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Cytochrome c oxidase is a membrane protein of the respiratory chain that consumes protons and molecular oxygen to produce water and uses the resulting energy to pump protons across the membrane. Our molecular dynamics simulations with an excess proton located at different positions in one of the proton-conducting channels, the K-channel, show a clear dependence of the number of water molecules inside the channel on the proton position. A higher hydration level facilitates the formation of hydrogen-bonded chains along which proton transfer can occur. However, a sufficiently high hydration level for such proton transport is observed only when the excess proton is located above S365, i.e., the lower third of the channel. From the channel entrance up to this point, proton transport is via water molecules as proton carriers. These hydronium ions move with their surrounding water molecules, up to K362, filling and widening the channel. The conformation of K362 depends on its own protonation state and on the hydration level, suggesting its role to be proton transport from a hydronium ion at the height of K362 to the upper part of the channel via a conformational change. The protonation-dependent conformational dynamics of E101 at the bottom of the channel renders proton transfer via E101 unlikely. Instead, its role is rather that of an amplifier of H96's proton affinity, suggesting H96 as the initial proton acceptor. [ABSTRACT FROM AUTHOR]

Lipids are major constituents of food but are also highly relevant substructures of drugs and are increasingly applied for the development of lipid-based drug delivery systems. Lipids are prone to oxidative degradation, thus affecting the quality of food or medicines. Therefore, analytical methods or tools that enable the degree of lipid oxidation to be assessed are of utmost importance to guarantee food and drug safety. Herein, we report the design, synthesis and application of the first-in-class fluorogenic triacylglycerols that enable dynamic monitoring of lipid oxidation via straightforward fluorescence readout. Our fluorogenic triacylglycerols can be used in both aqueous and lipid-based environments. Furthermore, we showed that the sensitivity of our fluorescent tracers towards oxidation could be tuned by incorporating either saturated or unsaturated acyl chains in their triacylglycerol core structure. With this, we provide a first proof of principle for the applicability of fluorescently labelled triacylglycerols as tracers to monitor the dynamics of lipid oxidation, thus paving the way for novel discoveries in the area of lipid analytics. [ABSTRACT FROM AUTHOR]

The S1 state geometries of benzonitrile, p-cyanophenol, o-cyanophenol, chlorobenzene, and p-chlorophenol were determined by Franck-Condon simulations and a fit of the geometry to the vibronic intensities and effective rotational constants in the harmonic limit based on ab initio force constants. © 2004 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Computation of phosphodiester hydrolysis in different models with one or two metal ions, representing typical active site architectures of nucleases, reveals an associative mechanism to be favorable in all of the cases studied in this work. Direct attack of the nucleophilic water molecule with proton transfer to the phosphate group is facilitated by an extra positive charge as provided by a metal ion located at the attack site or a positively charged histidine residue, whereas no such contribution can be observed on leaving group departure. A major catalytic effect is found by proton transfer from the nucleophilic water molecule to a histidine–aspartate cluster. Attack of the thus generated hydroxide ion on the phosphate group is just sufficiently stabilized by the metal ions to allow subsequent P–O bond dissociation. [ABSTRACT FROM AUTHOR]

