Mechanism of Interaction between the General Anesthetic Halothane and a Model Ion Channel Protein, III: Molecular Dynamics Simulation Incorporating a Cyanophenylalanine Spectroscopic Probe  Hongling Zou, Jing Liu, J. Kent Blasie  Biophysical Journal  Volume 96, Issue 10, Pages 4188-4199 (May 2009) DOI: 10.1016/j.bpj.2009.01.054 Copyright © 2009 Biophysical Society Terms and Conditions

Figure 1 Illustration of the chemical structure of nonbiological amino acid PheCN and the definition of the nitrile bond axis. The helix to which the PheCN residue is attached is shown in ribbon representation. Biophysical Journal 2009 96, 4188-4199DOI: (10.1016/j.bpj.2009.01.054) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 2 Ramachandran plot for residues 2–36 in all the helices of the apo protein hbAP1 (cross) and its mutant hbAP1-PheCN (circle) at the water-octane interface. The backbone conformation angles phi and psi are calculated as averages over the last 1 ns of the simulation. The dashed perimeter corresponds to the most favorable combinations of phi/psi values for a right-handed α-helical conformation, and the solid perimeter highlights the somewhat larger “Ramachandran-allowed” region. Biophysical Journal 2009 96, 4188-4199DOI: (10.1016/j.bpj.2009.01.054) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 3 Time evolution of the halothane's translational (a) and rotational (b) motion within the hbAP1-PheCN bundle. The halothane's translational displacement Z is defined as the distance from the center of the halothane molecule (Z) to the center of the four Ala19 residues (O) projected along the peptide bundle long axis. The halothane's rotational angle θ is defined as the angle between the C-C axis of the halothane molecule and the peptide bundle long axis. Biophysical Journal 2009 96, 4188-4199DOI: (10.1016/j.bpj.2009.01.054) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 4 Instantaneous configuration of the hbAP1-PheCN with the halothane binding to the cavity viewed from the top (a) and from the side (b). The peptide is shown in ribbon representation and the four PheCN residues in wire-frame representation. Biophysical Journal 2009 96, 4188-4199DOI: (10.1016/j.bpj.2009.01.054) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 5 Two possibilities were considered for hydrogen-bond formation. (a) hydrogen-bond formation between the halothane trifluorine atoms and the backbone hydrogen atoms. (b) Hydrogen-bond formation between the acidic hydrogen of the halothane and the nitrile nitrogen of the PheCN residue. Biophysical Journal 2009 96, 4188-4199DOI: (10.1016/j.bpj.2009.01.054) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 6 Correlations between the halothane's rotational motion (a) and the distance of approach of its relevant hydrogen-bond acceptor atoms to the cavity's backbone hydrogen atoms (b–d), which is shown in Fig. 5a. (a) Halothane's rotational angle θ is defined as in Fig. 3. (b–d) The distance between the halothane trifluorines (three fluorines are shown in three different colors) and the adjacent Ala (Ala139 in b and c, Ala99 in d) backbone amide-H (HN) or Cα-H (Hα). In a, the numbers 1–5 denotes the different phases of the halothane's orientation. Each phase is then matched well to the interaction between the halothane and the cavity backbone hydrogen atoms shown in the right panels of b–d. In the right panels, hydrogen bonds between the trifluorines and the HN or the Hα are represented in white dashed lines. Particularly, in right panel c, the same HN also forms a hydrogen bond with the backbone carbonyl oxygen, which is responsible for stabilizing the protein secondary structure. The peptide is shown in ribbon representation with backbone atoms in wire-frame representation. Nitrogen is colored in blue, oxygen in red, and carbon in silver. The halothane molecule is shown in CPK representation with trifluorines colored in cyan, bromine in yellow, chlorine in green, and hydrogen in white. Biophysical Journal 2009 96, 4188-4199DOI: (10.1016/j.bpj.2009.01.054) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 7 For two parallel simulations (simulation I, a and b; simulation II, c and d), correlations between the halothane's rotational motion (a and c) and the distance of approach of its relevant hydrogen-bond donor to the PheCN residues' nitrile-nitrogen (b and d), as presented in Fig. 5b, were studied with partial charges on nitrile groups included (left four panels) or removed (right four panels). (a and c) The halothane's orientational angle θ is defined as in Fig. 3. (b and d) The distance between acidic hydrogen of the halothane and the nitrile-nitrogen of the four PheCN residues are plotted, with each of them in a different gray scale. Biophysical Journal 2009 96, 4188-4199DOI: (10.1016/j.bpj.2009.01.054) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 8 Distribution of the aromatic ring's motion for two PheCN residues in the absence (gray bar) and presence of the halothane (black bar). χ is defined as the dihedral angle for Cα-Cβ-Cγ-Cδ, characterizing the “flipping” motion of the aromatic ring; θ is defined as the angle between Cβ-Cγ and the bundle long axis, characterizing the “tilting” motion of the aromatic ring. The residues in consideration are two PheCN residues that are closer to the halothane. The addition of halothane seems to have no apparent effect on the widths of the distributions for either of these motions of aromatic rings for either of the two PheCN residues, indicating the absence of an interaction between halothane and aromatic rings of PheCN probes. Biophysical Journal 2009 96, 4188-4199DOI: (10.1016/j.bpj.2009.01.054) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 9 Halothane's impact on the backbone structure of the hbAP1-PheCN peptide bundle. Shown here is a comparison of the computed RMSD of backbone Cα atoms in the absence (dashed line) and presence (solid line) of the halothane molecule, respectively. The equation for calculating RMSD is as follows: RMSD=[1M∑i=1M(Ri−Rieq)2]1/2, where Ri and Rieq are the coordinates of the ith backbone Cα of an instantaneous structure and the equilibrated structure, respectively. Biophysical Journal 2009 96, 4188-4199DOI: (10.1016/j.bpj.2009.01.054) Copyright © 2009 Biophysical Society Terms and Conditions

Figure 10 Comparison of the backbone dynamics of the hbAP1-PheCN peptide bundle in the absence (dashed line) and presence (solid line) of halothane. Shown here is the computed RMSF of backbone Cα atoms for each residue. The four helices in the bundle are named H1, H2, H3, and H4. H1 and H2 are within one dimer; H3 and H4 belong to the other dimer. The equation for calculating RMSF is as follows: RMSF=[1T∑tj=1T(Ri(tj)−Ri¯)2]1/2, where Ri(tj) and Ri¯ are the coordinates of the ith backbone Cα of an instantaneous structure at time tj and the coordinates averaged over time T, respectively. Note that the difference between RMSD and RMSF is that with the later the average is taken over time T, giving a value for each residue i; with RMSD the average is taken over the M residues, giving time specific values. Biophysical Journal 2009 96, 4188-4199DOI: (10.1016/j.bpj.2009.01.054) Copyright © 2009 Biophysical Society Terms and Conditions