Methods for estimation of pK_a values of residues in proteins were tested on a set of benchmark proteins with experimentally known pK_a values. The benchmark set includes 80 different residues (20 each for Asp, Glu, Lys, and His), half of which consists of significantly variant cases (ΔpK_a ≥ 1 pK_a unit from the amino acid in solution). The method introduced by Case and co-workers [J. Am. Chem. Soc. 2004, 126, 4167-4180], referred to as the molecular dynamics/generalized-Born/thermodynamic integration (MD/GB/TI) technique, gives a root-mean-square deviation (rmsd) of 1.4 pK_a units on the benchmark set. The use of explicit waters in the immediate region surrounding the residue was shown to generally reduce high errors for this method. Longer simulation time was also shown to increase the accuracy of this method. The empirical approach developed by Jensen and co-workers [Proteins 2005, 61, 704-721], PROPKA, also gives an overall rmsd of 1.4 pK_a units and is more or less accurate based on residue type—the method does very well for Lys and Glu, but less so for Asp and His. Likewise, the absolute deviation is quite similar for the two methods—5.2 for PROPKA and 5.1 for MD/ GB/TI. A comparison of these results with several prediction methods from the literature is presented. The error in pK_a prediction is analyzed as a function of variation of the pK_a from that in water and the solvent accessible surface area (SASA) of the residue. A case study of the catalytic lysine residue in 2-deoxyribose-5-phosphate aldolase (DERA) is also presented.
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