gramicidin-a and formal-glycol

Gramicidin is a hydrophobic peptide that assembles as a head-to-head dimer in lipid membranes to form water-filled channels selective to small monovalent cations. Two diastereoisomeric forms, respectively SS and RR, of chemically modified channels in which a dioxolane ring links the formylated N-termini of two gramicidin monomers, were shown to form ion channels. To investigate the structural basis underlying experimentally measured differences in proton conductance in the RR and SS channels, we construct atomic-resolution models of dioxolane-linked gramicidin dimers by analogy with the native dimer. A parametric description of the linker compatible with the CHARMM force field used for the peptide is derived by fitting geometry, vibrational frequencies, and energy to the results of ab initio calculations. The linker region of the modified gramicidin dimers is subjected to an extensive conformational search using high-temperature simulated annealing, and free-energy surfaces underlying the structural fluctuations of the channel backbone at 298K are computed from molecular dynamics simulations. The overall secondary structure of the beta-helical gramicidin pore is retained in both linked channels. The SS channel is found in a single conformation resembling that of the native dimer, with its peptide bonds undergoing rapid librations with respect to the channel axis. By contrast, its RR counterpart is characterized by local backbone distortions in which the two peptide bonds flanking the linker are markedly tilted in order to satisfy the pitch of the helix. In these distorted structures, each of the two carbonyl groups points either in or out of the lumen. Flipping these two peptides in and out involves thermally activated transitions, which results in four distinct conformational states at equilibrium with one another on a nanosecond time scale. This work opens the way to detailed comparative studies of structure-function relationships in biological proton ducts.

Topics: Computer Simulation; Dimerization; Dioxolanes; Energy Transfer; Gramicidin; Hot Temperature; Ion Channels; Macromolecular Substances; Models, Molecular; Motion; Protein Binding; Protein Conformation; Stereoisomerism; Stress, Mechanical

Proton conductance (g(H)) in single SS stereoisomers of dioxolane-linked gramicidin A (gA) channels were measured in different phospholipid bilayers at different HCl concentrations. In particular, measurements were obtained in bilayers made of 1,2-diphytanoyl 3-phosphocholine (DiPhPC) or its ethylated derivative 1,2-diphytanoyl 3-ethyl-phosphocholine (et-DiPhPC,). The difference between these phospholipids is that in et-DiPhPC one of the phosphate oxygens is covalently linked to an ethyl group and cannot be protonated. In relatively dilute acid solutions, g(H) in DiPhPC is significantly higher than in et-DiPhPC. At high acid concentrations, g(H) is the same in both diphytanoyl bilayers. Such differences in g(H) can be accounted for by surface charge effects at the membrane/solution interfaces. In the linear portion of the log g(H)-log [H] relationship, g(H) values in diphytanoyl bilayers were significantly larger (approximately 10-fold) than in neutral glyceryl monooleate (GMO) membranes. The slopes of the linear log-log relationships between g(H) and [H] in diphytanoyl and GMO bilayers are essentially the same (approximately 0.76). This slope is significantly lower than the slope of the log-log plot of proton conductivity versus proton concentration in aqueous solutions (approximately 1.00). Because the chemical composition of the membrane-channel/solution interface is strikingly different in GMO and diphytanoyl bilayers, the reduced slope in g(H)-[HCl] relationships may be a characteristic of proton transfer in the water wire inside the SS channel. Values of g(H) in diphytanoyl bilayers were also significantly larger than in membranes made of the more common biological phospholipids 1-palmitoyl 2-oleoyl phosphocholine (POPC) or 1-palmitoyl 2-oleoyl phosphoethanolamine (POPE). These differences, however, cannot be accounted for by different surface charge effects or by different internal dipole potentials. On the other hand, maximum g(H) measured in the SS channel does not depend on the composition of the bilayer and is determined essentially by the reduced mobility of protons in concentrated acid solutions. Finally, no experimental evidence was found in support of a lateral proton movement at the phospholipid/solution interface contributing to g(H) in single SS channels. Protein-lipid interactions are likely to modulate g(H) in the SS channel.