Cytochrome c Oxidase (CcO), a membrane protein of the respiratory chain, pumps protons against an electrochemical gradient by using the energy of oxygen reduction to water. The ("chemical") protons required for this reaction and those pumped are taken up via two distinct channels, named D-channel and K-channel, in a step-wise and highly regulated fashion. In the reductive phase of the catalytic cycle, both channels transport protons so that the pumped proton passes the D-channel before the "chemical" proton has crossed the K-channel. By performing molecular dynamics simulations of CcO in the O→E redox state (after the arrival of the first reducing electron) with various combinations of protonation states of the D- and K-channels, we analysed the effect of protonation on the two channels. In agreement with previous work, the amount of water observed in the D-channel was significantly higher when the terminal residue E286 was not (yet) protonated than when the proton arrived at this end of the D-channel and E286 was neutral. Since a sufficient number of water molecules in the channel is necessary for proton transport, this can be understood as E286 facilitating its own protonation. K-channel hydration shows an even higher dependence on the location of the excess proton in the K-channel. Also in agreement with previous work, the K-channel exhibits a very low hydration level that likely hinders proton transfer when the excess proton is located in the lower part of the K-channel, that is, on the N-side of S365. Once the proton has passed S365 (towards the reaction site, the bi-nuclear centre (BNC)), the amount of water in the K-channel provides hydrogen-bond connectivity that renders proton transfer up to Y288 at the BNC feasible. No significant direct effect of the protonation state of one channel on the hydration level, hydrogen-bond connectivity, or interactions between protein residues in the other channel could be observed, rendering proton conductivity in the two channels independent of each other. Regulation of the order of proton uptake and proton passage in the two channels such that the "chemical" proton leaves its channel last must, therefore, be achieved by other means of communication, such as the location of the reducing electron. [ABSTRACT FROM AUTHOR]

Distortions in the DNA sequence such as damages or mispairs are specifically recognized and processed by DNA repair enzymes. A particular challenge for the enzymatic specificity is the recognition of a wrongly-placed native nucleotide such as thymine in T:G mispairs. An important step of substrate binding which is observed in many repair proteins is the flipping of the target base out of the DNA helix into the enzyme's active site. In this work we investigate how much the intrinsic dynamics of mispaired DNA is changed compared to canonical DNA. Our molecular dynamics simulations of DNA with and without T:G mispairs show significant differences in the conformation of paired and mispaired DNA. The wobble pair T:G shows local distortions such as twist, shear and stretch which deviate from canonical B form values. Moreover, the T:G mispair is found to be kinetically less stable, exhibiting two states with respect to base opening: a closed state comparable to the canonical base pairs, and a more open state, indicating a proneness for base flip. In addition, we observe that the thymine base in a T:G mispair is significantly more probable to be flipped than thymine in a T:A pair or cytosine in a C:G pair. Such local deformations and in particular the existence of a second, more-open state can be speculated to help the target-site recognition by repair enzymes. [ABSTRACT FROM AUTHOR]

Understanding how the chemical environment modulatesthe predominantconformations and kinetics of flexible molecules is a core interestof biochemistry and a prerequisite for the rational design of syntheticcatalysts. This study combines molecular dynamics simulation and Markovstate models (MSMs) to a systematic computational strategy for investigatingthe effect of the chemical environment of a molecule on its conformationsand kinetics. MSMs allow quantities to be computed that are otherwisedifficult to access, such as the metastable sets, their free energies,and the relaxation time scales related to the rare transitions betweenmetastable states. Additionally, MSMs are useful to identify observablesthat may act as sensors for the conformational or binding state ofthe molecule, thus guiding the design of experiments. In the presentstudy, the conformation dynamics of UDP-GlcNAc are studied in vacuum,water, water + Mg2+, and in the protein UDP-GlcNAc 2-epimerase.It is found that addition of Mg2+significantly affectsthe conformational stability, thermodynamics, and kinetics of UDP-GlcNAc.In particular, the slowest structural process, puckering of the GlcNAcsugar, depends on the overall conformation of UDP-GlcNAc and may thusact as a sensor of whether Mg2+is bound or not. Interestingly,transferring the molecule from vacuum to water makes the protein-bindingconformations UDP-GlcNAc first accessible, while adding Mg2+further stabilizes them by specifically associating to binding-competentconformations. While Mg2+is not cocrystallized in theUDP-GlcNAc 2-epimerase complex, the selectively stabilized Mg2+/UDP-GlcNAc complex may be a template for the bound state,and Mg2+may accompany the binding-competent ligand conformationto the binding pocket. This serves as a possible explanation of theenhanced epimerization rate in the presence of Mg2+. Thisrole of Mg2+has previously not been described and opensthe question whether “binding co-factors” may be a conceptof general relevance for protein–ligand binding. [ABSTRACT FROM AUTHOR]

