Steric interactions of valines 1, 5, and 7 in [valine 5, D-alanine 8] gramicidin A channels.
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When the central valine residues 6, 7, and 8 of gramicidin A (gA) are shifted by one position, the resulting [Val(5), D-Ala(8)]gA forms right-handed channels with a single-channel conductance and average duration somewhat less than gA channels. The reduction in channel duration has been attributed to steric conflict between the side chains of Val(1) and Val(5) in opposing monomers (Koeppe, R. E. II, D. V. Greathouse, A. Jude, G. Saberwal, L. L. Providence, and O. S. Andersen. 1994. J. Biol. Chem. 269:12567-12576). To investigate the orientations and motions of valines in [Val(5), D-Ala(8)]gA, we have incorporated (2)H labels at Val 1, 5, or 7 and recorded (2)H-NMR spectra of oriented and nonoriented samples in hydrated dimyristoylphosphatidylcholine. Spectra of nonoriented samples at 4 degrees C reveal powder patterns that indicate rapid side chain "hopping" for Val(5), and an intermediate rate of hopping for Val(1) and Val(7) that is somewhat slower than in gA. Oriented samples of deuterated Val(1) and Val(7) show large changes in the methyl and C(beta)-(2)H quadrupolar splittings (Deltanu(q)) when Ala(5) in native gA is changed to Val(5). Three or more peaks for the Val(1) methyls with Deltanu(q) values that vary with the echo delay, together with an intermediate spectrum for nonoriented samples at 4 degrees C, suggest unusual side chain dynamics for Val(1) in [Val(5), D-Ala(8)]gA. These results are consistent with a steric conflict that has been introduced between the two opposing monomers. In contrast, the acylation of gA has little influence on the side chain dynamics of Val(1), regardless of the identity of residue 5. (+info)
Thermodynamics of alcohol-lipid bilayer interactions: application of a binding model.
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Several recent reports have provided evidence that interactions of small alcohols with lipid bilayer membranes are dominated by adsorption to the membrane-water interface. This mode of interaction is better modeled by binding models than solution theories. In the present study, alcohol-membrane interactions are examined by applying the 'solvent exchange model' [J.A. Schellmann, Biophys. Chem. 37 (1990) 121] to calorimetric measurements. Binding constants (in mole fraction units) for small alcohols to unilamellar liposomes of dimyristoyl phosphatidylcholine were found to be close to unity, and in contrast to partitioning coefficients they decrease through the sequence ethanol, 1-propanol, 1-butanol. Thus, the direct (intrinsic) affinity of the bilayer for these alcohols is lower the longer the acyl chain. A distinction between binding and partitioning is discussed, and it is demonstrated that a high concentration of solute in the bilayer (large partitioning coefficients) can be obtained even in cases of weak binding. Other results from the model suggest that the number of binding sites on the lipid bilayer interface is 1-3 times the number of lipid molecules and that the binding is endothermic with an enthalpy change of 10-15 kJ/mol. Close to the main phase transition of the lipid bilayer the results suggest the presence of two distinct classes of binding sites: 'normal' sites similar to those observed at higher temperatures, and a lower number of high-affinity sites with binding constants larger by one or two orders of magnitude. The occurrence of high-affinity sites is discussed with respect to fluctuating gel and fluid domains in bilayer membranes close to the main phase transition. (+info)
EDTA-induced self-assembly of cationic lipid-DNA multilayers near a monolayer-covered air-water interface.
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The presence of EDTA in the suspending buffer can induce the formation of multilayer structures from a mixture of the cationic lipid 3beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol and the zwitterionic 'helper' lipid 1, 2-dimyristoyl-sn-glycero-3-phosphocholine with DNA. The resulting structures consist of stacks of alternating sheets of lipid bilayer with intercalated DNA. In the absence of EDTA, only a single layer of DNA adsorbs to the lipid membrane. The buffer composition therefore influences the morphology of the lipid-aggregate/DNA assembly, which was not known to date. (+info)
Modification of liposomes with N-substituted polyacrylamides: identification of proteins adsorbed from plasma.
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Liposomes prepared from DMPC (80%) and cholesterol (20%) were modified with a series of hydrophobically modified N-substituted polyacrylamides, namely, poly[N-isopropylacrylamide] (PNIPAM), poly[N,N-bis(2-methoxyethyl) acrylamide] (PMEAM), and poly[(3-methoxypropyl)acrylamide] (PMPAM). The hydrophobic group, N-[4-(1-pyrenylbutyl)-N-n-octadecylamine was attached to one end of the polymer chains to serve as an anchor for incorporation into the liposome bilayer. Liposome-polymer interactions were confirmed using fluorescence spectroscopy and chemical analysis. Microscopy revealed differences in aggregation tendency between unmodified and polymer-modified liposomes. Proteins adsorbed to liposome surfaces during exposure to human plasma were identified by immunoblot analysis. It was found that both unmodified and polymer-modified liposomes adsorb a wide variety of plasma proteins. Contact phase coagulation proteins, complement proteins, cell-adhesive proteins, serine protease inhibitors, plasminogen, antithrombin III, prothrombin, transferrin, alpha(2)-microglobulin, hemoglobin, haptoglobin and beta-lipoprotein as well as the major plasma proteins were all detected. Some differences were found between the unmodified and polymer-modified liposomes. The unmodified liposomes adsorbed plasminogen mainly as the intact protein, whereas on the modified liposomes plasminogen was present in degraded form. Also, the liposomes modified with PNIPAM in its extended conformation (below the lower critical solution temperature) appeared to adsorb less protein than those containing the 'collapsed' form of PNIPAM (above the LCST). (+info)
Lipid-dependent activation of protein kinase C-alpha by normal alcohols.
