Proteins interact with ions in various ways. The surface of proteins has an innate capability to bind ions, and it is also influenced by the screening of the electrostatic potential owing to the presence of salts in the bulk solution. Alkali metal ions and chlorides interact with the protein surface, but such interactions are relatively weak and often transient. In this paper, computer simulations and analysis of protein structures are used to characterize the interactions between ions and the protein surface. The results show that the ion-binding properties of protein residues are highly variable. For example, alkali metal ions are more often associated with aspartate residues than with glutamates, whereas chlorides are most likely to be located near arginines. When comparing NaCl and KCl solutions, it was found that certain surface residues attract the anion more strongly in NaCl. This study demonstrates that protein–salt interactions should be accounted for in the planning and execution of experiments and simulations involving proteins, particularly if subtle structural details are sought after.

Methanol dehydrogenase (MDH) is an enzyme used by certain bacteria for the oxidation of methanol to formaldehyde, which is a necessary metabolic reaction. The discovery of a lanthanide-dependent MDH reveals that lanthanide ions (Ln(3+)) have a role in biology. Two types of MDH exist in methane-utilizing bacteria: one that is Ca2+-dependent (MxaF) and another that is Ln(3+)-dependent. Given that the triply charged Ln(3+) are strongly hydrated, it is not clear how preference for Ln(3+) is manifested and if the Ca2+-dependent MxaF protein can also bind Ln(3+) ions. A computational approach was used to estimate the Gibbs energy differences between the binding of Ln(3+) and Ca2+ to MDH using density functional theory. The results show that both proteins bind La3+ with higher affinity than Ca2+, albeit with a more pronounced difference in the case of Ln(3+)-dependent MDH. Interestingly, the binding of heavier lanthanides is preferred over the binding of La3+, with Gd3+ showing the highest affinity for both proteins of all Ln(3+) ions that were tested (La3+, Sm3+, Gd3+, Dy3+, and Lu3+). Energy decomposition analysis reveals that the higher affinity of La3+ than Ca2+ to MDH is due to stronger contributions of electrostatics and polarization, which overcome the high cost of desolvating the ion.

The capacity to transfer protons between surface groups is an innate property of many proteins. The transfer of a proton between donor and acceptor, located as far as 6−7 Å apart, necessitates the participation of water molecules in the process. In a previous study we investigated the mechanism of proton transfer (PT) between bulk exposed sites, a few ångströms apart, using as a model the proton exchange between the proton-binding sites of the fluorescein molecule in dilute aqueous solution.¹ The present study expands the understanding of PT reactions between adjacent sites exposed to water through the calculation the minimum energy pathways (MEPs) by the conjugate peak refinement algorithm² and a quantum-mechanical potential. The PT reaction trajectories were calculated for the fluorescein system with an increasing number of water molecules. The MEP calculations reveal that the transition state is highly strained and involves a supramolecular structure in which fluorescein and the interconnecting water molecules are covalently bonded together and the protons are shared between neighboring oxygens. These findings are in accord with the high activation energy, as measured for the reaction, and indicate that PT reactions on the surface proceed by a semi- or fully concerted rather than stepwise mechanism. A similar mechanism is assumed to be operative on the surface of proteins and renders water-mediated PT reactions as highly efficient as they are.

The role of the structural diversity of the widely used anticoagulant drug warfarin on its distribution in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayer membranes was investigated using a series of both restrained (umbrella sampling) and unrestrained molecular dynamics simulations. Data collected from unrestrained simulations revealed favorable positions for neutral isomers of warfarin, the open side chain form (OCO), and the cyclic hemiketal (CCO), along the bilayer normal close to the polar headgroup region and even in the relatively distant nonpolar lipid tails. The deprotonated open side chain form (DCO) was found to have lower affinity for the DOPC bilayer membrane relative to the neutral forms, with only a small fraction interacting with the membrane, typically within the polar headgroup region. The conformation of OCO inside the lipid bilayer was found to be stabilized by intramolecular hydrogen bonding thereby mimicking the structure of CCO. Differences in free energies, for positions of OCO and CCO inside the bilayer membrane, as compared to positions in the aqueous phase, were −97 and −146 kJ·mol^–1. Kinetic analysis based on the computed free energy barriers reveal that warfarin will diffuse through the membranes within hours, in agreement with experimental results on warfarin’s accumulation in the plasma, thus suggesting a passive diffusion mechanism. We propose that this membrane transport may be an isomerization-driven process where warfarin adapts to the various local molecular environments encountered under its journey through the membrane. Collectively, these results improve our understanding of the influence of warfarin’s structural diversity on the drug’s distribution and bioavailability, which in turn may provide insights for developing new formulations of this important pharmaceutical to better address its narrow therapeutic window.

