Oleamide-mediated sleep induction does not depend on perturbation of membrane homeoviscosity.
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To verify whether the sleep-inducing properties of oleamide were related to its ability to perturb membrane homeoviscosity, affecting 5-HT(2A) receptors, we compared the effects of oleamide and oleic acid, the latter lacking both the sleep-inducing effect and the action on 5-HT(2A) receptors. In binding studies the two compounds did not directly interact with rat brain cortex 5-HT(2A) receptors, nor did they increase the affinity of a 5-HT(2A) agonist, either in vitro or ex vivo. They had similar fluidizing effects, in vitro at high concentrations (>/=10 microM), and ex vivo after a dose of 100 mg/kg, and they reduced locomotor activity with similar potency. There thus appears to be no causal relationship between the fluidizing effects of oleamide and its sleep-inducing properties. (+info)
The crystal structure of a GroEL/peptide complex: plasticity as a basis for substrate diversity.
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The chaperonin GroEL is a double toriodal assembly that with its cochaperonin GroES facilitates protein folding with an ATP-dependent mechanism. Nonnative conformations of diverse protein substrates bind to the apical domains surrounding the opening of the double toroid's central cavity. Using phage display, we have selected peptides with high affinity for the isolated apical domain. We have determined the crystal structures of the complexes formed by the most strongly bound peptide with the isolated apical domain, and with GroEL. The peptide interacts with the groove between paired alpha helices in a manner similar to that of the GroES mobile loop. Our structural analysis, combined with other results, suggests that various modes of molecular plasticity are responsible for tight promiscuous binding of nonnative substrates and their release into the shielded cis assembly. (+info)
Forster excitation energy transfer in peridinin-chlorophyll-a-protein.
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Time-resolved fluorescence anisotropy spectroscopy has been used to study the chlorophyll a (Chl a) to Chl a excitation energy transfer in the water-soluble peridinin-chlorophyll a-protein (PCP) of the dinoflagellate Amphidinium carterae. Monomeric PCP binds eight peridinins and two Chl a. The trimeric structure of PCP, resolved at 2 A (, Science. 272:1788-1791), allows accurate calculations of energy transfer times by use of the Forster equation. The anisotropy decay time constants of 6.8 +/- 0.8 ps (tau(1)) and 350 +/- 15 ps (tau(2)) are respectively assigned to intra- and intermonomeric excitation equilibration times. Using the ratio tau(1)/tau(2) and the amplitude of the anisotropy, the best fit of the experimental data is achieved when the Q(y) transition dipole moment is rotated by 2-7 degrees with respect to the y axis in the plane of the Chl a molecule. In contrast to the conclusion of, Biochemistry. 23:1564-1571) that the refractive index (n) in the Forster equation should be equal to that of the solvent, n can be estimated to be 1.6 +/- 0.1, which is larger than that of the solvent (water). Based on our observations we predict that the relatively slow intermonomeric energy transfer in vivo is overruled by faster energy transfer from a PCP monomer to, e.g., the light-harvesting a/c complex. (+info)
An ultrafast time-resolved anisotropy study of bacteriochlorophyll a in pyridine.
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The transient absorption anisotropy spectrum of bacteriochlorophyll a (BChl a) in pyridine was measured in the wavelength interval 550-850 nm, 1 ps after optical excitation with a 792-nm femtosecond light pulse. In the wavelength region of Q(y) absorption and stimulated emission (775-825 nm), the anisotropy was found to be close to the theoretically expected value (0.4) for a two-level system. In the wavelength region 650-750 nm, where the transient absorption signal is dominated by excited state absorption, the anisotropy is reduced to approximately 0.18. Anisotropy kinetics were measured at several wavelengths and found to be constant within the time window 0-5 ps, showing that no internal dynamics of the BChl a molecule change the anisotropy on the time scale of tens of picoseconds. (+info)
Does membrane fluidity contribute to thermal compensation of beta-adrenergic signal transduction in isolated trout hepatocytes?
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The potential role of compensatory adjustments to membrane components in determining the function of the (&bgr;)-adrenergic receptor/adenylyl cyclase ((&bgr;)-AR/AC) signal-transduction system was studied in isolated hepatocytes of 5 degrees C- and 20 degrees C-acclimated rainbow trout Oncorhynchus mykiss. Rates of epinephrine-stimulated cyclic AMP (cAMP) production, although slowed (by a factor of 1.6- to 2.4-fold) by an acute drop in assay temperature from 20 to 5 degrees C, were significantly temperature-compensated, being approximately twofold higher in hepatocytes of 5 degrees C- than of 20 degrees C-acclimated trout. Membrane order in the bilayer interior of hepatocyte plasma membranes (as assessed by fluorescence polarization of 1, 6-diphenyl-1,3,5-hexatriene) was consistently lower in cold- than in warm-acclimated trout, reflecting an efficacy of homeoviscous adaptation of approximately 50 %. Temperature-induced changes in plasma membrane fatty acid composition (i.e. an increase in the proportions of polyunsaturated fatty acids with acclimation to 5 degrees C) were consistent with both the observed changes in the order of the bilayer interior and the extent of homeoviscous adaptation. However, isothermal fluidization of the bilayer interior by the addition of benzyl alcohol (30 mmol l(-)(1)) decreased rather than increased the rate of cAMP production. Significantly more (1. 81-fold) beta-adrenergic receptors were present in plasma membranes of hepatocytes from 5 degrees C-acclimated (6. 23x10(4)+/-4206 receptors per cell; mean +/- s.e.m., N=3) than 20 degrees C-acclimated fish (3.44x10(4)+/-4360 receptors per cell; N=3) when assayed at the acclimation temperature, whereas the equilibrium dissociation constants (K(d)) (13.73+/-4.33 nmol l(-)(1) at 5 degrees C; 9.75+/-3.29 nmol l(-)(1) at 20 degrees C; N=3) were similar. On the basis of a strong correlation between beta-adrenoceptor number and the rate of cAMP production (r(2)=0. 956), regardless of assay or acclimation temperature, we conclude that modulation of receptor number is the primary acclimatory response of this signal-transduction pathway to temperature change in trout liver. (+info)
Trp scanning analysis of Tet repressor reveals conformational changes associated with operator and anhydrotetracycline binding.
