Infrared spectroscopic studies on gramicidin ion-channels: relation to the mechanisms of anesthesia

Fourier transform infrared spectroscopic studies are reported on gramicidin ion-channels in phospholipid bilayers and the effects on the spectra of the anesthetics and related compounds (methoxyflurane, halothane, chloroform, carbon tetrachloride, n-pentane and n-decane) have been determined. The addition of anesthetics containing the 'acidic hydrogen' caused unique changes particularly on the amide I bands at 1639 cm-1 and 1670 cm-1. The 1639 cm-1 band became more intense while the intensity near 1670 cm-1 decreased dramatically. These effects were not observed with carbon tetrachloride, n-pentane and n-decane. The 1670 cm-1 band is interpreted as arising from the carbonyls involved in the head-to-head hydrogen-bonded dimerization where the relationship between chains is analogous to that of the antiparallel beta-pleated sheet structure and the anesthetics with 'acidic hydrogens' are considered to disrupt the hydrogen-bonded dimerization by competitive hydrogen bonding to the carbonyls at the head-to-head junction. As the dimer-monomer equilibrium is the 'on-off' mechanism for gramicidin ion-channel conductance, the results are considered in terms of the mechanism of action of anesthetics and are taken to suggest, for certain anesthetics, a hydrogen-bonding role to protein ion-channel components.     

Supramolecular organization of lysophosphatidylcholine-packaged Gramicidin A

Heat derived gramicidin A'/L-alpha-lysophosphatidylcholine complexes were separated on a sucrose gradient to form two fractions: Fraction A which had an approximately constant Gramicidin A' to phospholipid ratio of 8 to 10 lipid molecules per Gramicidin A' molecule and Fraction B which had a larger but variable ratio. Fluorescence and circular dichroism studies confirmed Fraction A to be a lipid-incorporated channel state. Electron microscopic studies, using uranyl acetate negative staining, showed fraction A to be a membranous state with the formation of bilayer vesicles, that is, the interaction of peptide and phospholipid micelles causes the lipid to reorganize into a bilayer structure. Freeze-fracture replicas of the channel incorporated state demonstrated the presence of a supramolecular organization of particles exhibiting a tendency to form rows with a 50-60 A periodicity along the row and with 70-80 A distance between rows. An idealized working model for the incorporated state is presented.     

Nuclear Overhauser effect and computational characterization of the beta-spiral of the polypentapeptide of elastin

The structure of the elastin polypentapeptide, poly(VPGVG), was studied by nuclear Overhauser effect experiments using perdeuterated Val1 and Val4 samples under the condition where intermolecular interactions are absent. More extensive interaction was found between the Val1 gamma CH and Pro2 beta CH protons than between the Val4 gamma CH and Pro2 beta CH protons. The Val1 gamma CH3-Pro2 beta CH interaction does not occur within the same pentamer as previously shown experimentally and as expected from steric considerations. The results are incompatible with the presence of a random chain network in poly(VPGVG) at room temperature but are readily explicable in terms of interturn interactions in a beta-spiral structure. More specifically, the results indicate that the beta-spiral conformation with 2.9 pentamers/turn is more prevalent than that with 2.7 pentamers/turn. Using conformations developed by molecular mechanics calculations, molecular dynamics simulations were carried out to compare the relative energies of these two variants of this class of beta-spiral structures. It was found in vacuo that the structure with 2.9 pentamers/turn is indeed more stable than that of 2.7 pentamers/turn by approximately 1 kcal/mole-pentamer.     

Nanometric design of extraordinary hydrophobic-induced pKa shifts for aspartic acid: Relevance to protein mechanisms

