Introduction: We have previously used molecular modeling combined with experimental data to visualize a plausible model of an anesthetic binding site within a LGIC.1 We have also previously shown a computational mechanism by which these LGICs may gate and postulated how this motion may be affected by the presence of anesthetics.2 The initial difficulty with these calculations concerns the 26000 atoms present in the receptor and the computing capabilities required to perform vibrational analyses on such a large construct. Here we show the successful application of an elastic network calculation on our previously published model of a glycine alpha one receptor (GlyRa1), now suspended in a fully hydrated lipid bilayer. Despite the presence of over 100,000 atoms , these calculations continue to demonstrate a symmetric motion of the ion channel protein that is consistent with the gating motion demonstrated in previous in vacuo work by us and others. Methods: Coordinates of the GlyRa1 model were obtained from our previous work. A 100x100A lipid bilayer matrix was constructed from POPC and then hydrated on both surfaces with water molecules using the VMD 1.86 software package (NCSA, Urbana, Ill.). Discovery Studio 1.7 (Accelrys, San Diego, CA) molecular modeling software was used to insert our GlyRa1 model into the lipid bilayer such that the known interfacial residue GLY 221 was at the POPC-water interface. All waters within 3.8A of the protein were removed as were all lipid molecules within 2A of the protein. Hydrogens were added followed by energy minimization of the entire system to remove energetically unfavorable contacts. The system was subsequently further hydrated within the GROMACS software suite and subjected to further energy equilibration via molecular dynamics simulation with periodic boundary conditions. Subsequent normal mode analysis was performed using an all atom elastic network model developed by Lindahl which takes advantage of a sparse matrix implementation for computational efficiency. Results: Despite the large size of the system, the introduction of water and lipid did not grossly distort the overall gating motion of the glyRa1 noted in previous works. Normal mode analysis revealed that the GlyRa1 in a fully hydrated bilayer environment continues to demonstrate an iris-like gating motion as a low frequency, high amplitude natural harmonic vibration. Furthermore, the introduction of periodic boundary conditions allowed simultaneous harmonic vibrations of lipid in sync with the protein gating motion that are compatible with reasonable lipid bilayer perturbations. Conclusions: This is among the first description of a normal mode calculation describing large-scale protein dynamics and ion channel gating in the presence of a fully hydrated lipid bilayer complex. This analysis was only possible on such a large system due to the computational efficiencies of the elastic network approximation. This model will hopefully provide a more accurate means of introducing anesthetics and alcohols into protein and lipid bilayer systems and allow us to discern their effects on LGIC gating. 1Bertaccini EJ, Shapiro J, Brutlag DL, Trudell JR: J Chem Inf Model 2005; 45: 128-35; 2Bertaccini EJ, Trudell JR, Lindahl E:J Chem Inf Model 2007; 47: 1572-9.
Anesthesiology 2008. October 20, 2008