Endonucleases, such as the restriction enzyme EcoRV, cleave the DNA backbone at a specific recognition sequence. We have investigated the catalytic mechanism of backbone phosphodiester hydrolysis by the restriction enzyme EcoRV by means of hybrid quantum mechanical/molecular mechanical calculations. An exhaustive computation of different reaction pathways is performed, thus generating a network of pathways. Comparison of the computed (AM1d/MM) enzymatic reaction pathways with an analogous mechanism for small-molecule model systems [AM1/d and B3LYP/6-31 + + G(d,p)] reveals that the transition barriers for associative hydrolysis, which is more probable in the model systems, are not lowered by the enzyme. Instead, a reaction mechanism which has mostly dissociative character is more likely. The protein environment is tuned to significantly electrostatically stabilize the transition state structures. The direct catalytic impact of essential residues is determined: The magnesium metal ion activates a water molecule, thus facilitating protonation of the leaving group. A reduction of the coordination number of the magnesium metal ion from six to four upon the positioning of the attacking water molecule explains why larger metal ions, such as calcium, are not catalytically active. The nucleophile is generated by the transfer of a proton from the attacking water molecule to a carboxylic oxygen atom of aspartate 90. The catalytic effect of lysine 92 involves proper positioning of the scissile phosphate group and, more importantly, stabilization of the metaphosphate intermediate in an orientation optimal for attack of the nucleophile. [ABSTRACT FROM AUTHOR]

Abstract: We model the hydrolytic cleavage of dimethylphosphate by hydroxide ion and water in the gas phase and in implicit water using density functional theory. In all cases the rate-determining step is the nucleophilic attack. The barrier for this nucleophilic attack in vacuum is much larger for the hydroxide than for the neutral nucleophile. However, in water the barriers are similar. The rate-determining step in the attack by the neutral nucleophile involves the concerted transfer of a proton from the water molecule to the phosphate ion and the formation of the P–O bond. [Copyright &y& Elsevier]

The vibrational frequencies of the electronic ground state of resorcinol have been determined via laser-induced dispersed fluorescence spectroscopy. An assignment based on comparison with ab initio calculations on the MP2 and B3LYP (6-311 G (d,p)) level is presented. [Copyright &y& Elsevier]

Carboxypeptidase is a Zn-dependent protease that specifically recognises and hydrolyses peptides with a hydrophobic side chain at the C-terminal residue. According to hydrolysis mechanisms proposed in the literature, catalysis requires a water molecule to be close to the Zn ion so as to be activated as a nucleophile. Among small molecules that resemble the slowly hydrolysed Gly-Tyr peptide, which have been previously designed as inhibitors and characterised structurally, a variant with the terminal amino acid in a D-configuration has been the most effective. Our molecular dynamics simulations of carboxypeptidase complexed with different variants of those inhibitor ligands as well as variants of the Gly-Tyr peptide show that the strength of the inhibitory effect is not related to the binding strength of the ligand. Our data rather support an earlier notion that the inhibition is, at least partially, due to blocking a coordination site at the Zn ion by the ligand coordinating the metal ion in a bidentate fashion. [ABSTRACT FROM AUTHOR]

Determination of proton uptake pathways in Cytochrome c Oxidase is difficult due to the complexity of the system. The transition networks approach allows sampling of proton transfer pathways without predefined reaction coordinate. Computation of the proton transfer pathways in a model of the D-channel of cytochrome c oxidase has been performed by a transition network approach that combines discrete, optimisation based and molecular dynamics based sampling. The optimal pathway involves an opening of the so-called asparagine gate, hydration of the asparagine region, the formation of a hydrogen-bonded chain, and finally concerted proton hole transport along this chain. The optimal pathway finds the protonation of residue H26 close to the channel entrance favourable for lowering the transition energies of subsequent steps, in particular, opening of the Asn gate and formation of a hydrogen-bonded chain. Residue Y33 plays an important role in shuttling the transferred proton hole. The optimal pathway found by the transition network approach shows the same important characteristics as pathways determined earlier by other methods. The computed barrier and reaction energies are also in good agreement with previous studies. The transition network approach provides an alternative to explore pathways in complex systems. The correct function of the enzyme as oxidase and proton pump depends on the interplay of several redox and proton transport steps. Understanding the proton transport mechanism is therefore key to understanding the protein's function. The complex nature of long- distances proton transfer through a protein requires a non-trivial simulation strategy. [ABSTRACT FROM AUTHOR]

