Continuum electrostatic analysis of preferred solvation sites around proteins in solution

The energetic contributions of individual DNA-contacting side chains to specific DNA recognition in the human papillomavirus 16 E2C-DNA complex is small (less than 1.0 kcal mol(-1)), independent of the physical and chemical nature of the interaction, and is strictly additive. The sum of the individual contributions differs 1.0 kcal mol(-1) from the binding energy of the wild-type protein. This difference corresponds to the contribution from the deformability of the DNA, known as "indirect readout." Thus, we can dissect the energetic contribution to DNA binding into 90% direct and 10% indirect readout components. The lack of high energy interactions indicates the absence of "hot spots," such as those found in protein-protein interfaces. These results are compatible with a highly dynamic and "wet" protein-DNA interface, yet highly specific and tight, where individual interactions are constantly being formed and broken.

Thymidylate synthase from Mycobacterium tuberculosis (MtbThyX) is a key enzyme and a promising target for drug development. While specific inhibitors are not yet available, research in this area is crucial. We used alchemical free energy simulations to quantify the stabilising effect of two conserved buried water molecules in the polar active site of MtbThyX, ensuring its structural integrity. Removing one of these localised water molecules quickly opens a lid, allowing another water molecule to enter and seal the active site. The conserved water sites are accessed through two distinct channels, with an arginine residue or the cofactor FAD serving as lids. The properties of these conserved water molecules differ significantly from bulk water, and their removal raises the free energy of the MtbThyX complex by as much as 12 kcal mol-1, triggering rapid lid opening without additional energy input. This reversible mechanism resembles prey capture in pitcher plants. With about 8 µs of sampling from multiple runs, we achieved good convergence and a statistical uncertainty of around 0.5 kcal mol-1 for the estimated free energies.

Calcium-dependent lectin I from Pseudomonas aeruginosa (PA-IL) binds specifically to oligosaccharides presenting an alpha-galactose residue at their nonreducing end, such as the disaccharides alphaGal1-2betaGalOMe, alphaGal1-3betaGalOMe, and alphaGal1-4betaGalOMe. This provides a unique model for studying the effect of the glycosidic linkage of the ligands on structure and thermodynamics of the complexes by means of experimental and theoretical tools. The structural features of PA-IL in complex with the three disaccharides were established by docking and molecular dynamics simulations and compared with those observed in available crystal structures, including PA-IL.alphaGal1-2betaGalOMe complex, which was solved at 2.4 A resolution and reported herein. The role of a structural bridge water molecule in the binding site of PA-IL was also elucidated through molecular dynamics simulations and free energy calculations. This water molecule establishes three very stable hydrogen bonds with O6 of nonreducing galactose, oxygen from Pro-51 main chain, and nitrogen from Gln-53 main chain of the lectin binding site. Binding free energies for PA-IL in complex with the three disaccharides were investigated, and the results were compared with the experimental data determined by titration microcalorimetry. When the bridge water molecule was included in the free energy calculations, the simulations predicted the correct binding affinity trends with the 1-2-linked disaccharide presenting three times stronger affinity ligand than the other two. These results highlight the role of the water molecule in the binding site of PA-IL and indicate that it should be taken into account when designing glycoderivatives active against P. aeruginosa adhesion.

We estimate both single ion hydration Gibbs free energies in water droplets, comprising from 50 to 1000 molecules, and water/vacuum surface potentials in pure water droplets comprising up to 10 000 molecules. We consider four ions, namely, Li+, NH4 +, F-, and Cl-, and we model their hydration process and water/water interactions using polarizable force fields based on an induced point dipole approach. We show both ion hydration Gibbs free energies and water surface potentials to obey linear functions of the droplet radius as soon as droplets comprising a few hundred water molecules. Moreover, we also show that the differences in anion/cation hydration Gibbs free energies in droplets obey a different regime in large droplets than in small clusters comprising no more than six water molecules, in line with the earlier results computed from standard additive point charge force fields. Hence, both point charge and more sophisticated induced point dipole molecular modeling approaches suggest that methods considering only the thermodynamical properties of small ion/water clusters to estimate the absolute proton hydration Gibbs free energy in solution are questionable. In particular, taking into account the data of large ion/water droplets may yield a proton hydration Gibbs free energy in solution value to be shifted by several kBT units compared to small clusters-based approaches.