A series of steady-state fluorescence anisotropy experiments has been performed to demonstrate the presence of a deprotonated open side chain form of warfarin in organic environments. We explain the observed emission-wavelength-dependent anisotropy of warfarin in ethanol, 2-propanol, and acetonitrile due to the coexistence of neutral isomers and deprotonated open side chain forms displaying different fluorescence decay kinetics. To investigate solvent-solute interactions in more detail, a series of molecular dynamics simulations was performed to study warfarin solvation and to predict the time scale of rotational diffusion displayed by this compound. Predictions obtained provide an explanation for the nonzero values in anisotropy observed for neutral isomers of warfarin associated with the short fluorescence lifetime (tau < 0.1 ns) and for an approximately zero anisotropy observed for the deprotonated open side chain form, which is associated with the longer fluorescence lifetime (tau = 0.5-1.6 ns). Finally, we address the potential use of fluorescence anisotropy for an increased understanding of the structural diversity of warfarin in protein binding pockets.

Archaea such as Sulfolobus acidocaldarius tolerate extreme temperatures and high acidity and can grow in the presence of toxic metals and low concentrations of Na⁺ or K⁺. It is believed that their unique tetraether membranes protect them from harsh environments and allow their survival under such conditions. We used molecular dynamics simulations to study membranes comprising glycerol dialkylnonitol tetraether lipids, which are the main component of S. acidocaldariusmembranes, in solutions containing different concentrations of NaCl and KCl or with Na⁺ or K⁺counterions (trace cations, 0 M). Anionic binding sites on the membranes were almost 50% occupied in the presence of counterions. The free energy of cation–phosphate complexation and the residence times of ions near the membranes were found to be both ion- and concentration-dependent. Sodium ions had more favorable interactions with the membranes and a longer residence time, whereas higher cation concentrations led to shorter ion residence times. When only counterions were present in the solutions, large residence times suggested that the membrane may function as a cation-attracting reservoir. The results suggested that the ions can be easily transferred to the cytoplasm as needed, explaining the growth curves of S. acidocaldarius under different salinities and pH.

The aberrant expression of kinases is often associated with pathologies such as cancer and autoimmune diseases. Like other types of enzymes, kinases can adopt active and inactive states, where a shift toward more stable active state often leads to disease. Dozens of kinase inhibitors are, therefore, used as drugs. Most of these bind to either the inactive or active state. In this work, we study the transitions between these two states in FLT₃, an important drug target in leukemias. Kinases are composed of two lobes (N- and C-terminal lobes) with the catalytic site in-between. Through projection of the largest motions obtained through molecular dynamics (MD) simulations, we show that each of the end-states (active or inactive) already possess the ability for transition as the two lobes rotate which initiates the transition. A targeted simulation approach known as essential dynamics sampling (EDS) was used to speed up the transition between the two protein states. Coupling the EDS to implicit-solvent MD was performed to estimate the free energy barriers of the transitions. The activation energies were found in good agreement with previous estimates obtained for other kinases. Finally, we identified FLT₃ intermediates that assumed configurations that resemble that of the c-Src nonreceptor tyrosine kinase. The intermediates show better binding to the drug ponatinib than c-Src and the inactive state of FLT3. This suggests that targeting intermediate states can be used to explain the drug-binding patterns of kinases and for rational drug design.