Chapter 11 pK a Calculations in Membrane Proteins from Molecular Dynamics Simulations Nuno F. B. Oliveira, Toma ´ s F. D. Silva, Pedro B. P. S. Reis, and Miguel Machuqueiro Abstract The conformational changes of membrane proteins are crucial to their function and usually lead to fluctuations in the electrostatic environment of the protein surface. A very effective way to quantify these changes is by calculating the pK a values of the protein’s titratable residues, which can be regarded as electrostatic probes. To achieve this, we need to take advantage of the fast and reliable pK a calculators developed for globular proteins and adapt them to include the explicit effects of membranes. Here, we provide a detailed linear response approximation protocol that uses our own software (PypKa) to calculate reliable pK a values from short MD simulations of membrane proteins. Key words Protonation, Conformation, Lipid bilayer, Poisson–Boltzmann, Monte Carlo, Linear response approximation 1 Introduction Membrane proteins (MPs) are key players in many biological pro- cesses and have been estimated to be 20 to 30% of all proteins encoded in the human genome [1]. These proteins can insert partially or present a full insertion across the lipid bilayer. It is remarkable how MPs live in such an anisotropic environment, which changes from very high dielectric water to polar head group region and very low dielectric region in the lipid tails, all over a few angstroms distance. These are the two-extreme media in terms of polarity, and MPs were evolved to take advantage of this scenario. The complexity of the embedding media provides ample possibilities for electrostatic, hydrogen bond, and Van der Waals- type interactions. It is no surprise that physicochemical properties like pH or ionic strength can also influence the MPs conformational Irina S. Moreira et al. (eds.), Computational Design of Membrane Proteins, Methods in Molecular Biology, vol. 2315, https://doi.org/10.1007/978-1-0716-1468-6_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021 185