Topics: Chlorides; Dioxolanes; Electric Conductivity; Gramicidin; Ion Channels; Ion Transport; Lipid Bilayers; Membrane Potentials; Phospholipids; Protons; Solutions; Stereoisomerism; Water

The association of two gramicidin A (gA) peptides via H-bonds in lipid bilayers causes the formation of an ion channel that is selective for monovalent cations only. In this study, two gAs were covalently linked with a dioxolane group (SS dimer). Some functional properties of natural gA channels were compared to that synthetic dimer in Na(+)- or Cs(+)-containing solutions. The SS dimer remained in the open configuration most of the time, while natural gA channels had a relatively brief mean open time. Single channel conductances to Na(+) (g(Na)) or Cs(+) (g(Cs)) in the SS dimer were smaller than in natural gA. However, g(Na) was considerably more attenuated than g(Cs). This probably results from a tight solvation of Na(+) by the dioxolane linker in the SS channel. In Cs(+) solutions, the SS had frequent closures. By contrast, in Na(+) solutions the synthetic dimer remained essentially in the open state. The mean open times of SS channels in different solutions (T(open, Na) > T(open,Cs) > T(open,H)) were inversely proportional to the single channel conductances (g(H) > g(Cs) > g(Na)). This suggests that ion occupancy inside the pore stabilizes the open configuration of the gA dimer. The mean closed time of the SS dimer was longer in Cs(+) than in H(+) solutions. Possible mechanisms for these effects are discussed.

Topics: Cesium; Dimerization; Dioxolanes; Gramicidin; Ion Channel Gating; Sodium; Solutions

Two different stereoisomers of the dioxolane-linked gramicidin A (gA) channels were individually synthesized (the SS and RR dimers;. Science. 244:813-817). The structural differences between these dimers arise from different chiralities within the dioxolane linker. The SS dimer mimics the helicity and the inter- and intramolecular hydrogen bonding of the monomer-monomer association of gA's. In contrast, there is a significant disruption of the helicity and hydrogen bonding pattern of the ion channel in the RR dimer. Single ion channels formed by the SS and RR dimers in planar lipid bilayers have different proton transport properties. The lipid environment in which the different dimers are reconstituted also has significant effects on single-channel proton conductance (g(H)). g(H) in the SS dimer is about 2-4 times as large as in the RR. In phospholipid bilayers with 1 M [H(+)](bulk), the current-voltage (I-V) relationship of the SS dimer is sublinear. Under identical experimental conditions, the I-V plot of the RR dimer is supralinear (S-shaped). In glycerylmonooleate bilayers with 1 M [H(+)](bulk), both the SS and RR dimers have a supralinear I-V plot. Consistent with results previously published (. Biophys. J. 73:2489-2502), the SS dimer is stable in lipid bilayers and has fast closures. In contrast, the open state of the RR channel has closed states that can last a few seconds, and the channel eventually inactivates into a closed state in either phospholipid or glycerylmonooleate bilayers. It is concluded that the water dynamics inside the pore as related to proton wire transfer is significantly different in the RR and SS dimers. Different physical mechanisms that could account for this hypothesis are discussed. The gating of the synthetic gA dimers seems to depend on the conformation of the dioxolane link between gA's. The experimental results provide an important framework for a detailed investigation at the atomic level of proton conduction in different and relatively simple ion channel structures.

Topics: Biological Transport; Dioxolanes; Gramicidin; Hydrogen Bonding; Models, Molecular; Porosity; Protein Multimerization; Protein Structure, Quaternary; Protons; Stereoisomerism; Water

The mobility of protons in a dioxolane-linked gramicidin A channel (D1) is comparable to the mobility of protons in aqueous solutions (Cukierman, S., E. P. Quigley, and D. S. Crumrine. 1997. Biophys. J. 73:2489-2502). Aliphatic alcohols decrease the mobility of H+ in aqueous solutions. In this study, the effects of methanol on proton conduction through D1 channels were investigated in different lipid bilayers and at different HCl concentrations. Methanol attenuated H+ currents in a voltage-independent manner. Attenuation of proton currents was also independent of H+ concentrations in solution. In phospholipid bilayers, methanol decreased the single channel conductance to protons without affecting the binding affinity of protons to bilayers. In glycerylmonooleate membranes, the attenuation of single channel proton conductances qualitatively resembled the decrease of conductivities of HCl solutions by methanol. However, in both types of lipid bilayers, single channel proton conductances through D1 channels were considerably more attenuated than the conductivities of different HCl solutions. This suggests that methanol modulates single proton currents through D1 channels. It is proposed that, on average, one methanol molecule binds to a D1 channel, and attenuates H+ conductance. The Gibbs free energy of this process (DeltaG0) is approximately 1.2 kcal/mol, which is comparable to the free energy of decrease of HCl conductivity in methanol solutions (1.6 kcal/mol). Apolar substances like urea and glucose that do not transport protons in HCl solutions and do not permeate D1 channels decreased solution conductivity and single channel conductance by a considerably larger proportion than methanol. Cs+ currents through D1 channels were considerably less (fivefold) attenuated by methanol than proton currents. It is proposed that methanol partitions inside the pore of gramicidin channels and delays the transfer of protons between water and methanol molecules, causing a significant attenuation of the single channel proton conductance. Gramicidin channels offer an interesting experimental model to study proton hopping along a single chain of water molecules interrupted by a single methanol molecule.

