Quantum Mechanical Study of Oxygen Ligands Protonation for the Stable States of the Laccase Active Site.

Laccases are enzymes catalyzing the oxidation of a wide range of organic and inorganic substrates accompanied by molecular oxygen reduction to water. Recently, oxygen reduction by laccases has been studied by single-crystal serial X-ray crystallography with increasing absorption doses at subatomic resolution. There were two determined structures corresponding to the reduced and oxidized stable states of the laccase active site. However, the protonation of the oxygen ligands involved cannot be determined even at subatomic resolution. In the present work, the protonation of oxygen ligands in the active site of laccase for the two stable states determined in the X-ray study was explored using quantum mechanical and continuum-electrostatics calculations. This is important for understanding the reaction of the oxygen reduction mechanism in laccases. The high precision of X-ray data at subatomic resolutions allowed us to optimize the quantum mechanical calculations.

Interaction of Synthetic Cannabinoid Receptor Agonists with Cannabinoid Receptor I: Insights into Activation Molecular Mechanism.

Synthetic cannabinoid receptor agonists (SCRAs) have become a wide group of new psychoactive substances since the 2010s. For the last few years, the X-ray structures of the complexes of cannabinoid receptor I (CB1) with SCRAs as well as the complexes of CB1 with its antagonist have been published. Based on those data, SCRA-CB1 interactions are analyzed in detail, using molecular modeling and molecular dynamics simulations. The molecular mechanism of the conformational transformation of the transmembrane domain of CB1 caused by its interaction with SCRA is studied. These conformational changes allosterically modulate the CB1-Gi complex, providing activation of the Gi protein. Based on the X-ray-determined structures of the CB1-ligand complexes, a stable apo conformation of inactive CB1 with a relatively low potential barrier of receptor activation was modeled. For that model, molecular dynamic simulations of SCRA binding to CB1 led to the active state of CB1, which allowed us to explore the key features of this activation and the molecular mechanism of the receptor's structural transformation. The simulated CB1 activation is in accordance with the previously published experimental data for the activation at protein mutations or structural changes of ligands. The key feature of the suggested activation mechanism is the determination of the stiff core of the CB1 transmembrane domain and the statement that the entire conformational transformation of the receptor to the active state is caused by a shift of alpha helix TM7 relative to this core. The shift itself is caused by protein-ligand interactions. It was verified via steered molecular dynamics simulations of the X-ray-determined structures of the inactive receptor, which resulted in the active conformation of CB1 irrespective of the placement of agonist ligand in the receptor's active site.

Poisson-Boltzmann model of electrolytes containing uniformly charged spherical nanoparticles.

Like-charged macromolecules typically repel each other in aqueous solutions that contain small mobile ions. The interaction tends to turn attractive if mobile ions with spatially extended charge distributions are added. Such systems can be modeled within the mean-field Poisson-Boltzmann formalism by explicitly accounting for charge-charge correlations within the spatially extended ions. We consider an aqueous solution that contains a mixture of spherical nanoparticles with uniform surface charge density and small mobile salt ions, sandwiched between two like-charged planar surfaces. We perform the minimization of an appropriate free energy functional, which leads to a non-linear integral-differential equation for the electrostatic potential that we solve numerically and compare with predictions from Monte Carlo simulations. Nanoparticles with uniform surface charge density are contrasted with nanoparticles that have all their charges relocated at the center. Our mean-field model predicts that only the former (especially when large and highly charged particles) but not the latter are able to mediate attractive interactions between like-charged planar surfaces. We also demonstrate that at high salt concentration attractive interactions between like-charged planar surfaces turn into repulsion.

Mediating role of multivalent cations in DNA electrostatics: an epsilon-modified Poisson-Boltzmann study of B-DNA-B-DNA interactions in mixture of NaCl and MgCl2 solutions.

