Molecular Dynamics Free Energy Calculations to Assess the Possibility of Water Existence in Protein Nonpolar Cavities  Masataka Oikawa, Yoshiteru Yonetani  Biophysical Journal  Volume 98, Issue 12, Pages 2974-2983 (June 2010) DOI: 10.1016/j.bpj.2010.01.029 Copyright © 2010 Biophysical Society Terms and Conditions

Figure 1 Distribution of volumes of 859 nonpolar cavities found using the MSP (38) (see Materials and Methods). (Inset) Enlarged view for the range from 60 to 120 Å3. Biophysical Journal 2010 98, 2974-2983DOI: (10.1016/j.bpj.2010.01.029) Copyright © 2010 Biophysical Society Terms and Conditions

Figure 2 Cavity locations of the four selected proteins (left) and the shapes of nonpolar cavities (right). Cavity surfaces are shown by green mesh. Side chains of residues, which construct the cavities, are shown by gray sticks. Among them, atoms exposed to the cavity surface are shown by gray spheres. The figures were created by using VMD (62). Biophysical Journal 2010 98, 2974-2983DOI: (10.1016/j.bpj.2010.01.029) Copyright © 2010 Biophysical Society Terms and Conditions

Figure 3 Cavity hydration free energies from the rigid-model calculations. For comparison, previous data from a spherical model (taken from Fig. 1 in (35)) are shown (solid circles). Results for Trp repressor are not shown, because water molecules got out of the cavity during the MD simulation. For the same reason, data for hemoglobin (n = 3 and 4) are missing from the figure. Details of the free energies are given in Table S1 in the Supporting Material. Biophysical Journal 2010 98, 2974-2983DOI: (10.1016/j.bpj.2010.01.029) Copyright © 2010 Biophysical Society Terms and Conditions

Figure 4 Cavity hydration free energies: comparison between the rigid- and flexible-model results. Data for IL-1β (n = 3 and 4) could not be evaluated because the water molecules got out of the cavity during the MD simulations. Errors of the free energies were estimated from three independent simulations performed with different random seed numbers for the Langevin thermostat. The errors were <0.3 kcal/mol in any case of the rigid-model results. Details of the free energies are given in Table S2 in the Supporting Material. Biophysical Journal 2010 98, 2974-2983DOI: (10.1016/j.bpj.2010.01.029) Copyright © 2010 Biophysical Society Terms and Conditions

Figure 5 Energy differences between flexible and rigid models. ΔG is the cavity hydration free energy (Figs. 3 and 4) and ΔE is in the potential energy due to the water-cavity wall interactions. Each point has a label for the protein name and the number of internal water molecules n. Data for Trp repressor were not obtained (see the caption in Fig. 3). Biophysical Journal 2010 98, 2974-2983DOI: (10.1016/j.bpj.2010.01.029) Copyright © 2010 Biophysical Society Terms and Conditions

Figure 6 (a) Number of hydrogen bonds associated with the internal water molecules. Water-water and protein-water hydrogen bonds are shaded and solid, respectively. These were obtained by averaging over 1-ns trajectories. (b) Typical snapshots from the rigid (left) and flexible (right) simulations of AvrPphB (n = 2). Hydrogen bonds are represented by orange dashed lines. Protein atoms near the cavity surface are shown. Main-chain carbonyl oxygen atoms of Leu185 and Ile244 form a hydrogen bond to the water molecule in the cavity (right). The figures were created by using VMD (62). Biophysical Journal 2010 98, 2974-2983DOI: (10.1016/j.bpj.2010.01.029) Copyright © 2010 Biophysical Society Terms and Conditions

Figure 7 Distribution of the volume of 5948 protein cavities; (a) polar and (b) nonpolar cavities. Cavities with and without water molecules are solid and shaded, respectively. One-hundred-eighty-three polar cavities, which were found to be filled with molecules other than water, are not shown. In the insets, ordinates are enlarged. Biophysical Journal 2010 98, 2974-2983DOI: (10.1016/j.bpj.2010.01.029) Copyright © 2010 Biophysical Society Terms and Conditions