This article reviews different formulations of the thermodynamic cycles used for the prediction of pK a values, their advantages, and disadvantages with special emphasis on the limitations resulting from the necessity of gas-phase calculations, which allow introducing some difficult cases that motivated alternative strategies. Before introducing the protocols that do not consider gas-phase calculations, the two current opinions available in the literature on the debate about the correct formalism for the calculation of free energies in solution are briefly introduced. Then, the isodesmic proton exchange reaction in solution is reviewed by analyzing its performance on difficult cases for thermodynamic cycles such as carbon acids and amino acids. The pK a values predicted by the isodesmic reaction for common acid species are also reviewed to compare their accuracy results in relation with those of thermodynamic cycles. Linear regressions between experimental pK a values and the calculated free energies obtained with the isodesmic reaction provide expressions for the dependence of the error in the calculated pK a s on the pK a difference between the studied acid and the reference species. Finally, it is shown that linear regressions correct the calculated free energies of the isodesmic reaction, when high constant precision is required in a large pK a range. The deviations from the expected behavior are equivalent to those reported previously for different pK a calculation protocols and are determined by the inaccuracies of continuum solvent models on the interactions with ionic species.

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Intrinsically disordered proteins (IDPs) are not well described by a single 3D conformation but by an ensemble of them, which makes their structural characterization especially challenging, both experimentally and computationally. Most all-atom force fields are designed for folded proteins and give too compact IDP conformations. α-Synuclein is a well-known IDP because of its relation to Parkinson’s disease (PD). To understand its role in this disease at the molecular level, an efficient methodology is needed for the generation of conformational ensembles that are consistent with its known properties (in particular, with its dimensions) and that is readily extensible to post-translationally modified forms of the protein, commonly found in PD patients. Herein, we have contributed to this goal by performing explicit-solvent, microsecond-long Replica Exchange with Solute Scaling (REST2) simulations of α-synuclein with the coarse-grained force field SIRAH, finding that a 30% increase in the default strength of protein–water interactions yields a much better reproduction of its radius of gyration. Other known properties of α-synuclein, such as chemical shifts, secondary structure content, and long-range contacts, are also reproduced. Furthermore, we have simulated a glycated form of α-synuclein to suggest the extensibility of the method to its post-translationally modified forms. The computationally efficient REST2 methodology in combination with coarse-grained representations will facilitate the simulations of this relevant IDP and its modified forms, enabling a better understanding of their roles in disease and potentially leading to efficient therapies.