gramicidin-a and methylamine

The accurate description of protein-ligand binding energies and configurations is an important problem in molecular biology with many applications in medicine and pharmacology. Molecular dynamics (MD) simulations provide the required accuracy but they are too slow for searching binding positions. Conversely, docking methods are much faster but have limited accuracy. An appropriate combination of the two methods could avoid the shortcomings associated with each, thus offering a better approach to the protein-ligand binding problem. Here we investigate the feasibility of such a combined docking-MD approach in a well-defined system: binding of organic cations to the gramicidin A channel. We use the AutoDock program to generate a set of protein-ligand binding configurations, which are then refined in MD simulations. For each system, we examine the binding configuration in detail and calculate the binding free energy by constructing the potential of mean force for the ligand. Our results show that AutoDock provides suitable initial configurations, which can be used profitably in MD simulations to obtain an accurate description of protein-ligand binding with a reasonable computational effort.

Topics: Cations; Computer Simulation; Gramicidin; Hydrogen Bonding; Methylamines; Models, Molecular; Molecular Conformation; Organic Chemicals; Quaternary Ammonium Compounds; Software; Tetraethylammonium

The free energy profiles for four organic cations in right-handed single-helix gramicidin A dimers were computed by using umbrella sampling molecular dynamics with CHARMM. Ion-water column translocations were facilitated by using a novel "water-tunnel" approach. The overlapping pieces of free energy profile for adjacent windows were selected from three trajectories that differed in initial ion rotation and were aligned by the method of umbrella potential differences. Neglected long-range electrostatic energies from the bulk water and the bilayer were computed with DelPhi and added to the profile. The approach was corroborated for the formamidinium-guanidinium pair by using perturbation dynamics at axial positions 0, 6, 12, and 15 A from the channel center. The barrier to ethylammonium entry was prohibitive at 21 kcal/mol, whereas for methylammonium it was 5.5 kcal/mol, and the profile was quite flat through the channel, roughly consistent with conductance measurements. The profile for formamidinium was very similar to that of methylammonium. Guanidinium had a high entry barrier (deltaF = +8.6 kcal/mol) and a narrow deep central well (deltaF = -2.6 kcal/mol), qualitatively consistent with predictions from voltage-dependent potassium current block measurements. Its deep central well, contrasting with the flat profile for formamidinium, was verified with perturbation dynamics and was correlated with its high propensity to form hydrogen bonds with the channel at the dimer junction (not shared by the other three cations). Analysis of the ensemble average radial forces on the ions demonstrates that all four ions undergo compressive forces in the channel that are at maximum at the center of the monomer and relieved at the dimer junction, illustrating increased flexibility of the channel walls in the center of the channel.

Topics: Amidines; Biophysical Phenomena; Biophysics; Cations; Dimerization; Gramicidin; Guanidine; Ion Channels; Methylamines; Models, Molecular; Protein Conformation; Quaternary Ammonium Compounds; Static Electricity; Thermodynamics; Water

Compared to the N-formyl gramicidin A (GA), the N-acetyl gramicidin A (NAG) channel has unchanged conductance in 1 M NH4+ (gamma NN/gamma GG = 1, conductance ratio) but reduced conductance in 1 M K+ (gamma NN/gamma GG = 0.6) methylammonium (gamma NN/gamma GG = 0.3), and formamidinium (gamma NN/gamma GG = 0.1) solutions. Except with formamidinium, "flicker blocks" are evident even at low cutoff frequencies. For all cations studied, channel lifetimes of N-acetyl homodimers (NN) are approximately 50-fold shorter than those of the GA homodimer (GG). The novel properties of GA channels in formamidinium solution (supralinear current-voltage relations and dimer stabilization (Seoh and Busath, 1993)) also appear in NN channels. The average single channel lifetime in 1 M formamidinium solution at 100 mV is 6-7-fold longer than in K+ and methylammonium solutions and, like in the GA channel, significantly decreases with increasing membrane potential. Experiments with mixtures of the two peptides, GA and NAG, showed three main conductance peaks. Oriented hybrids were formed utilizing the principle that monomers remain in one leaflet of the bilayer (O'Connell et al., 1990). With GA at the polarized side and NAG at the grounded side, at positive potentials (in which case hybrids were designated GN) and at negative potentials (in which case hybrids were designated NG), channels had the same conductances and channel properties at all potentials studied. Flicker blocks were not evident in the hybrid channels, which suggests that both N-acetyl methyl groups at the junction of the dimer are required to cause flickers. Channel lifetimes in hybrids are only approximately threefold shorter than those of the GG channels, and channel conductances are similar to those of GG rather than NN channels. We suggest that acetyl-acetyl crowding at the dimeric junction in NN channels cause dimer destabilization, flickers, and increased selectivity in N-acetyl gramicidin channels.

Topics: Amidines; Biophysical Phenomena; Biophysics; Electric Conductivity; Gramicidin; Hydrogen Bonding; Ion Channels; Lipid Bilayers; Membrane Potentials; Methylamines; Models, Molecular; Potassium; Protein Conformation; Quaternary Ammonium Compounds

The conductance properties of organic cations in single gramicidin A channels were studied using planar lipid bilayers. From measurements at 10 mM and at 27 mV the overall selectivity sequence was found to be NH4+ > K+ > hydrazinium > formamidinium > Na+ > methylammonium, which corresponds to Eisenman polyatomic cation sequence X'. Methylammonium and formamidinium exhibit self block, suggesting multiple occupancy and single filing. Formamidinium has an apparent dissociation constant (which is similar to those of alkali metal cations) for the first ion being 22 mM from the Eadie-Hofstee plot (G0 vs. G0/C), 12 mM from the rate constants of a three-step kinetic model. The rate-limiting step for formamidinium is translocation judging from supralinear I-V relations at low concentrations. 1 M formamidinium solutions yields exceptionally long single channel lifetimes, 20-fold longer than methylammonium, which yields lifetimes similar to those found with alkali metal cations. The average lifetime in formamidinium solution significantly decreases with increasing voltage up to 100 mV but is relatively voltage independent between 100 and 200 mV. At lower voltages (< or = 100 mV), the temperature and concentration dependences of the average lifetime of formamidinium were steep. At very low salt concentrations (0.01 M, 100 mV), there was no significant difference in average lifetime from that formed with 0.01 M methylammonium or hydrazinium. We conclude that formamidinium very effectively stabilizes the dimeric channel while inside the channel and speculate that it does so by affecting tryptophan-reorientation or tryptophan-lipid interactions at binding sites.

Topics: Amidines; Biophysical Phenomena; Biophysics; Cations; Electric Conductivity; Gramicidin; In Vitro Techniques; Ion Channels; Ion Transport; Kinetics; Lipid Bilayers; Membrane Potentials; Methylamines; Models, Biological; Permeability