Ion transport in the gramicidin channel: molecular dynamics study of single and double occupancy.

The structural and thermodynamic factors responsible for the singly and doubly occupied saturation states of the gramicidin channel are investigated with molecular dynamics simulations and free energy perturbation methods. The relative free energy of binding of all of the five common cations Li+, Na+, K+, Rb+, and Cs+ is calculated in the singly and doubly occupied channel and in bulk water. The atomic system, which includes the gramicidin channel, a model membrane made of neutral Lennard-Jones particles and 190 explicit water molecules to form the bulk region, is similar to the one used in previous work to calculate the free energy profile of a Na+ ion along the axis of the channel. In all of the calculations, the ions are positioned in the main binding sites located near the entrances of the channel. The calculations reveal that the doubly occupied state is relatively more favorable for the larger ions. Thermodynamic decomposition is used to show that the origin of the trend observed in the calculations is due to the loss of favorable interactions between the ion and the single file water molecules inside the channel. Small ions are better solvated by the internal water molecules in the singly occupied state than in the doubly occupied state; bigger ions are solvated almost as well in both occupation states. Water-channel interactions play a role in the channel response. The observed trends are related to general thermodynamical properties of electrolyte solutions.     

A molecular dynamics study of gating in dioxolane-linked gramicidin A channels.

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.     

Valence selectivity of the gramicidin channel: a molecular dynamics free energy perturbation study.

The valence selectivity of the gramicidin channel is examined using computer simulations based on atomic models. The channel interior is modeled using a gramicidin-like periodic poly (L,D)-alanine beta-helix. Free energy perturbation calculations are performed to obtain the relative affinity of K+ and Cl- for the channel. It is observed that the interior of the gramicidin channel provides an energetically favorable interaction site for a cation but not for an anion. Relative to solvation in bulk water, the carbonyl CO oxygens can provide a favorable interaction to stabilize K+, whereas the amide NH hydrogens are much less effective in stabilizing Cl-. The results of the calculations demonstrate that, as a consequence of the structural asymmetry of the backbone charge distribution, a K+ cation can partition spontaneously from bulk water to the interior of the gramicidin channel, whereas a Cl- anion cannot.     

Solid-state nuclear magnetic resonance derived model for dynamics in the polypeptide backbone of the gramicidin A channel.

The dynamics of the backbone of the gramicidin A transmembrane cation channel in dimyristoylphosphatidylcholine bilayers have been investigated using solid state 15N nuclear magnetic resonance (n.m.r.) spectroscopy. With the temperature-dependent fluidity of the bilayer, the rates of motions in the helical gramicidin channel can be modulated. It is shown that in the gel phase, all substantial motions of the channel are slow on the timescale of the n.m.r. experiment (3.5 kHz). The use of oriented samples in which the axis of global channel rotation is aligned parallel to the magnetic field enables separation of global and local dynamics. Spectra obtained from oriented bilayer samples containing single-site 15N-labeled gramicidin at 8 degrees C are analyzed to yield a spatial model for local backbone motion. This model includes the axis of motion, the mean orientation, and the maximum amplitude of displacement for individual peptide planes. Specific sites in the first turn of the amino terminus were investigated, with emphasis on the Ala3 and Leu4 linkages, for which the orientation of the 15N chemical shift tensor with respect to the molecular frame has been determined. The effect of two well-characterized bilayer defect structures, parabolic focal conics and oily streaks, is included in the spectral simulations. It is found that only relatively small amplitude motions are possible at the two sites, with amplitudes of not more than +/- 8 degrees and +/- 15 degrees for the Ala3 and Leu4 sites, respectively. Detailed characterization of the bilayer surface geometry in the oriented samples is presently the major limiting factor in the use of this technique for probing the spatial extent of local motions in integral membrane proteins.     

Ab initio molecular dynamics study of proton transfer in a polyglycine analog of the ion channel gramicidin A.