Sampling the shallow free energy surface of hydrated atmospheric molecular clusters is a significant challenge. Using computational methods, we present an efficient approach to obtain minimum free energy structures for large hydrated clusters of atmospheric relevance. We study clusters consisting of two to four sulfuric acid (sa) molecules and hydrate them with up to five water (w) molecules. The structures of the "dry" clusters are obtained using the ABCluster program to yield a large pool of low-lying conformer minima with respect to free energy. The conformers (up to ten) lowest in free energy are then hydrated using our recently developed systematic hydrate sampling technique. Using this approach, we identify a total of 1145 unique (sa)2-4(w)1-5 cluster structures. The cluster geometries and thermochemical parameters are calculated at the ωB97X-D/6-31++G(d,p) level of theory, at 298.15 K and 1 atm. The single-point energy of the most stable clusters is calculated using a high-level DLPNO-CCSD(T0)/aug-cc-pVTZ method. Using the thermochemical data, we calculate the equilibrium hydrate distribution of the clusters under atmospheric conditions and find that the larger (sa)3 and (sa)4 clusters are significantly more hydrated than the smaller (sa)2 cluster or the sulfuric acid (sa)1 molecule. These findings indicate that more than five water molecules might be required to fully saturate the sulfuric acid clusters with water under atmospheric conditions. The presented methodology gives modelers a tool to take the effect of water explicitly into account in atmospheric particle formation models based on quantum chemistry.

The mechanism of forming an oxime from nucleophilic addition of a hydroxylamine to a ketone is taught early on in most courses of organic chemistry. Here I subject the first step of this reaction to form a tetrahedral intermediate to quantum mechanical scrutiny. The first decision is to decide which atom of the hydroxylamine acts as the nucleophile. Reaction 1 shows the oxygen and reaction 2 the nitrogen. The text books will tell you that nitrogen nucleophiles are better than oxygen ones. This is because nitrogen is less electronegative than oxygen (it has a smaller nuclear charge) and so binds its single lone pair less tightly than oxygen does its two lone pairs. Sometimes the Klopman-Salem equation is invoked, which tells you that the reactivity is directly proportional to the overlap between the donor MO and the (empty) acceptor MO, and inversely proportional to the energy gap between these two orbitals. Nitrogen wins out because its lone pair is “larger” and hence overlaps better, and because its donor MO energy is higher than oxygen and hence the energy gap between it and the π* C=O acceptor is lower. Reality check: We need to construct a suitable transition state for both possibilities, and then compare their free energies. There is a choice of choosing a stepwise pathway (the one shown in all the text books) in which the bond from N or O to C is formed in an initial step, and then followed by a step often just labelled PT to transfer the proton using solvent molecules. These two steps can also be conflated into a single concerted mechanism involving a 6-membered ring transition state. Quantum mechanically, this latter option has the advantage of avoiding any great build up of charge separation at any stage in the mechanism, but has the disadvantage that the entropic loss at the transition state is greater (although “borrowing” a water molecule from a bulk solvent for this purpose is easier than doing so from an infinite distance away). Shown below is a ωB97XD/6-311G(d,p)/SCRF=water calculation of the transition state for N-attack. It has a dipole moment of 6.2D, which is really quite small, and far from that expected for the zwitterionic intermediate shown in the stepwise mechanism (that would be between 15-30D). Cyclic transition state for N-attack. Click for animation of transition mode. The intrinsic reaction coordinate shows a concerted reaction with quite a small barrier. It is small because the nitrogen is in fact a super-nucleophile, its nucleophilicity has been augmented over that of a simple amine by a so-called α-effect from the adjacent two pairs of lone pairs on the oxygen activating the nitrogen lone pair by lone-pair repulsions. The gradient norm along the coordinate also shows an almost synchronous reaction. The only blip occurs at around IRC +1.3, and this corresponds to the transfer of a proton from NH to a water molecule. An earlier proton transfer from water to the carbonyl oxygen was essentially synchronous with formation of the N-C bond. This synchronicity is what helps avoid any large build up of charge separation. For this reason, I cannot help but feel that the text books could absorb this lesson and show a cyclic concerted reaction mechanism as a probable alternative to two stepwise processes. Next, O-attack. The IRC for this isomeric mode shows a significantly higher barrier compared to N (the computed relative free energies show the O to be higher by 8.3 kcal/mol than the N) and smaller exothermicity. It reveals even greater synchrony of the two proton transfers with the O-C bond formation. So we have a reality check of the text-books on this point in the form of an energy difference, which is always useful. O-attack. Click for animation of reaction mode. Now that our proton transfers are involved in the mechanism, it is time to take a closer look at the geometry of these transfers. On this point, the text books tell us that the most favourable geometry for a proton transfer is having the proton co-linear with the two oxygens. Whilst this is largely true for the geometries shown above, the resulting 6-membered ring as a result adopts a triangular shape, which is not ideal for the bond angles. This could be solved by incorporating a second water molecule, to give us model 3 shown above. A second water molecule can be placed in two alternative positions. The first simply solvates the 6-ring transition state. The second actively participates via an enlarged 8-membered ring transition state. It turns out that the latter is lower by 4.5 kcal/mol in free energy, largely due to the far better bond angles and the almost exactly linear proton transfers now possible. O-Transition state with two water molecules, one merely hydrogen bonding to the 6-ring (magenta arrow). O-Transition state with two water molecules, both part of a cyclic transition state. So the following is our best model. It is 10.4 kcal/mol lower in free energy than the isomeric O-attack transition state. The timing of the bonds shows that N-C formation coincides with the first proton transfer to the carbonyl oxygen, followed by an O to O proton transfer and finally N to O. The dipole moment at the transition state is 5.9D, revealing little explicit charge separation. N-attack via an 8-ring transition state. Click to view animation of reaction mode. It is worth concluding this exploration by reiterating that the models above are not complete. A bulk solvent would allow (statistical) participation of more than just two solvent molecules, and the dynamics of such a (very complex) process has yet to be explored. But I hope what you see here is a bit closer to “reality” than many a text-book author has when they illustrate their books. doi:10042/a3uxl

