Assessment of water accessibility of the gating pore in Shaker channels.
(A) Left: 3-D structure of the Shaker VSD obtained after 100 ns of a MD simulation in which water was allowed to equilibrate inside the intracellular and extracellular vestibules. The protein backbone is represented in cyan/transparent, while the side chains of the gating pore residues are shown in licorice representation in green. Finally, water molecules are in van der Waals (VdW) representation in red. Right: Same as the structure shown to the left, but this time the water molecules from 60 consecutive frames of the simulation (one every 0.1 ns) are superimposed in order to define the WA region of the VSD. Note that only a very short region inside the gating pore is totally inaccessible to water. (B) Plot of the mean number of water molecules inside the gating pore region (defined as specified in C) as a function of the simulation time. (C) Plot of the mean number of water molecules in the gating pore. Error bars in B and C represent the SD. Water molecules were counted when residing inside a cylinder of 5 Å radius and 7 Å high, extending from −1 Å to 6 Å with respect to the z component of the center of mass of the F290 aromatic ring (see inset). (D) Plot of the distance between the centers of mass of the two closest intracellular and extracellular water molecules as a function of the simulation time. If the radius of a water molecule is 1.4 Å (gray regions), a WI region of 2.5 Å is estimated. (E) 3-D structure of the Shaker VSD obtained after 20-ns MD simulation in which water was allowed to equilibrate inside the intracellular and extracellular vestibules. The protein backbone is represented in cyan/transparent, while the side chains of the gating pore residues are shown in VdW representation in green for the three residues forming the region totally WI and orange for the remaining seven residues forming the remaining part of the gating pore, which is partially WA. Two water molecules inside the gating pore are also shown (red oxygens and white hydrogens).