Proton transfer in biological systems is thought to often proceed through hydrogen-bonded chains of water molecules. The ion channel, gramicidin A (gA), houses within its helical structure just such a chain. Using the density functional theory based ab initio molecular dynamics Car-Parrinello method, the structure and dynamics of proton diffusion through a polyglycine analog of the gA ion channel has been investigated. In the channel, a proton, which is initially present as hydronium (H3O+), rapidly forms a strong hydrogen bond with a nearest neighbor water, yielding a transient H5O2+ complex. As in bulk water, strong hydrogen bonding of this complex to a second neighbor solvation shell is required for proton transfer to occur. Within gA, this second neighbor shell included not only a channel water molecule but also a carbonyl of the channel backbone. The present calculations suggest a transport mechanism in which a priori carbonyl solvation is a requirement for proton transfer.     

Structure and dynamics of hydronium in the ion channel gramicidin A.

The effects of the hydronium ion, H(3)0+, on the structure of the ion channel gramicidin A and the hydrogen-bonded network of waters within the channel were studied to help elucidate a possible mechanism for proton transport through the channel. Several classical molecular dynamics studies were carried out with the hydronium in either the center of a gramicidin monomer or in the dimer junction. Structural reorganization of the channel backbone was observed for different hydronium positions, which were most apparent when the hydronium was within the monomer. In both cases the average O-O distance between the hydronium ion and its nearest neighbor water molecule was found to be approximately 2.55 A, indicating a rather strong hydrogen bond. Importantly, a subsequent break in the hydrogen-bonded network between the nearest neighbor and the next-nearest neighbor(approximately 2.7 -3.0 A) was repeatedly observed. Moreover, the carbonyl groups of gramicidin A were found to interact with the charge on the hydronium ion, helping in its stabilization. These facts may have significant implications for the proton hopping mechanism. The presence of the hydronium ion in the channel also inhibits to some degree the reorientational motions of the channel water molecules.     

Water permeation through gramicidin A: desformylation and the double helix: a molecular dynamics study

Multinanosecond molecular dynamics simulations of gramicidin A embedded in a dimyristoylphosphatidylcholine bilayer show a remarkable structural stability for both experimentally determined conformations: the head-to-head helical dimer and the double helix. Water permeability was found to be much higher in the double helical conformation, which is explained by lower hydrogen bond-mediated enthalpic barriers at the channel entrance and its larger pore size. Free-energy perturbation calculations show that the double helical structure is stabilized by the positive charges at the N termini introduced by the desformylation, whereas the helical dimer is destabilized. Together with the recent experimental observation that desformyl gramicidin conducts water hundredfold better than gramicidin, this suggests that desformyl gramicidin A predominantly occurs in the double helical conformation.     

Raman and infrared spectroscopic study of gramicidin A conformations

Raman scattering and infrared spectroscopic techniques were used to study the vibrational spectrum and conformation of the membrane channel protein gramicidin A in the solid state, in organic solutions and, using Raman scattering only, in a phospholipid environment. The investigation also includes measurements on head- and tail-group-modifled gramicidin A and a potassium thiocyanate-gramicidin A complex. Tentative identification of the molecular vibrations is proposed on the basis of the data on model compounds. The existence of four distinct conformations of the gramicidin A chain is established: conformation I present in the solid state, and CH₃OH and CD₃OD solutions; conformation II present in films cast from CHCl₃ solution; conformation III present in (CH₃)₂SO and (CD₃)₂SO solutions at concentrations below 0.5 m gramicidin A; and conformation IV present in the potassium thiocyanate-gramicidin A complex. The data obtainable on a gramicidin A-phospholipid suspension indicate a gramicidin A conformation in this environment corresponding either to the conformation I or II. The details of the spectra in the amide I region are shown to be consistent with a β-parallel hydrogen-bonded π_LD helix for conformational I, in terms of the polypeptide vibrational calculations of Nevskaya and co-workers. Conformation II is found to be consistent with an antiparallel double-stranded π_LD helix, while conformations III and IV probably have π-helical structures with larger channel diameters. The data on head- and tail-modified gramicidin A molecules indicate that their conformations are only slightly different from that of gramicidin A in conformation I.     