Commonly a key element enabling proteins to function is an amino acid residue or residues with functional side chains having shifted pKa values. This article reports the results on a set of protein-based polymers (model proteins) that exhibit hydrophobic folding and assembly transitions, and that have been designed for the purpose of achieving large hydrophobic-induced pKa shifts by selectively replacing Val residues by Phe residues. The high molecular weight polypentapeptides, actually poly (tricosapeptides) with six varied pentamers in fixed sequence, were designed and synthesized to have the same amino acid compositions but different proximities between a single aspartic acid residue and 5 Phe residues per 30 residues. With the 5 Phe residues distal from the Asp residue, the observed pKa shift was 2.9 when compared to the Val-containing reference. With the 5 Phe residues within 1 nm of the Asp residue, the pKa shift was 6.2. This represents a free energy of interaction of 8 kcal/moles. To our knowledge, this is the largest pKa shift documented for an Asp residue in a polypeptide– or protein–water system. Data are reviewed that do not support the usual electrostatic arguments for pKa shifts of charge–charge repulsion and/or unfavorable ion self-energies arising from displacement of water by hydrophobic moieties, but rather the data are interpreted to indicate the presence of an apolar–polar repulsive free energy of hydration, which results from a potentially highly cooperative competition between apolar and polar species for hydration. © 1994 John Wiley & Sons, Inc.     

Use of synthetic gramicidins in the determination of channel structure and mechanism

The syntheses of (1-13C) Trp9 gramicidin A (GA), (1-13C) Trp11 GA, (1-13C) Trp13 GA, (1-13C) Trp15 GA, and D . Leu2 GA are verified by means of high performance liquid chromatography, carbon-13 nuclear magnetic resonance, circular dichroism and characterization of transport properties. The use of these and other synthetic gramicidins is discussed in terms of determining ion binding sites within the channel, helix sense of the channel, the basis of monovalent vs divalent cation selectivity, and means of modulating channel conductance.     

Receptor functions for the integrin VLA-3: fibronectin, collagen, and laminin binding are differentially influenced by Arg-Gly-Asp peptide and by divalent cations

The capability of the integrin VLA-3 to function as a receptor for collagen (Coll), laminin (Lm), and fibronectin (Fn) was addressed using both whole cell adhesion assays and ligand affinity columns. Analysis of VLA-3-mediated cell adhesion was facilitated by the use of a small cell lung carcinoma line (NCI-H69), which expresses VLA-3 but few other integrins. While VLA-3 interaction with Fn was often low or undetectable in cells having both VLA-3 and VLA-5, NCI-H69 cells readily attached to Fn in a VLA-3-dependent manner. Both Arg-Gly-Asp (RGD) peptide inhibition studies, and Fn fragment affinity columns suggested that VLA-3, like VLA-5, may bind to the RGD site in human Fn. However, unlike Fn, both Coll and Lm supported VLA-3-mediated adhesion that was not inhibited by RGD peptide, and was totally unaffected by the presence of VLA-5. In addition, VLA-3-mediated binding to Fn was low in the presence of Ca++, but was increased 6.6-fold with Mg++, and 30-fold in the presence of Mn++. In contrast, binding to Coll was increased only 1.2-fold with Mg++, and 1.7-fold in Mn++, as compared to the level seen with Ca++. Together, these experiments indicate that VLA-3 can bind Coll, Lm, and Fn, and also show that (a) VLA-3 can recognize both RGD-dependent and RGD-independent ligands, and (b) different VLA-3 ligands have distinctly dissimilar divalent cation sensitivities.     

Characterization of micellar-packaged gramicidin A channels.

The effects of heating, on an aqueous gramicidin A lysolecithin system, were examined by carbon-13 nuclear magnetic resonance (¹³C-NMR), circular dichroism (CD), and sodium-23 nuclear magnetic resonance (²³Na-NMR), and the results are collectively interpreted to indicate micellar-packaging of gramicidin channels and cation occupancy in the channel. ¹³C-NMR of the gramicidin-lysolecithin system demonstrates a decrease in mobility of the micellar lipid on heating which is indicative of incorporation of gramicidin into the hydrophobic core of the micelle. A unique and reproducible CD spectrum is obtained for the heat incorporated state. Sodium-23 spin-lattice relaxation times (T₁) demonstrated sodium interaction to be dependent on heat incorporation. The T₁ identified interaction is blocked by silver ion which is known to block sodium transport through the channel in lipid bilayer studies. The temperature dependence of the sodium-23 line width defines an exchange process with an activation energy of 6.8 kcal/mole which is essentially the same as the activation energy reported for transport through the channel in lecithin bilayer studies, and the sodium exchange process is blocked by thallium ion which is also known to block sodium transport through the channel.