Supramolecular recognition of nucleobases and short sequences is an emerging research field focusing on possible applications to treat many diseases. Controlling the affinity and selectivity of synthetic receptors to target desired nucleotides or short sequences is a highly challenging task. Herein, we elucidate the effect of substituents in the phenyl ring of the anthracene‐benzene azacyclophane on the recognition of nucleoside triphosphates (NTPs) and double‐stranded DNA. We show that introducing phenyl rings increases the affinity for NTPs 10‐fold and implements groove and intercalation binding modes with double‐stranded DNA. NMR studies and molecular modeling calculations support the ability of cyclophanes to encapsulate nucleobases as part of nucleotides. [ABSTRACT FROM AUTHOR]

Pro-drugs, which ideally release their active compound only at the site of action, i.e., in a cancer cell, are a promising approach towards an increased specificity and hence reduced side effects in chemotherapy. A popular form of pro-drugs is esters, which are activated upon their hydrolysis. Since carboxylesterases that catalyse such a hydrolysis reaction are also abundant in normal tissue, it is of great interest whether a putative pro-drug is a probable substrate of such an enzyme and hence bears the danger of being activated not just in the target environment, i.e., in cancer cells. In this work, we study the binding mode of carboxylesters of the drug molecule camptothecin, which is an inhibitor of topoisomerase I, of varying size to human carboxylesterase 2 (HCE2) by molecular docking and molecular dynamics simulations. A comparison to irinotecan, known to be a substrate of HCE2, shows that all three pro-drugs analysed in this work can bind to the HCE2 protein, but not in a pose that is well suited for subsequent hydrolysis. Our data suggest, moreover, that for the irinotecan substrate, a reactant-competent pose is stabilised once the initial proton transfer from the putative nucleophile Ser202 to the His431 of the catalytic triad has already occurred. Our simulation work also shows that it is important to go beyond the static models obtained from molecular docking and include the flexibility of enzyme–ligand complexes in solvents and at a finite temperature. Under such conditions, the pro-drugs studied in this work are unlikely to be hydrolysed by the HCE2 enzyme, indicating a low risk of undesired drug release in normal tissue. [ABSTRACT FROM AUTHOR]

Examines the resonance enhanced multiphoton ionization (REMPI) of laser desorbed guanosines. Observation of fundamental properties of deoxyribonucleic acid (DNA) building blocks; Nucleosides in the gas phase; Guanine as chromophore in DNA; Laser desorption jet cooling REMPI spectrometry.

The transfer of protons through proton translocating channels is a complex process, for which direct samplings of different protonation states and side chain conformations in a transition network calculation provide an efficient, bias-free description. In principle, a new transition network calculation is required for every unsampled change in the system of interest, e.g. an unsampled protonation state change, which is associated with significant computational costs. Transition networks void of or including an unsampled change are termed unperturbed or perturbed, respectively. Here, we present a prediction method, which is based on an extensive coarse-graining of the underlying transition networks to speed up the calculations. It uses the minimum spanning tree and a corresponding sensitivity analysis of an unperturbed transition network as initial guess and refinement parameter for the determination of an unknown, perturbed transition network. Thereby, the minimum spanning tree defines a sub-network connecting all nodes without cycles and minimal edge weight sum, while the sensitivity analysis analyzes the stability of the minimum spanning tree towards individual edge weight reductions. Using the prediction method, we are able to reduce the calculation costs in a model system by up to 80%, while important network properties are maintained in most predictions. [ABSTRACT FROM AUTHOR]