Noncontact dipole effects on channel permeation. II. Trp conformations and dipole potentials in gramicidin A

The four Trp dipoles in the gramicidin A (gA) channel modulate channel conductance, and their side chain conformations should therefore be important, but the energies of different conformations are unknown. A conformational search for the right-handed helix based on molecular mechanics in vacuo yielded 46 conformations within 20 kcal/mol of the lowest energy conformation. The two lowest energy conformations correspond to the solid-state and solution-state NMR conformations, suggesting that interactions within the peptide determine the conformation. For representative conformations, the electrostatic potential of the Trp side chains on the channel axis was computed. A novel application of the image-series method of. Biophys. J. 9:1160-1170) was introduced to simulate the polarization of bulk water by the Trp side chains. For the experimentally observed structures, the CHARm toph19 potential energy (PE) of a cation in the channel center is -1.65 kcal/mol without images. With images, the PE is -1.9 kcal/mol, demonstrating that the images further enhance the direct dipole effect. Nonstandard conformations yielded less favorable PEs by 0.4-1.1 kcal/mol.     

Orientation of gramicidin A transmembrane channel. Infrared dichroism study of gramicidin in vesicles.

Polarized infrared spectroscopy has been used to investigate the orientation of gramicidin A incorporated in dimyristoylphosphatidylcholine liposomes. Dichroism measurements of the major lipid (C = O ester, PO2-, CH2) and peptide (amide A, I, II) bands were performed on liposomes (with or without gramicidin) oriented by air-drying. The mean orientation of the lipid groups and of the pi LD helix chain in the gramicidin has been determined. It can be inferred from infrared frequencies of gramicidin that the dominant conformation of the peptide in liposomes cannot be identified to the antiparallel double-helical dimer found in organic solution. No shift in lipid frequencies was observed upon incorporation of gramicidin in the liposomes. However, a slight reorganization of the lipid hydrocarbon chains which become oriented more closely to the normal to the bilayer is evidenced by a change in the dichroism of the CH2 vibrations. The infrared dichroism results of gramicidin imply a perpendicular orientation of the gramicidin transmembrane channel with the pi LD helix axis at less than 15 degrees with respect to the normal to the bilayer.     

Test of molecular dynamics force fields in gramicidin A

The force fields commonly used in molecular dynamics simulations of proteins are optimized under bulk conditions. Whether the same force fields can be used in simulations of membrane proteins is not well established, although they are increasingly being used for such purposes. Here we consider ion permeation in the gramicidin A channel as a test of the AMBER force field in a membrane environment. The potentials of mean force for potassium ions are calculated along the channel axis and compared with the one deduced from the experimental conductance data. The calculated result indicates a rather large central barrier similar to those obtained from other force fields, which are incompatible with the conductance data. We suggest that lack of polarizability is the most likely cause of this problem, and, therefore, urge development of polarizable force fields for simulations of membrane proteins.     

The binding site of sodium in the gramicidin A channel: comparison of molecular dynamics with solid-state NMR data.

The location of the main binding site for sodium in the gramicidin A (GA) channel was investigated with molecular dynamics simulations, using an atomic model of the channel embedded in a fully hydrated dimyristoyl phosphatidycholine (DMPC) bilayer. Twenty-four separate simulations in which a sodium was restrained at different locations along the channel axis were generated. The results are compared with carbonyl 13C chemical shift anisotropy solid-state NMR experimental data previously obtained with oriented GA:DMPC samples. Predictions are made for other solid-state NMR properties that could be observed experimentally. The combined information from experiment and simulation strongly suggests that the main binding sites for sodium are near the channel's mouth, approximately 9.2 A from the center of the dimer channel. The 13C chemical shift anisotropy of Leu10 is the most affected by the presence of a sodium ion in the binding site. In the binding site, the sodium ion is lying off-axis, making contact with two carbonyl oxygens and two single-file water molecules. The main channel ligand is provided by the carbonyl group of the Leu10-Trp11 peptide linkage, which exhibits the largest deviation from the ion-free channel structure. Transient contacts with the carbonyl group of Val8 and Trp15 are also present. The influence of the tryptophan side chains on the channel conductance is examined based on the current information about the binding site.     

Ion-water and ion-polypeptide correlations in a gramicidin-like channel. A molecular dynamics study.

