Internal
water molecules in proteins are conceivably part of the
protein structure, not exchanging easily with the bulk. We present
a detailed molecular dynamics study of the water molecule bound to
the green fluorescent protein (GFP) chromophore that conducts its
proton following photoexcitation. It readily exchanges above 310 K
through a hole that forms between strands 7 and 10, due to fluctuations
in the 6–7 loop. As the hole widens, rapid succession of water
exchange events occur. The exiting water molecule passes three layers
of atoms, constituting the binding, internal, and surface sites. Along
this pathway, hydrogen bonding protein residues are replaced with
water molecules. The mean squared displacement along this pathway
is initially subdiffusive, becomes superdiffusive as the water traverses
the protein wall in a flip-flop motion, and reverts to normal diffusion
in the bulk. The residence correlation function for the bound state
decays biexponentially, supporting this three-site scenario. For a
favorable orientation of the Thr203 side-chain, the hole often fills
with a single file of water molecules that could indeed rapidly conduct
the photodissociated proton outside the protein. The activation enthalpy
for its formation, 26 kJ/mol, agrees with the experimental value for
a protein conformation change suggested to gate proton escape
