Extensive Assessment of Various Computational Methods for Aspartate's pKa Shift.

A series of computational methods for pKa shift prediction are extensively tested on a set of benchmark protein systems, aiming at identifying pitfalls and evaluating their performance on high variants. Including 19 ASP residues in 10 protein systems, the benchmark set consists of both residues with highly shifted pKa values as well as those varying little from the reference value, with an experimental RMS free energy differences of 2.49 kcal/mol with respect to blocked amino acid, namely the RMS pKa shift being 1.82 pKa units. The constant pH molecular dynamics (MD), alchemical methods, PROPKA3.1, and multiconformation continuum electrostatics give RMSDs of 1.52, 2.58, 1.37, and 3.52 pKa units, respectively, on the benchmark set. The empirical scoring method is the most accurate one with extremely low computational cost, and the pH-dependent model is also able to provide accurate results, while the accuracy of MD sampling incorporating alchemical free energy simulation is prohibited by convergence achievement and the performance of conformational search incorporating multiconformation continuum electrostatics is bad. Former research works did not define statistical uncertainty with care and yielded the questionable conclusion that alchemical methods perform well in most benchmarks. In this work the traditional alchemical methods are thoroughly tested for high variants. We also performed the first application of nonequilibrium alchemical methods to the pKa cases.