This work describes a molecular dynamics study of ion-water and ion-polypeptide correlation in a model gramicidin-like channel (the polyglycine analogue) based upon interaction between polarizable, multipolar groups. The model suggests that the vicinity of the dimer junction and of the ethanolamine tail are regions of unusual flexibility. Cs+ binds weakly in the mouth of the channel: there it coordinates five water molecules and the #11CO group with which it interacts strongly and is ideally aligned. In the channel interior it is generally pentacoordinate; at the dimer junction, because of increased channel flexibility, it again becomes essentially hexacoordinate. The ion is also strongly coupled to the #13 CO but not to either #9 or #15, consistent with 13C NMR data. Water in the channel interior is strikingly different from bulk water; it has a much lower mean dipole moment. This correlates with our observation (which differs from that of previous studies) that water-water angular correlations do not persist within the channel, a result independent of ion occupancy or ionic polarity. In agreement with streaming potential measurements, there are seven single file water molecules associated with Cs+ permeation; one of these is always in direct contact with bulk water. At the mouth of an ion-free channel, there is a pattern of dipole moment alteration among the polar groups. Due to differential interaction with water, exo-carbonyls have unusually large dipole moments whereas those of the endo-carbonyls are low. The computed potential of mean force for CS+ translocation is qualitatively reasonable. However, it only exhibits a weakly articulated binding site and it does not quantitatively account for channel energetics. Correction for membrane polarization reduces, but does not eliminate, these problems.     

Water transport and ion-water interaction in the gramicidin channel

The diffuse permeability and the diffusion coefficient of water (Dw) in the gramicidin channel is determined from the osmotic water permeability of the channel and "single file" pore theory. Dw is about 7% of the self-diffusion coefficient of bulk water. The diffusion coefficient of a single water molecule alone in the channel is also determined and is about equal to the value in bulk water. This provides an estimate of the mobility of water on the channel walls in the absence of water-water interaction. Since the gramicidin channel walls should be representative of uncharged polar protein surfaces, this result provides direct evidence that the presence of a cation in the channel reduces the hydraulic water permeability by a factor ranging from 60 for Tl+ to 5 for Na+. The diffusion coefficient of a cation (Dc) in the channel is estimated and compared with Dw. For Na+ it is found that Dc approximately equal to Dw, which implies that the movement of the row of water molecules through the channel determines the local mobility of Na+. Thus, it seems that short range ion-wall interactions are not important in determining the channel conductance for Na+. In contrast, for Li+, local ion-wall interactions probably do limit the conductance.     

Binding of thallium and other cations to the gramicidin A channel. Equilibrium dialysis study of gramicidin in phosphatidylcholine vesicles

Gramicidin A is a linear peptide antibiotic which forms dimer transmembrane channels selective for small monovalent cations, including thallium ions (Tl⁺) which are strongly bound. While there is great interest in the number of ion-binding sites per channel and the affinities of the sites for the various cations, measurements of the kinetics of ion permeation yield these equilibrium parameters only as indirect estimates dependent on the model assumed for the channel. Sonicated lipid vesicles. containing 1 mole of gramicidin per 30 moles of dimyristoylphosphatidylcholine. can be prepared with 5 mm-gramicidin. Evidence from our previous spectroscopic studies strongly supports the belief that this gramicidin is in the form of symmetrical dimer channels. Lipid vesicles containing gramicidin were dialyzed against control vesicles without gramicidin in the presence of a constant amount of radioactive ²⁰¹Tl⁺ and increasing amounts of non-radioactive Tl⁺. The ratio of ²⁰¹Tl⁺ free in solution to ²⁰¹Tl⁺ bound to the channel was measured after equilibrium (≥ 48 h) at 23 °C, and this ratio was plotted as a function of the free Tl⁺ concentration. The inverse of the slope yielded 0.8 to 1.1 for the maximum number of simultaneously occupied highest affinity sites per channel, and the inverse of the intercept yielded a highest affinity constant of 500 to 1000 m^?1 for each site. It appears that direct electrostatic repulsion prevents ions from binding simultaneously to the identical channel ends for thallium ion concentrations up to 20 mm. Estimates of the highest affinity constants for Rb⁺ and Na⁺ were also obtained.     