Potentials of mean force acting between two ions in SPC/E water have been determined via molecular dynamics simulations using the spherical cavity approach ( J. Phys. Chem. B 2006 , 110 , 10878 ). The potentials were obtained for Me(2+)-Me(+) pairs, where Me(2+) means cations Mg(2+) and Ca(2+) and Me(+) denotes monovalent ions Li(+), Na(+), and K(+). The hard-core interaction distance for effective Me(2+)-Me(+) potentials appears to be of about 5 A that looks like a sum of the effective radii of a Me(2+) ion (3 A) and of an alkali metal ion Me(+) (about 2 A). These ion-ion interaction parameters were used in the epsilon-Modified Poisson-Boltzmann (epsilon-MPB) calculations ( J. Phys. Chem. B 2007 , 111 , 5264 ) of ionic distributions around DNA generalized for the arbitrary mixture of different ion species. Ionic distributions around an all-atom geometry model of B-DNA in solution of a mixture of NaCl and MgCl(2) were obtained. It was found that even a small fraction of ions Mg(2+) led to sharp condensation of Mg(2+) near the phosphate groups of DNA due to polarization deficiency of cluster [Mg(H(2)O)(6)](2+) in an external field. The epsilon-MPB calculations of the B-DNA-B-DNA interaction energies suggest that adding 1 mM of Mg(2+) to 50 mM solution of NaCl notably affects the force acting between the two macromolecules. Being compared to Poisson-Boltzmann results and to MPB calculations for the primitive model of ions, the epsilon-MPB results also indicate an important contribution of dielectric saturation effects to the mediating role of divalent cations in the DNA-DNA interaction energies.

The subatomic resolution study of laccase inhibition by chloride and fluoride anions using single-crystal serial crystallography: insights into the enzymatic reaction mechanism.

Laccases are enzymes that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of molecular oxygen to water. Here, a subatomic resolution X-ray crystallographic study of the mechanism of inhibition of the laccase from the basidiomycete fungus Steccherinum murashkinskyi by chloride and fluoride ions is presented. Three series of X-ray diffraction data sets were collected with increasing doses of absorbed X-ray radiation from a native S. murashkinskyi laccase crystal and from crystals of complexes of the laccase with chloride and fluoride ions. The data for the native laccase crystal confirmed the previously deduced enzymatic mechanism of molecular oxygen reduction. The structures of the complexes allowed the localization of chloride and fluoride ions in the channel near the T2 copper ion. These ions replace the oxygen ligand of the T2 copper ion in this channel and can play the role of this ligand in the enzymatic reaction. As follows from analysis of the structures from the increasing dose series, the inhibition of laccases by chloride and fluoride anions can be explained by the fact that the binding of these negatively charged ions at the position of the oxygen ligand of the T2 copper ion impedes the reduction of the T2 copper ion.

Electrostatics of B-DNA in NaCl and CaCl2 solutions: ion size, interionic correlation, and solvent dielectric saturation effects.

The epsilon-modified Poisson-Boltzmann (-MPB) equations ( J. Phys. Chem. B, 2007, 111, 5264) have been solved on a three-dimensional grid for an all-atom geometry model of B-DNA. The approach is based on the implicit solvent model including finite sizes of hydrated ions and a dielectric approximation of the ion hydration shell. Results were obtained for the detailed geometry model of B-DNA in dilute and moderately concentrated solutions of NaCl and CaCl(2). All -MPB parameters of ions and dielectric medium were extracted from published results of all-atom molecular dynamics simulations. The study allows evaluations of the ion size, interionic correlation, and the solvent dielectric saturation effects on the ion distributions around DNA. It unambiguously suggests that the difference between the -MPB and Poisson-Boltzmann distributions of ions is low for Na(+) counterions. Such a difference in the case of divalent counterions Ca(2+) is dramatic: the dielectric saturation of the ion hydration shell leads to point-like adsorption of Ca(2+) on the phosphate groups of DNA. The -MPB equations were also applied to calculate the energy of interaction between two B-DNA molecules. Results agree with previously published simulations and experimental data. Some aspects of ion specificity of polyelectrolyte properties are discussed.

Dielectric saturation of the ion hydration shell and interaction between two double helices of DNA in mono- and multivalent electrolyte solutions: foundations of the epsilon-modified Poisson-Boltzmann theory.

Potentials of mean force between single Na+, Ca2+, and Mg2+ cations and a highly charged spherical macroion in SPC/E water have been determined using molecular dynamics simulations. Results are compared to the electrostatic energy calculations for the primitive polarization model (PPM) of hydrated cations describing the ion hydration shell as a dielectric sphere of low permittivity (Gavryushov, S.; Linse, P. J. Phys. Chem. B 2003, 107, 7135). Parameters of the ion dielectric sphere and radius of the macroion/water dielectric boundary were extracted by means of this comparison to approximate the short-range repulsion of ions near the interface. To explore the counterion distributions around a simplified model of DNA, the obtained PPM parameters for Na+ and Ca2+ have been substituted into the modified Poisson-Boltzmann (MPB) equations derived for the PPM and named the epsilon-MPB (epsilon-MPB) theory. epsilon-MPB results for DNA suggest that such polarization effects are important in the case of 2:1 electrolyte and highly charged macromolecules. The three-dimensional implementation of the epsilon-MPB theory was also applied to calculation of the energies of interaction between two parallel macromolecules of DNA in solutions of NaCl and CaCl2. Being compared to results of MPB calculations without the ion polarization effects, it suggests that the ion hydration shell polarization and inhomogeneous solvent permittivity might be essential factors in the experimentally known hydration forces acting between charged macromolecules and bilayers at separations of less than 20 A between their surfaces.

