positively charged regions of both C2A and C2B are essential for the strong Ca²⁺-dependent binding of synaptotagmin to the bilayer.     

Interaction of GAPR-1 with lipid bilayers is regulated by alternative homodimerization

Golgi-Associated Plant Pathogenesis-Related protein 1 (GAPR-1) is a mammalian protein that belongs to the superfamily of plant pathogenesis related proteins group 1 (PR-1). GAPR-1 is a peripheral membrane-binding protein that strongly associates with lipid-enriched microdomains at the cytosolic leaflet of Golgi membranes. Little is known about the mechanism of GAPR-1 interaction with membranes. We previously suggested that dimerization plays a role in the function of GAPR-1 and here we report that phytic acid (inositol hexakisphosphate) induces dimerization of GAPR-1 in solution. Elucidation of the crystal structure of GAPR-1 in the presence of phytic acid revealed that the GAPR-1 dimer differs from the previously published GAPR-1 dimer structure. In this structure, one of the monomeric subunits of the crystallographic dimer is rotated by 28.5°. To study the GAPR-1 dimerization properties, we investigated the interaction with liposomes in a light scattering assay and by flow cytometry. In the presence of negatively charged lipids, GAPR-1 caused a rapid and stable tethering of liposomes. [D81K]GAPR-1, a mutant predicted to stabilize the IP6-induced dimer conformation, also caused tethering of liposomes. [A68K]GAPR-1 however, a mutant predicted to stabilize the non-rotated dimer conformation, is capable of binding to liposomes but did not cause liposome tethering. Our combined data suggest that the charge properties of the lipid bilayer can regulate GAPR-1 dynamics as a potential mechanism to modulate GAPR-1 function.     

Interaction of nonionic PEO-PPO diblock copolymers with lipid bilayers

The relationship between the molecular architecture of a series of poly(ethylene oxide)-b-poly(propylene oxide) (PEO-PPO) diblock copolymers and the nature of their interactions with lipid bilayers has been studied using small- and wide-angle X-ray scattering (SAXS and WAXS) and differential scanning calorimetry (DSC). The number of molecular repeat units in the hydrophobic PPO block has been found to be a critical determinant of the nature of diblock copolymer-lipid bilayer association. For dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-based biomembrane structures, polymers whose PPO chain length approximates that of the acyl chains of the lipid bilayer yield highly ordered, expanded lamellar structures consistent with well-integrated (into the lipid bilayer) PPO blocks. Shorter diblock copolymers produce mixed lamellar and nonlamellar mesophases. The thermotropic phase behavior of the polymer-doped membrane systems is highly influenced by the presence and molecular architecture of the diblock copolymer, as evidenced by shifting of the main phase transition to higher temperatures, broadening of the main transition, and the appearance of other features. Studies of temperature-induced changes in the mesophase structure for compositions prepared with well-integrated PEO-PPO polymers indicate that they undergo reversible changes to a nonlamellar structure as the temperature is lowered. Increasing either the number of repeat units in the PEO block or the polymer concentration promotes a greater degree of structural ordering.     

Quantitative detection of a DNA ligase reaction on a quartz-crystal microbalance.

We report here kinetic analyses of DNA ligation by using a DNA-immobilized quartz-crystal microbalance (QCM), which enables in situ real-time monitoring of both the binding of ligase and ligation reaction on DNA strands, as mass changes.     

Replacement-free mass-amplified sandwich assay with 180-MHz electrodeless quartz-crystal microbalance biosensor

This study describes a sensitivity-amplified detection method in a replacement-free electrodeless quartz-crystal microbalance (QCM) biosensor. A sandwich assay is proposed for detecting C-reactive proteins (CRP), where the biotinated second anti-CRP antibody is weighted by streptavidin for sensitivity amplification. Because the first CRP antibody was immobilized nonspecifically on naked quartz surfaces, the sandwich assay was repeated using the same sensor chip, making possible the replacement-free assay. The mass-amplified sandwich assay detected a CRP solution of 0.1 ng/ml. A methodology for determining the molecular mass of the injected protein is also proposed.     

Interaction of a two-transmembrane-helix peptide with lipid bilayers and dodecyl sulfate micelles

To probe structural changes that occur when a membrane protein is transferred from lipid bilayers to SDS micelles, a fragment of bacteriorhodopsin containing transmembrane helical segments A and B was studied by fluorescence spectroscopy, molecular dynamics (MD) simulation, and stopped flow kinetics. In lipid bilayers, F?rster resonance energy transfer (FRET) was observed between tyrosine 57 on helix B and tryptophans 10 and 12 on helix A. FRET efficiency decreased substantially when the peptide was transferred to SDS. MD simulation showed no evidence for significant disruption of helix-helix interactions in SDS micelles. However, a cluster of water molecules was observed to form a hydrogen-bonded network with the phenolic hydroxyl group of tyrosine 57, which probably causes the disappearance of tyrosine-to-tryptophan FRET in SDS. The tryptophan quantum yield decreased in SDS, and the change occurred at nearly the same rate as membrane solubilization. The results provide a clear example of the importance of corroborating distance changes inferred from FRET by using complementary methods.     