Recent advances in operating and manipulating DNA have provided unique experimental possibilities in many fields of DNA research, especially in gene therapy. Researchers have deployed many techniques, experimental and theoretical, to study the DNA structure changes due to external perturbation. It is crucial to understand the structural and dynamical changes in the DNA molecules in a confined state to understand and control the self-assembly of DNA confined in a chamber or nano-channel for various applications. In the current manuscript, we extend the work study the effect of confinement on the thermal stability and the structural properties of duplex DNA. The present work is an extension of our previous research works (Maity et al., 2019; 2020). For our study we have considered a 1 BNA chain that is confined in a cylindrical geometry. How the geometry of the confinement affects the opening and other structural parameters of DNA molecule is the objective of this manuscript. We have used a statistical model(PBD model) and Molecular dynamics simulations for our purpose. [ABSTRACT FROM AUTHOR]

Distortions in the DNA sequence, such as damage or mispairs, are specifically recognized and processed by DNA repair enzymes. Many repair proteins and, in particular, glycosylases flip the target base out of the DNA helix into the enzyme's active site. Our molecular dynamics simulations of DNA with intact and damaged (oxidized) methyl-cytosine show that the probability of being flipped is similar for damaged and intact methyl-cytosine. However, the accessibility of the different 5-methyl groups allows direct discrimination of the oxidized forms. Hydrogen-bonded patterns that vary between methyl-cytosine forms carrying a carbonyl oxygen atom are likely to be detected by the repair enzymes and may thus help target site recognition. [ABSTRACT FROM AUTHOR]

Highlights: [•] First principles simulation of prototypical metabolite molecule. [•] Free energy barrier of proton transfer reaction underlying chemical exchange saturation transfer experiments. [•] Spontaneous proton back transfer. [•] Proton readily releases on biological time scales. [Copyright &y& Elsevier]

Abstract: The restriction endonuclease EcoRV binds two magnesium ions. One of these ions, , binds to the phosphate group where the cleavage occurs and is required for catalysis, but the role of the other ion, is debated. Here, multiple independent molecular dynamics simulations suggest that is crucial for achieving a tightly bound protein–DNA complex and stabilizing a conformation that allows cleavage. In the absence of in all simulations the protein–DNA hydrogen bond network is significantly disrupted and the sharp kink at the central base pair step of the DNA, which is observed in the two-metal complex, is not present. Also, the active site residues rearrange in such a way that the formation of a nucleophile, required for DNA hydrolysis, is unlikely. [Copyright &y& Elsevier]

The Diels-Alderase ribozyme, an in vitro-evolved ribonucleic acid enzyme, accelerates the formation of carbon-carbon bonds between an anthracene diene and a maleimide dienophile in a [4 + 2] cycloaddition, a reaction with broad application in organic chemistry. Here, the Diels-Alderase ribozyme is examined via molecular dynamics (MD) simulations in both crystalline and aqueous solution environments. The simulations indicate that the catalytic pocket is highly dynamic. At low Mg2+ ion concentrations, inactive states with the catalytic pocket closed dominate. Stabilization of the enzymatically active, open state of the catalytic pocket requires a high concentration of Mg2+ ions (e.g., 54 mM), with cations binding to specific phosphate sites on the backbone of the residues bridging the opposite strands of the pocket. The free energy profile for pocket opening at high Mg2+ cation concentration exhibits a double minimum, with a barrier to opening of ∼5.5 kJ/mol and the closed state ∼3 kJ/mol lower than the open state. Selection of the open state on substrate binding leads to the catalytic activity of the ribozyme. The simulation results explain structurally the experimental observation that full catalytic activity depends on the Mg2+ ion concentration. [ABSTRACT FROM AUTHOR]

A density functional study of [VO(O2)2(lm)]- (1, Im = imidazole) is presented, calling special attention to effects of dynamics and solvation on the 51V chemical shift. According to Car-Parrinello molecular dynamics simulations, rotation of the Im ligand can be fast in the gas phase, but is more hindered in aqueous solution. In the latter, bonding between Im and V is reinforced, and dynamic averaging of GlAO- B3LYP magnetic shieldings affords a gas-to-liquid shift of ca. -100 ppm for δ(51V). A complete catalytic cycle has been characterized for olefin epoxidation mediated by 1, using H2O2 as oxidant. The rate-determining step is indicated to be initial oxygen atom transfer from 1 to the substrate via a spiro-like transition state. Substituent effects on this barrier are examined, and a significant decrease (by 2-6 kcal/ mol) is revealed upon removal of the Im proton or upon complexation with a H-bond acceptor. Implications for the mechanism of the oxidative chemistry of vanadium-dependent haloperoxidases and requirements for prospective biomimetic analogues are discussed. [ABSTRACT FROM AUTHOR]