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Significant stimulation of protein kinase C-alpha (PKCalpha) by n-alcohols was observed in characterized lipid systems composed of phosphatidylcholine/phosphatidylserine/dioleoylglycerol (PC/PS/DO). The logarithm of the alcohol concentrations to achieve half-maximal PKC stimulation (ED(50)) and of the maximal PKC stimulation by alcohols were both linear functions of alcohol chain length, consistent with the Meyer-Overton effect. Binding of phorbol esters to PKC was not significantly affected by octanol. Octanol increased, up to 4-fold, the affinity of PKC binding to the lipid bilayers in both the absence and presence of DO. However, octanol increased PKC activity much more significantly than it enhanced binding of the enzyme to the lipid bilayers, suggesting that the stimulation of PKC is not merely a reflection of the increase in PKC bilayer binding affinity. (31)P NMR experiments did not reveal formation of non-lamellar phases with octanol. Differential scanning calorimetry suggested that alcohols, like diacylglycerol, induce formation of compositionally distinct domains and the maximal enzyme activity with alcohol resided roughly in the putative domain-coexistence region. These results suggest that alcohols are mimicking diacylglycerol in activating PKC, not by binding to the high affinity phorbol ester binding site, but by altering lipid structure and by enhancing PKC-bilayer binding. (+info)
NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin: applicability of solution (19)F NMR.
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We report high resolution solution (19)F NMR spectra of fluorine-labeled rhodopsin mutants in detergent micelles. Single cysteine substitution mutants in the cytoplasmic face of rhodopsin were labeled by attachment of the trifluoroethylthio (TET), CF(3)-CH(2)-S, group through a disulfide linkage. TET-labeled cysteine mutants at amino acid positions 67, 140, 245, 248, 311, and 316 in rhodopsin were thus prepared. Purified mutant rhodopsins (6-10 mg), in dodecylmaltoside, were analyzed at 20 degrees C by solution (19)F NMR spectroscopy. The spectra recorded in the dark showed the following chemical shifts relative to trifluoroacetate: Cys-67, 9.8 ppm; Cys-140, 10.6 ppm; Cys-245, 9.9 ppm; Cys-248, 9.5 ppm; Cys-311, 9.9 ppm; and Cys-316, 10.0 ppm. Thus, all mutants showed chemical shifts downfield that of free TET (6.5 ppm). On illumination to form metarhodopsin II, upfield changes in chemical shift were observed for (19)F labels at positions 67 (-0.2 ppm) and 140 (-0.4 ppm) and downfield changes for positions 248 (+0.1 ppm) and 316 (+0.1 ppm) whereas little or no change was observed at positions 311 and 245. On decay of metarhodopsin II, the chemical shifts reverted largely to those originally observed in the dark. The results demonstrate the applicability of solution (19)F NMR spectroscopy to studies of the tertiary structures in the cytoplasmic face of intact rhodopsin in the dark and on light activation. (+info)
Persistence of phase coexistence in disaturated phosphatidylcholine monolayers at high surface pressures.
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Prior reports that the coexistence of the liquid-expanded (LE) and liquid-condensed (LC) phases in phospholipid monolayers terminates in a critical point have been compromised by experimental difficulties with Langmuir troughs at high surface pressures and temperatures. The studies reported here used the continuous interface of a captive bubble to minimize these problems during measurements of the phase behavior for monolayers containing the phosphatidylcholines with the four different possible combinations of palmitoyl and/or myristoyl acyl residues. Isothermal compression produced surface pressure-area curves for dipalmitoyl phosphatidylcholine (DPPC) that were indistinguishable from previously published data obtained with Langmuir troughs. During isobaric heating, a steep increase in molecular area corresponding to the main LC-LE phase transition persisted for all four compounds to 45 mN/m, at which collapse of the LE phase first occurred. No other discontinuities to suggest other phase transitions were apparent. Isobars for DPPC at higher pressures were complicated by collapse of the monolayer, but continued to show evidence up to 65 mN/m for at least the onset of the LC-LE transition. The persistence of the main phase transition to high surface pressures suggests that a critical point for these monolayers of disaturated phospholipids is either nonexistent or inaccessible at an air-water interface. (+info)
The structure of the membrane-binding 38 C-terminal residues from bovine PP3 determined by liquid- and solid-state NMR spectroscopy.
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The secondary structure and membrane-associated conformation of a synthetic peptide corresponding to the putative membrane-binding C-terminal 38 residues of the bovine milk component PP3 was determined using 1H NMR in methanol, CD in methanol and SDS micelles, and 15N solid-state NMR in planar phospholipid bilayers. The solution NMR and CD spectra reveal that the PP3 peptide in methanol and SDS predominantly adopts an alpha-helical conformation extending over its entire length with a potential bend around residue 19. 15N solid-state NMR of two PP3 peptides 15N-labelled at the Gly7 and Ala32 positions, respectively, and dissolved in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol phospholipid bilayers shows that the peptide is associated to the membrane surface with the amphipathic helix axis oriented parallel to the bilayer surface. (+info)