AMOEBA Force Field Trajectories Improve Predictions of Accurate pKa Values of the GFP Fluorophore: The Importance of Polarizability and Water Interactions.

Precisely quantifying the magnitude, direction, and biological functions of electric fields in proteins has long been an outstanding challenge in the field. The most widely implemented experimental method to measure such electric fields at a particular residue in a protein has been through changes in pKa of titratable residues. While many computational strategies exist to predict these values, it has been difficult to do this accurately or connect predicted results to key structural or mechanistic features of the molecule. Here, we used experimentally determined pKa values of the fluorophore in superfolder green fluorescent protein (GFP) with amino acid mutations made at position Thr 203 to evaluate the pKa prediction ability of molecular dynamics (MD) simulations using a polarizable force field, AMOEBA. Structure ensembles from AMOEBA were used to calculate pKa values of the GFP fluorophore. The calculated pKa values were then compared to trajectories using a conventional fixed charge force field (Amber03 ff). We found that the position of water molecules included in the pKa calculation had opposite effects on the pKa values between the trajectories from AMOEBA and Amber03 force fields. In AMOEBA trajectories, the inclusion of water molecules within 35 Å of the fluorophore decreased the difference between the predicted and experimental values, resulting in calculated pKa values that were within an average of 0.8 pKa unit from the experimental results. On the other hand, in Amber03 trajectories, including water molecules that were more than 5 Å from the fluorophore increased the differences between the calculated and experimental pKa values. The inaccuracy of pKa predictions determined from Amber03 trajectories was caused by a significant stabilization of the deprotonated chromophore's free energy compared to the result in AMOEBA. We rationalize the cutoffs for explicit water molecules when calculating pKa to better predict the electrostatic environment surrounding the fluorophore buried in GFP. We discuss how the results from this work will assist the prospective prediction of pKa values or other electrostatic effects in a wide variety of folded proteins.