Description. It was recently demonstrated [26,27] that the one-dimensional free energy associated with the principal curve represents a significant advancement over the minimum-free-energy-pathway in the conventional string method. The one-dimensional free energy can be calculated from confined or restrained simulations. A recent approach involves sampling in the Voronoi tessellation with reflective boundaries [26,27]. Here we present an alternative approach. By invoking a local linear approximation, we employ traditional one-dimensional umbrella sampling to calculate the free energy along the principal curve. Our method is conceptually simple, computationally efficient, and relatively convenient to implement. We thus use this technique to map a free energy profile in the conformational space visited by the unrestrained simulations, thereby supplementing the free conformational dynamics of AdK with the thermodynamic energetics.Methods Unrestrained SimulationsWe constructed two simulation systems with AdK initially in the open and Bexagliflozin supplier closed conformations, respectively. The atomic coordinates of the protein were taken from monomer A in the crystal structures (PDB ID: 4AKE [7] and 1AKE [6] with the bound nucleotide analog removed) for the open and closed states, respectively. We adopted the standard protonation states at pH 7 for all residues. In particular, all His residues are neutral, with the proton at the E position. For both systems, the protein was solvated by adding 8,900 1315463 water molecules in a cubic box. 21 K+ and 17 Cl2 were also added to each system, mimicking a KCl concentration of ,0.1 M. In both cases the simulation system (Fig. 1) contains a total of 30,079 atoms. For each system, we first fixed the entire protein and equilibrated the water and ions for 1 ns. Next, we relaxed the protein and applied harmonic restraints on the Ca atoms only, and further equilibrated the system for 2 ns. We then selected seven and eight snapshots from the open- and closed-state simulation trajectories above, respectively. Starting from the selected snapshots in the open-state trajectory, we performed seven simulations (O1 7), in which the entire system is subject to no restraint and is free to evolve. Similarly, we initiated eight unrestrained simulations (C1 8) from the closed state. The fifteen unrestrained simulations were each run for 100 ns, with four of them extended to 200 ns, as will be described in Results. All simulations were performed using the CHARMM (Ver. c36) protein force field [28?0], the TIP3P water model [31], and the NAMD2 (Ver. 2.9) program [32], with a time step of 2 fs. All bond lengths Title Loaded From File involving hydrogen atoms were constrained using the SHAKE [33] and SETTLE [34] algorithms. We adopted a cutoff ?distance of 12 A for nonbonded interactions, with a smooth ?switching function taking effect at 10 A. Full electrostatics was calculated every 4 fs using the particle-mesh Ewald method [35]. Temperature was maintained at 300 K by Langevin dynamics with a damping coefficient of 0.1 ps21. A constant pressure ofAdenylate Kinase Conformation!Avg in the group. These average coordinates X k (k = 0,…,99) thus delineate a curve that lies at the center of the conformational space visited by the protein. To obtain a smooth pathway through the average coordinates above, we applied multidimensional curve fitting [24,37]. The fitted curve is of the form 3N P PP !Avg !Avg ! !Avg tz wij sin pt?^i , e with X X 0 z X M {Xi 1 jM = 99 and ^i the.Description. It was recently demonstrated [26,27] that the one-dimensional free energy associated with the principal curve represents a significant advancement over the minimum-free-energy-pathway in the conventional string method. The one-dimensional free energy can be calculated from confined or restrained simulations. A recent approach involves sampling in the Voronoi tessellation with reflective boundaries [26,27]. Here we present an alternative approach. By invoking a local linear approximation, we employ traditional one-dimensional umbrella sampling to calculate the free energy along the principal curve. Our method is conceptually simple, computationally efficient, and relatively convenient to implement. We thus use this technique to map a free energy profile in the conformational space visited by the unrestrained simulations, thereby supplementing the free conformational dynamics of AdK with the thermodynamic energetics.Methods Unrestrained SimulationsWe constructed two simulation systems with AdK initially in the open and closed conformations, respectively. The atomic coordinates of the protein were taken from monomer A in the crystal structures (PDB ID: 4AKE [7] and 1AKE [6] with the bound nucleotide analog removed) for the open and closed states, respectively. We adopted the standard protonation states at pH 7 for all residues. In particular, all His residues are neutral, with the proton at the E position. For both systems, the protein was solvated by adding 8,900 1315463 water molecules in a cubic box. 21 K+ and 17 Cl2 were also added to each system, mimicking a KCl concentration of ,0.1 M. In both cases the simulation system (Fig. 1) contains a total of 30,079 atoms. For each system, we first fixed the entire protein and equilibrated the water and ions for 1 ns. Next, we relaxed the protein and applied harmonic restraints on the Ca atoms only, and further equilibrated the system for 2 ns. We then selected seven and eight snapshots from the open- and closed-state simulation trajectories above, respectively. Starting from the selected snapshots in the open-state trajectory, we performed seven simulations (O1 7), in which the entire system is subject to no restraint and is free to evolve. Similarly, we initiated eight unrestrained simulations (C1 8) from the closed state. The fifteen unrestrained simulations were each run for 100 ns, with four of them extended to 200 ns, as will be described in Results. All simulations were performed using the CHARMM (Ver. c36) protein force field [28?0], the TIP3P water model [31], and the NAMD2 (Ver. 2.9) program [32], with a time step of 2 fs. All bond lengths involving hydrogen atoms were constrained using the SHAKE [33] and SETTLE [34] algorithms. We adopted a cutoff ?distance of 12 A for nonbonded interactions, with a smooth ?switching function taking effect at 10 A. Full electrostatics was calculated every 4 fs using the particle-mesh Ewald method [35]. Temperature was maintained at 300 K by Langevin dynamics with a damping coefficient of 0.1 ps21. A constant pressure ofAdenylate Kinase Conformation!Avg in the group. These average coordinates X k (k = 0,…,99) thus delineate a curve that lies at the center of the conformational space visited by the protein. To obtain a smooth pathway through the average coordinates above, we applied multidimensional curve fitting [24,37]. The fitted curve is of the form 3N P PP !Avg !Avg ! !Avg tz wij sin pt?^i , e with X X 0 z X M {Xi 1 jM = 99 and ^i the.