Topics: Biophysical Phenomena; Biophysics; Cesium; Dioxolanes; Electric Conductivity; Glucose; Gramicidin; In Vitro Techniques; Ion Channels; Lipid Bilayers; Membrane Potentials; Methanol; Protons; Solutions; Urea

Gramicidin A (gA) molecules were covalently linked with a dioxolane ring. Dioxolane-linked gA dimers formed ion channels, selective for monovalent cations, in planar lipid bilayers. The main goal of this study was to compare the functional single ion channel properties of natural gA and its covalently linked dimer in two different lipid bilayers and HCl concentrations (10-8000 mM). Two ion channels with different gating and conductance properties were identified in bilayers from the product of dimerization reaction. The most commonly observed and most stable gramicidin A dimer is the main object of this study. This gramicidin dimer remained in the open state most of the time, with brief closing flickers (tau(closed) approximately 30 micros). The frequency of closing flickers increased with transmembrane potential, making the mean open time moderately voltage dependent (tau(open) changed approximately 1.43-fold/100 mV). Such gating behavior is markedly different from what is seen in natural gA channels. In PEPC (phosphatidylethanolamine-phosphatidylcholine) bilayers, single-channel current-voltage relationships had an ohmic behavior at low voltages, and a marked sublinearity at relatively higher voltages. This behavior contrasts with what was previously described in GMO (glycerylmonooleate) bilayers. In PEPC bilayers, the linear conductance of single-channel proton currents at different proton concentrations was essentially the same for both natural and gA dimers. g(max) and K(D), obtained from fitting experimental points to a Langmuir adsorption isotherm, were approximately 1500 pS and 300 mM, respectively, for both the natural gA and its dimer. In GMO bilayers, however, proton affinities of gA and the dioxolane-dimer were significantly lower (K(D) of approximately 1 and 1.5 M, respectively), and the g(max) higher (approximately 1750 and 2150 pS, respectively) than in PEPC bilayers. Furthermore, the relationship between single-channel conductance and proton concentration was linear at low bulk concentrations of H+ (0.01-2 M) and saturated at concentrations of more than 3 M. It is concluded that 1) The mobility of protons in gramicidin A channels in different lipid bilayers is remarkably similar to proton mobilities in aqueous solutions. In particular, at high concentrations of HCl, proton mobilities in gramicidin A channel and in solution differ by only 25%. 2) Differences between proton conductances in gramicidin A channels in GMO and PEPC cannot be expl

Topics: Amino Acid Sequence; Dimerization; Dioxolanes; Electric Conductivity; Glycerides; Gramicidin; Hydrogen-Ion Concentration; Ion Channel Gating; Ion Channels; Lipid Bilayers; Membrane Potentials; Molecular Sequence Data; Molecular Structure; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Protons; Static Electricity

The gating transition of the RR and SS dioxolane ring-linked gramicidin A channels were studied with molecular dynamics simulations using a detailed atomic model. It was found that the probable reaction path, describing the transition of the ring from the exterior to the interior of the channel where it blocked the permeation pathway, involved several steps including the isomerization of the transpeptide plane dihedral angle of Val1. Reaction coordinates along this pathway were defined, and the transition rates between the stable conformers were calculated. It was found, in good accord with experimental observations, that the calculated blocking rate for the RR-linked channel was 280/s with a mean blocking time of 0.04 ms, whereas such blocking did not occur in the case of the SS-linked channel. An important observation is that the resulting lifetime for the blocked state of the RR-linked channel was in good accord with the experimental observations only when the calculations were performed in the presence of a potassium ion inside the channel.

Topics: Dioxolanes; Gramicidin; Hydrogen Bonding; Ion Channel Gating; Kinetics; Models, Biological; Models, Molecular; Models, Theoretical; Protein Conformation; Thermodynamics