The heterotrimeric factor e/aIF2 plays a central role in eukaryotic/archaeal initiation of translation. By delivering the initiator methionyl-tRNA to the ribosome, e/aIF2 ensures specificity of initiation codon selection. The three subunits of aIF2 from the hyperthermophilic archaeon Pyrococcus abyssi could be overproduced in Escherichia coli. The beta and gamma subunits each contain a tightly bound zinc. The large gamma subunit is shown to form the structural core for trimer assembly. The crystal structures of aIF2gamma, free or complexed to GDP-Mg(2+) or GDPNP-Mg(2+), were resolved at resolutions better than 2 A. aIF2gamma displays marked similarities to elongation factors. A distinctive feature of e/aIF2gamma is a subdomain containing a zinc-binding knuckle. Examination of the nucleotide-complexed aIF2gamma structures suggests mechanisms of action and tRNA binding properties similar to those of an elongation factor. Implications for the mechanism of translation initiation in both eukarya and archaea are discussed. In particular, positioning of the initiator tRNA in the ribosomal A site during the search for the initiation codon is envisaged.

Energetics of Storage and Diffusion of Water and Cyclo-Octasulfur for a Nonpolar Cavity of RHCC Tetrabrachion by Molecular Dynamics Simulations

Eukaryotic cytochromes c contain a buried water molecule (Wat166) next to the heme that is associated through a network of hydrogen bonds to three invariant residues: tyrosine 67, asparagine 52, and threonine 78. Single-site mutations to two of these residues (Y67F, N52I, N52A) and the double-site mutation (Y67F/N52I) were introduced into Saccharomyces cerevisiae iso-1-cytochrome c to disrupt the hydrogen bonding network associated with Wat166. The N52I and Y67F/N52I mutations lead to a loss of Wat166 while N52A and Y67F modifications lead to the addition of a new water molecule (Wat166) at an adjacent site (Berghuis, A. M., Guillemette, J. G., McLendon, G., Sherman, F., Smith, M., and Brayer, G. D. (1994) J. Mol. Biol. 236, 786-799; Berghuis, A. M., Guillemette, J. G., Smith, M., and Brayer, G. D. (1994) J. Mol. Biol. 235, 1326-1341; Rafferty, S. P., Guillemette, J. G., Berghuis, A. M., Smith, M., Brayer, G. D., and Mauk, A. G. (1996) Biochemistry, 35, 10784-10792). We used differential scanning calorimetry (DSC) to determine the change in heat capacity (DeltaCp) and the temperature dependent enthalpy (DeltaHvH) for the thermal denaturation of both the oxidized and reduced forms of the iso-1 cytochrome c variants. The relative stabilities were expressed as the difference in the free energy of denaturation (DeltaGD) between the wild type and mutant proteins in both redox states. The disruption of the hydrogen bonding network results in increased stability for all of the mutant proteins in both redox states with the exception of the reduced Y67F variant which has approximately the same stability as the reduced wild type protein. For the oxidized proteins, DeltaGD values of 1.3, 4.1, 1.5, and 5.8 kcal/mol were determined for N52A, N52I, Y67F, and Y67F/N52I, respectively. The oxidized proteins were 8.2-11.5 kcal/mol less stable than the reduced proteins due to a redox-dependent increase in the entropy of unfolding.