Molecular dynamics simulations of Trp side-chain conformational flexibility in the gramicidin A channel

Gramicidin A (gA) is prototypical peptide antibiotic and a model ion channel former. Configured in the solid-state NMR beta(6.5)-helix channel conformation, gA was subjected to 1-ns molecular dynamics (MD) gas phase simulations using the all-atom charmm22 force field to ascertain the conformational stability of the Trp side chains as governed by backbone and neighboring side-chain contacts. Three microcanonical trajectories were computed using different initial atomic velocities for each of twenty different initial structures. For each set, one of the four Trp side chains in each monomer was initially positioned in one of the five non-native conformations (A. E. Dorigo et al., Biophysical Journal, 1999, Vol. 76, 1897-1908), the other Trps being positioned in the native state, o1. In three additional control simulations, all Trps were initiated in the native conformation. After equilibration, constraints were removed and subsequent conformational changes of the initially constrained Trp were measured. The chi(1) was more flexible than chi(2.1). The energetically optimal orientation, o1 (Dorigo et al., 1999), was the most stable in all four Trp positions (9, 11, 13, 15) and remained unchanged for the entire 1 ns simulation in 19 of 24 trials. Changes in chi(1) from each of the 5 suboptimal states occur readily. Two of the non-native conformations reverted readily to o1, whereas the other three converted to an intermediate state, i2. There were frequent interconversions between i2 and o1. We speculate that experimentally observed Trp stability is caused by interactions with the lipid-water interface, and that stabilization of one of the suboptimal conformations in gA, such as i2, by lipid headgroups could produce a secondary, metastable conformational state. This could explain recent experimental studies of differences in the channel conductance dispersity between gA and a Trp-to-Phe gA analog, gramicidin M (gM, J. C. Markham et al., Biochimica et Biophysica Acta, 2001, Vol. 1513, 185-192).     

Side-chain structure and dynamics at the lipid-protein interface: Val1 of the gramicidin A channel.

High resolution dynamics and structural information has been resolved from 2H solid-state NMR spectra of the Val-1 side-chain of the gramicidin channel in a lipid bilayer. Both powder pattern lineshapes and spectra from uniformly aligned samples of gramicidin in lipid bilayers have been analyzed to achieve a fully consistant interpretation of the data. Torsional motions about the C alpha C beta axis (chi 1) are shown to be three-state jumps in which the occupancy of the states is given by the ratio, 75:15:10 for the chi 1 angles of 184 degrees:304 degrees:64 degrees. The dominant conformer is also the most common conformation observed for valines in well defined protein structures. The distribution of conformational substates that represents the chi 1 dynamics appears to be largely independent of the lipid phase transition and the hydration of the sample. However, there is evidence that the residence time between jumps is dependent on the lipid phase transition. Although this time is shown to be approximately 1 microseconds below the phase transition temperature, it is in the fast exchange limit above the transition temperature.     

Investigating the size and dynamics of voltage-gated sodium channel fenestrations: A molecular dynamics study

Eukaryotic voltage-gated sodium channels (VGSCs) are essential for the initiation and propagation of action potentials in electrically excitable cells, and are important pharmaceutical targets for the treatment of neurological disorders such as epilepsy, cardiac arrhythmias, and chronic pain. Evidence suggests that small, hydrophobic, VGSC-blocking drugs can gain access to binding residues within the central cavity of these channels by passing through lateral, lipid-filled “fenestrations” which run between the exterior of the protein and its central pore. Here, we use molecular dynamics simulations to investigate how the size and shape of fenestrations change over time in several bacterial VGSC models and a homology model of Nav1.4. We show that over the course of the simulations, the size of the fenestrations is primarily influenced by rapid protein motions, such as amino acid side-chain rotation, and highlight that differences between fenestration bottleneck-contributing residues are the primary cause of variations in fenestration size between the 6 bacterial models. In the eukaryotic channel model, 2 fenestrations are wide, but 2 are narrow due to differences in the amino acid sequence in the 4 domains. Lipid molecules are found to influence the size of the fenestrations by protruding acyl chains into the fenestrations and displacing amino acid side-chains. Together, the results suggest that fenestrations provide viable pathways for small, flexible, hydrophobic drugs.