Interaction of sporidesmin,a mycotoxin fromPithomyces chartarum,with lipid bilayers

Sporidesmin, a mycotoxin fromPithomyces chartarum is a hydrophobic molecule. It can therefore be easily incorporated in the cell membrane, where it is likely to cause changes in the bilayer organization and the properties of membrane proteins. In order to understand the redox behaviour of sporidesmin in a hydrophobic environment, we have investigated the effects of oxidized and reduced sporidesmin on the phase transition properties of bilayers and on the susceptibility of bilayers to pancreatic phospholipase A₂ (PLA₂). The changes induced by sporidesmin in the thermotropic phase transition profiles of dimyristoyl-sn-3-phosphatidyl choline (DMPC) bilayers were similar to those caused by solutes known to localize in the glycerol-backbone region of the lipid bilayer, suggesting a similar localization for oxidized and reduced sporidesmin. Neither form of toxin disrupt the bilayer or membrane organization even at relatively high mole fractions. At concentrations <10 mole% both forms partitioned equally well in the gel and liquid-crystalline phases, whereas at higher concentrations (30 mole%) reduced sporidesmin is preferentially localized in the liquid-crystalline phase. These effects of sporidesmin on the phase properties of DMPC vesicles were also reported by the fluorescence behavior of 10-pyrenedecanoic acid (PDA). The effects of oxidized and reduced sporidesmins on PLA₂ kinetics are consistent with their ability to perturb bilayer organisation.     

IgG binding kinetics to oligo B protein A domains on lipid layers immobilized on a 27 MHz quartz-crystal microbalance

Although molecular recognitions between membrane receptors and their soluble ligands have been analyzed using their soluble proteins in bulk solutions, molecular recognitions of membrane receptors should be studied on lipid membranes considering their orientation and dynamics on membrane surfaces. We employed Staphylococcal Protein A (SpA) oligo B domains with long trialkyl-tags from E. coli (LppBx, x = 1, 2, and 5) and immobilized LppBx on lipid layers using hydrophobic interactions from the trialkyl-tag, while maintaining the orientation of B domain-chains on a 27 MHz quartz-crystal microbalance (QCM; AT-cut shear mode). The binding of IgG Fc regions to LppBx on lipid layers was detected by frequency decreases (mass increases) on the QCM. The maximum amount bound (Delta m(max)), association constants (K(a)), association and dissociation rate constants (k(1) and k(-1), respectively) were obtained. Binding kinetics of IgG to LppB2 and LppB5 were quite similar, showing a simple 1:1 binding of the IgG Fc region to the B domain, when the surface coverage of LppB2 and LppB5 on the lipid surface is low (1.4%). When LppB5 was immobilized at the high surface coverage of 3.5%, the complex bindings of IgG such as one IgG bound to one or two LppB5 on the membrane could be observed. IgG-LppB1 binding was largely restricted because of steric hindrance on lipid surfaces. This gives a suggestion why Protein A has five IgG binding domains.     

Interaction of diverse voltage sensor homologs with lipid bilayers revealed by self-assembly simulations

Homologous pairing and braiding (supercoiling) have crucial effects on genome organization, maintenance, and evolution. Generally, the pairing and braiding processes are discussed in different contexts, independently of each other. However, analysis of electrostatic interactions between DNA double helices suggests that in some situations these processes may be related. Here we present a theory of DNA braiding that accounts for the elastic energy of DNA double helices as well as for the chiral nature of the discrete helical patterns of DNA charges. This theory shows that DNA braiding may be affected, stabilized, or even driven by chiral electrostatic interactions. For example, electrostatically driven braiding may explain the surprising recent observation of stable pairing of homologous double-stranded DNA in solutions containing only monovalent salt. Electrostatic stabilization of left-handed braids may stand behind the chiral selectivity of type II topoisomerases and positive plasmid supercoiling in hyperthermophilic bacteria and archea.     

Interaction of glycosylated human myelin basic protein with lipid bilayers

Myelin basic protein (MBP), isolated from normal human myelin, was glycosylated with UDP-N-acetyl-D-galactosamine and a glycosyltransferase isolated from porcine submaxillary glands. MBP containing 0.85 mol of N-acetyl-D-galactosamine per mole of protein was oxidized at carbon 6 by galactose oxidase and complexed with a spin-label, Tempoamine, in order to study its interactions with lipids. When the spin-labeled MBP was reacted with lipid vesicles consisting of DSPG, DPPG, and DMPG, most of the spin-label was motionally restricted in the gel phase, with a correlation time greater than 10(-8)s. The motion increased with increasing temperature and was sensitive to the lipid phase transition. Interaction with the gel phase of DPPA caused much less motional restriction of the probe. However, melting of the lipid allowed increased interaction and motional restriction of the probe, which was only partially reversed on cooling back to the gel phase. The motional restriction of the probe in these lipids is attributed to its penetration partway into the lipid bilayer in both the gel and liquid-crystalline phases. The fact that the probe bound to the protein can penetrate partway into the bilayer suggests that other hydrophobic side chains and residues of the protein can similarly penetrate into the bilayer. Additional evidence for penetration was provided by digestion of the lipid-bound protein with endoproteinase Lys-C. When nonglycosylated and glycosylated MBP in solution was treated with Lys-C, extensive digestion occurred. A single radioactive peptide which eluted at 25 min was identified as residues 92-105.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of tea catechins on lipid metabolism and body fat accumulation

Long-term feeding of tea catechins suppressed body fat accumulation in high-fat diet-induced obesity in mice, and that their effects might be attributed, at least in part, to the activation of hepatic lipid metabolism. Consecutive intake of tea catechins (588 mg/day) reduced body fat, especially abdominal fat in humans. These results demonstrate that intake of tea catechins is beneficial for body fat accumulation.