Thymine DNA Glycosylase (TDG) is an enzyme of the base excision repair mechanism and removes damaged or mispaired bases from DNA via hydrolysis of the glycosidic bond. Specificity is of high importance for such a glycosylase, so as to avoid the damage of intact DNA. Among the substrates reported for TDG are mispaired uracil and thymine but also formyl-cytosine and carboxyl-cytosine. Methyl-cytosine and hydroxylmethyl-cytosine are, in contrast, not processed by the TDG enzyme. We have in this work employed molecular dynamics simulations to explore the conformational dynamics of DNA carrying a formyl-cytosine or carboxyl-cytosine and compared those to DNA with the non-cognate bases methyl-cytosine and hydroxylmethyl-cytosine, as amino and imino tautomers. Whereas for the mispairs a wobble conformation is likely decisive for recognition, all amino tautomers of formyl-cytosine and carboxyl-cytosine exhibit the same Watson–Crick conformation, but all imino tautomers indeed form wobble pairs. The conformational dynamics of the amino tautomers in free DNA do not exhibit differences that could be exploited for recognition, and also complexation to the TDG enzyme does not induce any alteration that would indicate preferable binding to one or the other oxidised methyl-cytosine. The imino tautomers, in contrast, undergo a shift in the equilibrium between a closed and a more open, partially flipped state, towards the more open form upon complexation to the TDG enzyme. This stabilisation of the more open conformation is most pronounced for the non-cognate bases methyl-cytosine and hydroxyl-cytosine and is thus not a likely mode for recognition. Moreover, calculated binding affinities for the different forms indicate the imino forms to be less likely in the complexed DNA. These findings, together with the low probability of imino tautomers in free DNA and the indifference of the complexed amino tautomers, suggest that discrimination of the oxidised methyl-cytosines does not take place in the initial complex formation. [ABSTRACT FROM AUTHOR]

Brr2 is an essential Ski2-like RNA helicase that exhibits a unique structure among the spliceosomal helicases. Brr2 harbors a catalytically active N-terminal helicase cassette and a structurally similar but enzymatically inactive C-terminal helicase cassette connected by a linker region. Both cassettes contain a nucleotide-binding pocket, but it is unclear whether nucleotide binding in these two pockets is related. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and determine whether occupancy of one nucleotide-binding site may influence nucleotide binding at the other cassette. Our results show that Brr2 exhibits high specificity for adenine nucleotides, with both cassettes binding ADP tighter than ATP. Adenine nucleotide affinity for the inactive C-terminal cassette is more than two orders of magnitude higher than that of the active Nterminal cassette, as determined by slow nucleotide release. Mutations at the intercassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Moreover, we found that abrogation of nucleotide binding at the C-terminal cassette reduces nucleotide binding at the N-terminal cassette 70 Å away. Molecular dynamics simulations identified structural communication lines that likely mediate these long-range allosteric effects, predominantly across the intercassette interface. Together, our results reveal intricate networks of intramolecular interactions in the complex Brr2 RNA helicase, which fine-tune its nucleotide affinities and which could be exploited to regulate enzymatic activity during splicing. [ABSTRACT FROM AUTHOR]

