All-atom, explicit solvent MD simulations of the selected targets were conducted. All simulations were conducted in keeping with our core MD protocols and simulation methods that are published elsewhere [1, 2]. The simulations used all-atom (i.e. hydrogens were included) fully flexible representations of protein and solvent in the Levitt et al. force field with standard parameters [3]. Non-bonded interactions between atoms in charge groups separated by three bonds were included and scaled by 0.4.

In the MD simulations, waters were represented explicitly by using the flexible three center (F3C) water model of Levit et al., as it has demonstrated success in reproducing a large number of experimental observables across a range of temperatures [4, 5]. Simulations were performed in the microcanonical ensemble where the number of atoms, unit cell volume, and total energy were conserved (NVE), as well as linear momentum (NVEp). A two femtosecond (fs) timestep was used such that each picosecond (ps) of simulation time required 500 steps. The unit cell was periodic with minimum image conventions employed [6]. Non-bonded interaction lists were used for no more than 6 fs (i.e., three steps).

The simulations were performed with in lucem Molecular Mechanics (ilmm, © Beck, D. A. C., Alonso, D. O. V., Daggett V, University of Washington, 2006). A variety of computational resources were employed for the simulations. Most of the simulations were conducted on Seaborg – the Department of Energy’s National Energy Research Supercomputing Council’s flagship 6,080 processor supercomputer.  Each supercomputer node (IBM Nighthawk) has 16 POWER3 processors operating at 375 Mhz.

For each target six or more simulations were performed. A simulation at 298 Kelvin (K) was conducted for at least 21 nanoseconds (ns). For the intact structures this is the functional biological native state, while for domains of larger proteins the simulation assesses the stability of the domain in isolation. Disulfide bonds were included in the native state simulation where appropriate. A 10 Ångstrom (Å) cutoff range was used for the 298 K simulations [2]. Structures were saved from the trajectory at one ps resolution for later analyses and full restart files with velocities and accelerations at the limits of machine precision where saved one per ns.

To elucidate the unfolding / folding pathway of the Dynameomics targets, at least five thermal unfolding simulations at 498 K were conducted. This minimum number was established by a previous study [7]. Two of the thermal unfolding runs were at least 21 ns in length with the remaining 3 (or more) at least 2 ns in length. Where the native state had disulfide bonds, these bonds where removed and the cysteines protonated. An 8 Å non-bonded cutoff range was used. For the first 2 ns of the majority of 498 K trajectories, structures were saved at 0.2 ps intervals to aid in the identification of the protein folding transition state. Subsequently, structures were saved at 1 ps intervals. As with the 298 K simulations, full velocities and accelerations were saved once per ns.

The starting structure for each simulation was derived from the corresponding crystal or NMR structure from the PDB. In the case of NMR structures, the first model was used. When multiple conformations for specific residues were present in the PDB structure file, the first conformation was chosen. Protons were then added. Then the coordinates of the structure were minimized with respect to the force field’s potential energy with steepest descents (SD) minimization for 1000 steps or until the potential energy converged. The minimized structure was solvated in a box of F3C water extending at least 10 Å from any protein atom such that no waters were added within 1.8 Å of any protein atom. The box size was adjusted slightly such that its density matched that of experiment at the desired temperature: 0.997 g/ml for 298 K [8] and 0.829 g/ ml for 498 K [9]. The water network was then smoothed by 1000 steps of SD minimization of water only, followed by 1 ps of dynamics of the water only where the temperature of the water was raised through approximately 40 K.  The water only was then SD minimized for 500 steps and, finally, the protein was SD minimized for 500 steps.

As a quality control measure, the resulting simulation systems were individually inspected. Problems such as minimization or preparation failures (e.g. failure of the total energy to decrease during minimization, typically a result of omission of a disulfide bond, etc.), and large structural changes in the protein were identified and corrected. To ensure that all systems were correct, each simulation’s starting configuration was examined by hand.

References

1. Beck, D.A.C. and V. Daggett, Methods for Molecular Dynamics Simulations of Protein Folding / Unfolding in Solution. Methods, 2004. 34(1): p. 112-120.

2. Beck, D.A.C., R. Armen, and V. Daggett, Cutoff size need not strongly influence molecular dynamics results on solvated polypeptides. Biochemistry, 2005. 44(2): p. 609-616.
3. Levitt, M., M. Hirshberg, R. Sharon, and V. Daggett, Potential-Energy Function and Parameters for Simulations of the Molecular-Dynamics of Proteins and Nucleic-Acids in Solution. Computer Physics Communications, 1995. 91(1-3): p. 215-231.
4. Levitt, M., M. Hirshberg, R. Sharon, K.E. Laidig, and V. Daggett, Calibration and testing of a water model for simulation of the molecular dynamics of proteins and nucleic acids in solution. Journal of Physical Chemistry B, 1997. 101(25): p. 5051-5061.
5. Beck, D.A.C., D.O. Alonso, and V. Daggett, A microscopic view of peptide and protein solvation. Biophys Chem, 2003. 100(1-3): p. 221-237.
6. Allen, M.P. and D.J. Tildesley, Computer Simulation of Liquids. 1987, Oxford: Oxford University Press.
7. Day, R. and V. Daggett, Ensemble versus single-molecule protein unfolding. Proceedings of the National Academy of Sciences of the United States of America, 2005. 102(38): p. 13445-13450.
8. Kell, G.S., J. Chem. Eng. Data, 1967. 12: p. 66.
9. Haar, L., J.S. Gallagher, and G.S. Kell, NBS/NRC Steam Tables. 1984, New York: Hemisphere.