The present article discusses some aspects concerning the inclusion of proton isomerism in simulations of the global protonation equilibrium of protein molecules. In the context of continuum electrostatic methods, the usual basis for these simulations, this isomerism can be treated as a coexistence of tautomeric forms in equilibrium in a rigid structure; furthermore, it can be formally extended to nontitrable sites with proton isomerism, such as alcohol groups and water molecules. We follow the previously adopted approach of transforming the real system of tautomeric sites into a thermodynamically equivalent one of nontautomeric pseudosites, establishing a proper relation between the two systems. The necessary energetic and entropic modifications of model compound pK a values are also discussed. Additionally, we discuss the new entropy term, named tautomeric entropy, that results from the explicit inclusion of tautomerism in the simulations and how it can be computed together with the occupational entropy. Simulations using tautomerism were done for hen egg white lysozyme (HEWL) using a simple set of tautomers at dihedral energy minima. A very good overall prediction of pK a values was obtained, presumably the best in the literature for HEWL, using a high value for the dielectric constant assigned to the protein region, p . The explicit inclusion of water molecules treated under the extended tautomer formalism further improved the prediction, in contrast with previous works using rigid water molecules. In all calculations performed, the region with p ≈ 20 is shown the to be the optimal one. Some aspects of the somewhat controversial issue of the "proper" p value are also discussed.

The phase behavior of PPO (sn-1,2-dipalmitoyl-sn-3-oleoylglycerol) and POP (sn-1,3-dipalmitoyl-sn-2-oleoylglycerol) binary mixture was examined using differential scanning calorimetry, conventional and synchrotron radiation X-ray diffraction (XRD), and highly pure samples. A molecular compound, βC, was formed at the 1:1 concentration ratio of PPO and POP, giving rise to two monotectic phases of PPO/compound and compound/POP in a juxtapositional way. βC has a long spacing value of 4.1 nm of double chain length structure. In the PPO/compound region, the DSC melting peak increased with increasing PPO concentration, whereas the DSC melting peak increased with increasing POP concentration in the compound/POP region. The melting point of βC was lowest at 31.2 °C. Time-resolved XRD study unveiled the formation of molecular compounds in metastable forms, αC and β‘C, having the same PPO/POP concentration ratio as βC. αC and β‘C exhibited monotectic phases with corresponding metastable forms of the pure components. In αC, two hexagonal packing XRD short spacing peaks were obtained for αC, corresponding to a differently packed hexagonal subcell of two leaflets. A structure model of the PPO/POP molecular compound is proposed, involving separation of palmitoyl chain leaflet and palmitoyl−oleoyl mixed-acid chain leaflet in the double chain length structure. This model justifies the structure of βC of SOS/SSO proposed by Engstrom (Engstrom, L. J. Fat Sci. Technol. 1992 94, 173−181), in which the saturated and unsaturated chains are packed in the same leaflet of the double chain length. The present work additionally proved that the formation of the molecular compound was present in the αC and β‘C metastable forms, as directly proved by dynamic X-ray diffraction study using synchrotron radiation.