Evaluating Molecular Mechanical Potentials for Helical Peptides and Proteins

Multiple variants of the AMBER all-atom force field were quantitatively evaluated with respect to their ability to accurately characterize helix-coil equilibria in explicit solvent simulations. Using a global distributed computing network, absolute conformational convergence was achieved for large ensembles of the capped A21 and Fs helical peptides. Further assessment of these AMBER variants was conducted via simulations of a flexible 164-residue five-helix-bundle protein, apolipophorin-III, on the 100 ns timescale. Of the contemporary potentials that had not been assessed previously, the AMBER-99SB force field showed significant helix-destabilizing tendencies, with beta bridge formation occurring in helical peptides, and unfolding of apolipophorin-III occurring on the tens of nanoseconds timescale. The AMBER-03 force field, while showing adequate helical propensities for both peptides and stabilizing apolipophorin-III, (i) predicts an unexpected decrease in helicity with ALA→ARG+ substitution, (ii) lacks experimentally observed 310 helical content, and (iii) deviates strongly from average apolipophorin-III NMR structural properties. As is observed for AMBER-99SB, AMBER-03 significantly overweighs the contribution of extended and polyproline backbone configurations to the conformational equilibrium. In contrast, the AMBER-99φ force field, which was previously shown to best reproduce experimental measurements of the helix-coil transition in model helical peptides, adequately stabilizes apolipophorin-III and yields both an average gyration radius and polar solvent exposed surface area that are in excellent agreement with the NMR ensemble

Chronic ethanol attenuates circadian photic phase resetting and alters nocturnal activity patterns in the hamster

Acute ethanol (EtOH) administration impairs circadian clock phase resetting, suggesting a mode for the disruptive effect of alcohol abuse on human circadian rhythms. Here, we extend this research by characterizing the chronobiological effects of chronic alcohol consumption. First, daily profiles of EtOH were measured in the suprachiasmatic nucleus (SCN) and subcutaneously using microdialysis in hamsters drinking EtOH. In both cases, EtOH peaked near lights-off and declined throughout the dark-phase to low day-time levels. Drinking bouts preceded EtOH peaks by ∼20 min. Second, hamsters chronically drinking EtOH received a light pulse during the late dark phase [Zeitgeber time (ZT) 18.5] to induce photic phase advances. Water controls had shifts of 1.2 ± 0.2 h, whereas those drinking 10% and 20% EtOH had much reduced shifts (0.5 ± 0.1 and 0.3 ± 0.1 h, respectively; P < 0.001 vs. controls). Third, incremental decreases in light intensity (270 lux to 0.5 lux) were used to explore chronic EtOH effects on photic entrainment and rhythm stability. Activity onset was unaffected by 20% EtOH at all light intensities. Conversely, the 24-h pattern of activity bouts was disrupted by EtOH under all light intensities. Finally, replacement of chronic EtOH with water was used to examine withdrawal effects. Water controls had photic phase advances of 1.1 ± 0.3 h, while hamsters deprived of EtOH for 2–3 days showed enhanced shifts (2.1 ± 0.3 h; P < 0.05 vs. controls). Thus, in chronically drinking hamsters, brain EtOH levels are sufficient to inhibit photic phase resetting and disrupt circadian activity. Chronic EtOH did not impair photic entrainment; however, its replacement with water potentiated photic phase resetting

Structural sampling of apolipophorin-III per residue.

<p>Probabilities of sampling helix (H), turn (T), random coil (C), and polyproline type II (P) states when simulated using the AMBER-94, AMBER-99φ, AMBER-03, and AMBER-99SB force fields are shown. The schematic at the top represents the NMR model that was used to initiate all simulations, with turns shown in green and coil regions shown in pink to match the color coded state sampling plots below, which show probability ranges from 0.0 (black) to 1.0 (color).</p

Free energy landscapes projected onto the Ramachandran map.

<p>These maps represent equilibrium sampling of the F_s peptide in the AMBER force fields evaluated, which have been ordered to match <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010056#pone-0010056-g001" target="_blank">Figure 1</a> in (a) through (d). Each map consists of backbone torsional values binned in 3° intervals for all residues, and contours represent <i>k</i>T units at 305 K, the midpoint temperature of the helical peptide.</p

Ensemble averaged equilibrium structural properties for the F_s and A₂₁ peptides.

<p>RMSD (all-atom root-mean-square deviation), R_g (radius of gyration), N_helix (number of α-helical residues), N₃₁₀ (number of 3₁₀-helical residues), N_seg (number of helical segments), and N_cont (length of helical segments).</p

Convergence of mean helical content for the (a) F_s and (b) A₂₁ ensembles.

<p>The AMBER-03 (red), AMBER-99SB (green), and AMBER-99φ (blue) potentials are shown, where helix> represents the number of helical residues averaged across all runs in a given ensemble of 1,000 simulations. Dotted and solid lines represent simulation ensembles initiated from the fully random coil and fully helical states, respectively. Other structural properties listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010056#pone-0010056-t002" target="_blank">Table 2</a> show similar convergence. Noise near the 100 ns regime is the result of a limited number of simulations reaching those times following the ensemble convergence that occurs prior to the 40 ns timepoint.</p

Ribbon views of the NMR model of apolipophorin-III.

<p>NMR model 1 of this 164-residue, five-helix-bundle protein (PDB 1LS4) was used to start our simulations in the noted AMBER force fields. Bright green and black unstructured regions represent turn and random coil regions, respectively. Helices are colored from blue (helix 1) to green (helix 5). The bottom view is rotated toward the reader to provide an axial view down the helical bundle central core region.</p

Simulated ensemble statistics for the F_s and A₂₁ peptides.

<p>*Each force field was sampled using 1,000 trajectories starting in the fully helical state (H) and 1,000 trajectories starting in the random coil state (C) with no structured residues.</p><p>Max (longest individual trajectory), Total time (total ensemble simulation time), and >EQ (total equilibrium simulation time) are shown for each data set.</p