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We analysed the conformational states of free, tet operator-bound and anhydrotetracycline-bound Tet repressor employing a Trp-scanning approach. The two wild-type Trp residues in Tet repressor were replaced by Tyr or Phe and single Trp residues were introduced at each of the positions 162-173, representing part of an unstructured loop and the N-terminal six residues of alpha-helix 9. All mutants retained in vivo inducibility, but anhydrotetracycline-binding constants were decreased up to 7.5-fold when Trp was in positions 169, 170 and 173. Helical positions (168-173) differed from those in the loop (162-167) in terms of their fluorescence emission maxima, quenching rate constants with acrylamide and anisotropies in the free and tet operator-complexed proteins. Trp fluorescence emission decreased drastically upon atc binding, mainly due to energy transfer. For all proteins, either free, tet operator bound or anhydrtetracycline-bound, mean fluorescence lifetimes were determined to derive quenching rate constants. Solvent-accessible surfaces of the respective Trp side chains were calculated and compared with the quenching rate constants in the anhydrotetracycline-bound complexes. The results support a model, in which residues in the loop become more exposed, whereas residues in alpha-helix 9 become more buried upon the induction of TetR by anhydrotetracycline. (+info)
Selective destabilization of acidic phospholipid bilayers performed by the hepatitis B virus fusion peptide.
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A peptide corresponding to the N-terminal region of the S protein of hepatitis B virus (Met-Glu-Asn-Ile-Thr-Ser-Gly-Phe-Leu-Gly-Pro-Leu-Leu-Val-Leu-Gln) has been previously demonstrated to perform aggregation and destabilization of acidic liposome bilayers and to adopt a highly stable beta-sheet conformation in the presence of phospholipids. The changes in the lipid moiety produced by this peptide have been followed by fluorescence depolarization and electron microscopy. The later was employed to determine the size and shape of the peptide-vesicle complexes, showing the presence of highly aggregated and fused structures only when negatively charged liposomes were employed. 1,6-Diphenyl-1,3,5-hexatriene depolarization measurements showed that the interaction of the peptide with both negatively charged and zwitterionic liposomes was accompanied by a substantial reduction of the transition amplitude without affecting the temperature of the gel-to-liquid crystalline phase transition. These data are indicative of the peptide insertion inside the bilayer of both types of liposomes affecting the acyl chain order, though only the interaction with acidic phospholipids leads to aggregation and fusion. This preferential destabilization of the peptide towards negatively charged phospholipids can be ascribed to the electrostatic interactions between the peptide and the polar head groups, as monitored by 1-(4-(trimethylammoniumphenyl)-6-phenyl-1,3, 5-hexatriene fluorescence depolarization analysis. (+info)
A 130-kDa membrane protein of sperm flagella is the receptor for asterosaps, sperm-activating peptides of starfish Asterias amurensis.
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Spermatozoa of the starfish, Asterias amurensis, have a specific receptor for asterosap, a sperm-activating peptide isolated from the jelly coat of homologous eggs. We characterized the receptor by using several asterosap derivatives. Analysis of equilibrium binding of radioactive di-iodinated Bolton-Hunter reagent-labeled asterosap ((125)I(2)-BHP15) to the spermatozoa indicated that the cell has 1.1 x 10(5) binding sites of high affinity (K(d) = 57 pM), and also the receptor showed positive cooperativity for asterosap binding. When spermatozoa were treated with fluorophore-labeled asterosap, the sperm flagella were labeled, indicating that the receptors are mostly localized in the sperm tail. When spermatozoa were reacted with radioactive asterosap prelabeled with photoaffinity cross-linkers, a single 130-kDa membrane protein of sperm flagella was specifically radiolabeled. This result was reproducible regardless of the length of spacer arm of cross-linkers so far studied. Therefore, the 130-kDa protein is likely to be the receptor for asterosaps. Modification of asterosap at the N-terminal region with bulky molecules such as carboxyfluorescein did not affect the activity of asterosap, suggesting that the N-terminus of asterosap is not involved in the ligand-receptor interaction. On the other hand, S-alkylated asterosaps did not compete with (125)I(2)-BHP15 for binding to the receptor, indicating that disulfide linkage of asterosap is essential for the ligand-receptor interaction. The properties of the receptor, high affinity and high concentration, enabled us to apply the fluorescence polarization technique to study the molecular interaction between asterosap and the receptor. Using this method, we performed binding experiments in almost real time and found that divalent cations are significantly involved in the interaction between asterosap and the receptor. (+info)