Effective interaction potentials for alkali and alkaline earth metal ions in SPC/E water and polarization model of hydrated ions.

In the first paper (J. Phys. Chem. B, 2006, 110, 10878), effective ion-ion potentials in SPC/E water were obtained for Me-Me, Me-Cl-, and Cl(-)-Cl- pairs, where Me is Li+, Na+, K+, Mg2+, Ca2+, Sr2+, and Ba2+ cations. In this second part of the study of effective interionic potentials, ion-ion distribution functions obtained from implicit-water Monte Carlo simulations of electrolyte solution with these potentials have been explored. This analysis verifies the range of applicability of the primitive model of electrolyte. It is shown that this approximation can be applied to monovalent electrolyte solutions in a wide range of concentrations, whereas the nature of ion-ion interactions is notably different for 2:1 electrolytes. An improved model of ions is discussed. The model includes approximations of the ion hydration shell polarization and specific short-range ion-ion interaction. It allows approximation of the potential of mean force acting on ions in strong electric fields of highly charged macromolecules and bilayers.

Effective interaction potentials for alkali and alkaline earth metal ions in SPC/E water and prediction of mean ion activity coefficients.

The potential of mean force (PMF) acting between two simple ions surrounded by SPC/E water have been determined by molecular dynamics (MD) simulations using a spherical cavity approach. Such effective ion-ion potentials were obtained for Me-Me, Me-Cl-, and Cl(-)-Cl- pairs, where Me is a Li+, Na+, K+, Mg2+, Ca2+, Sr2+, and Ba2+ cation. The ionic sizes estimated from the effective potentials are not pairwise additive, a feature in the frequently used primitive model for electrolytes. The effective potentials were used in Monte Carlo (MC) simulations with implicit water to calculate mean ion activity coefficients of LiCl, NaCl, KCl, MgCl2, CaCl2, SrCl2, and BaCl2. Predicted activities were compared with experimental ones in the electrolyte concentration range 0.1-1 M. A qualitative agreement for LiCl and a satisfactory agreement for NaCl were found, whereas the predictions for KCl by two K+ models were less coherent. In the case of alkaline earth metal ions, all experimental activities were successfully reproduced at c = 0.1 M. However, at higher concentrations, similar deviations occurred for all divalent cations, suggesting that the dependence of the permittivity on the salt concentration and the polarization deficiency arising from the ordering of water molecules in the ion hydration shells are important in such systems.

Water sorption and glass transition of pig gastric mucin studied by QCM-D.

Hydration of films of pig gastric mucin was studied using a quartz crystal microbalance with dissipation monitoring (QCM-D) equipped with a humidity module. As a prerequisite, the water adsorption isotherm of a clean silica surface was determined. Atomic force microscopy was used to characterize the changes occurring on the silica surface after repeated sorption/desorption and cleaning cycles. The water sorption isotherms of several hundreds of nanometers thick mucin films were obtained in QCM-D experiments using analysis of overtone behavior. The results show that the sorption isotherms are not dependent on the film thicknesses and are in good agreement with sorption calorimetric data on mucin in the bulk phase. Moreover, hydration-induced changes of rheological properties of mucin films were investigated using a model-free approach. The ratio of G'/G″ was evaluated as a function of relative humidity. The transition from solidlike behavior to liquidlike behavior was observed in the same humidity range as in sorption calorimetric experiments. Thus, ability of QCM-D to monitor glass transition in biopolymers was demonstrated.

Substrate specificity of thymidine phosphorylase of E. coli: role of hydroxyl groups.

Substrate specificity of E. coli thymidine phosphorylase to pyrimidine nucleoside modified at 5'-, 3'-, and 2'-positions of sugar moiety has been studied. Equilibrium (K(eq)) and kinetics constants of phosphorolysis reaction of nucleosides were measured. The most important hydrogen bonds in enzyme-substrate complex have been determined.