Localized water molecules in the binding pockets of proteins play an important role in noncovalent association of proteins and small drug compounds. At times, the dominant contribution to the binding free energy comes from the release of localized water molecules in the binding pockets of biomolecules. Therefore, to quantify the energetic importance of these water molecules for drug design purposes, we have used the double-decoupling approach to calculate the standard free energy of tying up a water molecule in the binding pockets of two protein complexes. The double-decoupling approach is based on the underlying principle of statistical thermodynamics. We have calculated the standard free energies of tying up the water molecule in the binding pockets of these complexes to be favorable. These water molecules stabilize the protein-drug complexes by interacting with the ligands and binding pockets. Our results offer ideas that could be used in optimizing protein-drug interactions, by designing ligands that are capable of targeting localized water molecules in protein binding sites. The resulting free energy of ligand binding could benefit from the potential free energy gain accompanying the release of these water molecules. Furthermore, we have examined the theoretical background of the double-decoupling method and its connection to the molecular dynamics thermodynamic integration techniques.

The thermodynamic stability of a clathrate hydrate has been investigated by examining the free energy of formation of clathrate hydrate II encaging propane. The total free energy has been divided into several contributions—the interaction between water and guest propane molecules, the entropic contribution arising from the combinations of cage occupancy, and also the free energy due to intermolecular vibrations. The present method avoids some of the fundamental assumptions in the van der Waals and Platteeuw theory. This enables us to assess separately the factors which have a bearing on the thermodynamic stability of the hydrate. Kinetic stability has also been investigated by calculating molecular dynamics trajectories having initially excited several characteristic vibrational modes. We show, for propane in large cages, that the potential energy surface of the guest molecule in a cage has a single minimum and molecular motions can be approximated accurately to a collection of harmonic oscillators. It is found that the intermolecular vibrational modes for water molecules shift toward the higher frequency regions in the presence of the guest molecules. This shift of the vibrational motions of water molecules gives rise to an increase in the chemical potential of water compared with that evaluated under the fixed lattice sites approximation where only vibrations of the guest molecules are taken into account. The empty clathrate hydrate structures melt into liquid water within a few picoseconds when several of the lowest frequency modes are excited. On the other hand, the guest molecules are found to prevent the clathrate structures from melting even 10 ps after the excitation of the same frequency modes.

Abstract: The newly synthesized fluorinated chalcone derivatives are observed to possess antioxidant potential. Two new fluorinated chalcone compounds were effectively synthesized using the Claisen- Schmidt condensation reaction and were recrystallized using the slow evaporation method. The single crystal structure of the compounds was determined and refined through the X-ray single crystal diffraction method. All compounds were subjected to computational structural characterization and Hirshfeld surface analysis. The compounds were then further characterized through the Ultravioletvisible (UV-Vis) spectroscopic study. The chalcone derivatives were further analysed with biological experimentation and simulation such as in vitro antioxidant (DPPH) assay, molecular docking and in silico ADMET study. The crystal packing revealed that the molecules in the compounds were linked together through the intermolecular C—H···O and C—H···π interactions. Hirshfeld surface analysis validated the presence of intermolecular interactions in crystal packing. The UV-Vis spectroscopic study revealed that the absorption wavelength of the compounds that range from 421.79 to 428.98 nm was within the visible region with the energy gap value of 2.58 to 2.62 eV. The DPPH assay disclosed weak antioxidant activity of both compounds (-31 to 20 %, 10000 μg/mL) compared to the standard ascorbic acid (94.5 %, 50 μg/mL). The binding energy of the docked complex inside the target protein, 2CAG was within the range of -7.3 to -7.5 kcal/mol. In the silico model, SwissADME predicted that the two compounds have overall good drug-like properties. Different substituents, more planar configuration and high intramolecular interactions in the crystal packing played their role in increasing the antioxidant activities, binding energy and drug likeliness of the synthesized compounds.

