What Is the Protonation\nState of Proteins in Crystals?\nInsights from Constant pH Molecular Dynamics Simulations

X-ray crystallography is an important technique to determine\nthe\npositions of atoms in a protein crystal. However, because the native\nenvironment in which proteins function, is not a crystal, but a solution,\nit is not a priori clear if the crystal structure represents the functional\nform of the protein. Because the protein structure and function often\ndepend critically on the pH, the question arises whether proton affinities\nare affected by crystallization. X-ray diffraction usually does not\nreveal protons, which makes it difficult to experimentally measure\np<i>K</i><sub>a</sub> shifts in crystals. Here, we investigate\nwhether this challenge can be addressed by performing in silico titration\nwith constant pH molecular dynamics (MD) simulations. We compare the\ncomputed p<i>K</i><sub>a</sub> values of proteins between\nsolution and crystal environment and analyze these differences in\nthe context of molecular interactions. For the proteins considered\nin this work, p<i>K</i><sub>a</sub> shifts were mostly found\nfor residues at the crystal interfaces, where the environment is more\napolar in the crystal than in water. Although convergence was an obstacle,\nour simulations suggest that in principle it is possible to apply\nconstant pH MD to protein crystals routinely and assess the effect\nof crystallization on protein function more systematically than with\nstandard MD simulations. We also highlight technical challenges that\nneed to be addressed to make MD simulations of crystals more reliable.