gramicidin-a and dodecylphosphocholine

M2delta, one of the transmembrane segments of the nicotinic acetylcholine receptor, is a 23-amino-acid peptide, frequently used as a model for peptide-membrane interactions. In this and the companion article we describe studies of M2delta-membrane interactions, using two different computational approaches. In the present work, we used continuum-solvent model calculations to investigate key thermodynamic aspects of its interactions with lipid bilayers. M2delta was represented in atomic detail and the bilayer was represented as a hydrophobic slab embedded in a structureless aqueous phase. Our calculations show that the transmembrane orientation is the most favorable orientation of the peptide in the bilayer, in good agreement with both experimental and computational data. Moreover, our calculations produced the free energy of association of M2delta with the lipid bilayer, which, to our knowledge, has not been reported to date. The calculations included 10 structures of M2delta, determined by nuclear magnetic resonance in dodecylphosphocholine micelles. All the structures were found to be stable inside the lipid bilayer, although their water-to-membrane transfer free energies differed by as much as 12 kT. Although most of the structures were roughly linear, a single structure had a kink in its central region. Interestingly, this structure was found to be the most stable inside the lipid bilayer, in agreement with molecular dynamics simulations of the peptide and with the recently determined structure of the intact receptor. Our analysis showed that the kink reduced the polarity of the peptide in its central region by allowing the electrostatic masking of the Gln13 side chain in that area. Our calculations also showed a tendency for the membrane to deform in response to peptide insertion, as has been previously found for the membrane-active peptides alamethicin and gramicidin. The results are compared to Monte Carlo simulations of the peptide-membrane system, as presented in the accompanying article.

Topics: Alamethicin; Amino Acid Sequence; Binding Sites; Cell Membrane; Gramicidin; Lipid Bilayers; Micelles; Models, Molecular; Molecular Sequence Data; Monte Carlo Method; Peptides; Phosphorylcholine; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Receptors, Cholinergic; Software; Solvents; Thermodynamics

Based on the malonyl gramicidin A structure of a single-stranded head-to-head hydrogen bonded right-handed, beta 6.3-helix in dodecyl phosphocholine (DPC) lipid micelles (Jing et al. (1994) Biophys. J. 66, A353), the determination of cation binding sites for gramicidin A (GA) in DPC micelles becomes a significant step in the study of ion transport through the model channel. First, the investigation of cation binding sites in DPC micellar packaged gramicidin A was achieved by 13C-NMR experiments at 30 degrees C using four C-13 labeled GA samples. Then, the analyses based on two different equations, one for single and one for double occupancy, were employed to evaluate the correct occupancy model for GA in DPC micelles. The results clearly indicate double occupancy to be correct for Na+ ion as well as for K+, Rb+, Cs+, and Tl+ ions. Finally, the binding constants for Na+ ion were also estimated by the measurement of the longitudinal relaxation time (T1) using 23Na-NMR of the same sample at the same ffmperature as used for the 13C-NMR study. The binding constants obtained from 23Na-NMR are essentially equivalent to those determined from the 13C-chemical shifts.

Topics: Amino Acid Sequence; Binding Sites; Cesium; Gramicidin; Magnetic Resonance Spectroscopy; Micelles; Molecular Sequence Data; Phosphorylcholine; Potassium; Protein Conformation

The two independent NMR experiments were performed to investigate the interaction between CaCl2 and the gramicidin A (GA) ion transport channel, using 13C-enriched GA and GA molecules incorporated into dodecylphosphocholine (DPC) micelles. The chemical shifts of C-13 labeled carbonyl carbons vs. CaCl2 concentration demonstrate that Ca2+ and Cl- ions interact as an ion pair within the GA structure with the Cl- ion located near the position of the carbonyl group of the Trp11 residue some 5.5 A from the mouth of the GA helix, and the Ca2+ ion bound at the position of the carbonyl group of the Trp15 residue some 2.5 A from the entrance to the helical pore. The measurements of the 35Cl line-widths and transverse relaxation times illustrate that the interaction occurs between Cl- ions and GA in DPC when in CaCl2 solution, that no interaction is detected between Cl- ions and GA in DPC when in NaCl solution, and that the interaction between Cl- ions and GA in DPC when in MgCl2 solution is much weaker than in CaCl2 solution. In short, a Cl- ion can enter the GA when it is paired with a divalent Ca2+ ion; and Ca2+ and Cl- ions as a pair exchange rapidly with sites of the GA dimer.

Topics: Amino Acid Sequence; Calcium; Chlorides; Circular Dichroism; Gramicidin; Magnetic Resonance Spectroscopy; Micelles; Molecular Sequence Data; Phosphorylcholine