Molecular selection, ion exclusion, and water permeation are well known regulatory mechanisms in aquaporin. Water permeability was found to be diverse in different subgroups of plasma membrane intrinsic proteins (PIPs), even though the residues surrounding the water holes remained the same across the subgroups. Upon homology modeling and structural comparison, a conserved Ala/Ile(Val) residue difference was identified in helix 2 that affected the conformation of the NPA region and consequently influenced the water permeability. The residue difference was found to be conservative within the two subgroups of PIPs in rice as well as in other plants. Functional tests further confirmed the prediction via site-directed mutagenesis where replacement of Ala(103) or Ala(102) in respective OsPIP1;1 or OsPIP1;3 with Val yielded 7.0- and 2.2-fold increases in water transportation, and substitution of Ile(98) or Val(95) in respective OsPIP2;3 or OsPIP2;7 with Ala resulted in 73 or 52% reduction of water transportation. Based on structural analyses and molecular dynamics simulations, we proposed that the difference in water permeability was attributed to the orientation variations of helix 2 that modified water-water and water-protein interactions.

Surface active agents (usually referred to as surfactants) are amphipathic molecules consisting of a nonpolar hydrophobic portion, usually a straight or branched hydrocarbon or fluorocarbon chain containing 8–18 carbon atoms, which is attached to a polar or ionic portion (hydrophilic). The hydrophilic portion can, therefore, be nonionic, ionic or zwitterionic, accompanied by counter ions in the last two cases. The hydrocarbon chain interacts weakly with the water molecules in an aqueous environment, whereas the polar or ionic head group interacts strongly with water molecules via dipole or ion‐dipole interactions. It is this strong interaction with the water molecules that renders the surfactant soluble in water. However, the cooperative action of dispersion and hydrogen bonding between the water molecules tends to squeeze the hydrocarbon chain out of the water and hence these chains are referred to as hydrophobic. The balance between hydrophobic and hydrophilic part of the molecule gives these systems their special properties, eg, accumulation at various interfaces and association in solution (to form micelles). The driving force for surfactant adsorption is the lowering of the free energy of the phase boundary. The interfacial free energy per unit area is the amount of work required to expand the interface. This interfacial free energy, referred to as surface or interfacial tension, γ, is given in mJm −2 or mNm −1 . Adsorption of surfactant molecules at the interface lowers γ and the higher the surfactant adsorption (ie, the more dense the layer is) the larger the reduction in γ. The degree of surfactant adsorption at the interface depends on surfactant structure and the nature of the two phases that meet the interface. Surface active agents also aggregate in solution forming micelles. The driving force micelle formation (or micellization) is the reduction of contact between the hydrocarbon chain and water, thereby reducing the free energy of the system. In the micelle, the surfactant hydrophobic groups are directed towards the interior of the aggregate and the polar head groups are directed towards the solvent. These micelles are in dynamic equilibrium and the rate of exchange between a surfactant molecule and the micelle may vary by orders of magnitude, depending on the structure of the surfactant molecule. Surfactants find application in almost every chemical industry of which the following may be worth mentioning: detergents, paints, dyestuffs, cosmetics, pharmaceuticals, agrochemicals, fibers, plastics, etc. Moreover, surfactants play a major role in the oil industry, for example, in enhanced and tertiary oil recovery. They are also occasionally used for environmental protection, eg, in oil slick dispersants. Therefore, a fundamental understanding of the physical chemistry of surface active agents, their unusual properties and their phase behavior is essential for most industrial chemists. In addition, an understanding of the basic phenomena involved in the application of surfactants, such as in the preparation of emulsions and suspensions and their subsequent stabilization, in microemulsions, in wetting spreading and adhesion, etc, is of vital importance in arriving at the right composition and control of the system involved. This is particularly the case with many formulations in the chemical industry mentioned above. It should be stated that commercially produced surfactants are not pure chemicals, and within each chemical type there can be tremendous variation. This can be understood, since surfactants are prepared from various feedstocks, namely petrochemicals, natural vegetable oils and natural animal fats. It is important to realize that in every case the hydrophobic group exists as a mixture of chains of different lengths. Products that may be given the same generic name could vary a great deal in their properties and the formulation chemist should bear this in mind when choosing a surfactant from a particular manufacturer.

Thermodynamic stability of water molecules in the bacteriorhodopsin proton channel: a molecular dynamics free energy perturbation study