The vibrational spectrum of the Ala-Leu-Ala-Leu peptide in solution, computed from first-principles simulations, shows a prominent band in the amide I region that is assigned to stretching of carbonyl groups. Close inspection reveals combined but slightly different contributions by the three carbonyl groups of the peptide. The shift in their exact vibrational signature is in agreement with the different probabilities of these groups to form hydrogen bonds with the solvent. The central carbonyl group has a hydrogen bond probability intermediate to the other two groups due to interchanges between different hydrogen-bonded states. Analysis of the interaction energies of individual water molecules with that group shows that shifts in its frequency are directly related to the interactions with the water molecules in the first hydration shell. The interaction strength is well correlated with the hydrogen bond distance and hydrogen bond angle, though there is no perfect match, allowing geometrical criteria for hydrogen bonds to be used as long as the sampling is sufficient to consider averages. The hydrogen bond state of a carbonyl group can therefore serve as an indicator of the solvent's effect on the vibrational frequency. [ABSTRACT FROM AUTHOR]

Phosphoserine phosphatase (PSP), a member of the haloacid dehalogenase (HAD) superfamily that comprises the vast majority of phosphotransferases, is likely a steady-state regulator of the level of d-serine in the brain. The proposed catalytic cycle of PSP consists of a two-step mechanism: formation of a phospho-enzyme intermediate by phosphate transfer to Asp11 and its subsequent hydrolysis. Our combined quantum mechanical/molecular mechanical (QM/MM) calculations of the reaction pathways favour a dissociative mechanism of nucleophilic substitution via a trigonal-planar metaphosphate-like configuration for both steps, associated with proton transfer to the leaving group or from the nucleophile. This proton transfer is facilitated by active site residue Asp13 that acts as both a general base and a general acid. Free energy calculation on the reaction pathways further support the structural role of the enzymatic environment and the active site architecture. The choice of a proper reaction coordinate along which to bias the free energy calculations can be guided by a projection of the canonical reaction coordinate obtained from a chain-of-state optimisation onto important internal coordinates. [ABSTRACT FROM AUTHOR]

Proton transfer in cytochrome c oxidase (CcO) from the cellular inside to the binuclear redox centre as well as proton pumping through the membrane takes place through proton entrance via two distinct pathways, the D- and K-channel. Both channels show a dependence of their hydration level on the protonation states of their key residues, K362 for the K-channel, and E286 or D132 for the D-channel. In the oxidative half of CcO's catalytic cycle the D-channel is the proton-conducting path. For this channel, an interplay of protonation state of the D-channel residues with the water and hydrogen-bond dynamics has been observed in molecular dynamics simulations of the CcO protein, embedded in a lipid bi-layer, modelled in different protonation states. Protonation of residue E286 at the end of the D-channel results in a hydrogen-bonded network pointing from E286 to N139, that is against proton transport, and favouring N139 conformations which correspond to a closed asparagine gate (formed by residues N121 and N139). Consequently, the hydration level is lower than with unprotonated E286. In those models, the Asn gate is predominantly open, allowing water molecules to pass and thus increase the hydration level. The hydrogen-bonded network in these states exhibits longer life times of the Asn residues with water than other models and shows the D-channel to be traversable from the entrance, D132, to exit, E286. The D-channel can thus be regarded as auto-regulated with respect to proton transport, allowing proton passage only when required, that is the proton is located at the lower part of the D-channel (D132 to Asn gate) and not at the exit (E286). [ABSTRACT FROM AUTHOR]

We have combined infrared (IR) experiments with molecular dynamics (MD) simulations in solution at finite temperature to analyse the vibrational signature of the small floppy peptide Alanine-Leucine. IR spectra computed from first-principles MD simulations exhibit no distinct differences between conformational clusters of α -helix or β -sheet-like folds with different orientations of the bulky leucine side chain. All computed spectra show two prominent bands, in good agreement with the experiment, that are assigned to the stretch vibrations of the carbonyl and carboxyl group, respectively. Variations in band widths and exact maxima are likely due to small fluctuations in the backbone torsion angles. [ABSTRACT FROM AUTHOR]

Proton transfer reactions are complex transitions due to the size and flexibility of the hydrogen-bonded networks along which the protons may “hop”. The combination of molecular dynamics based sampling of water positions and orientations with direct sampling of proton positions is an efficient way to capture the interplay of these degrees of freedom in a transition network. The energetically most favourable pathway in the proton transfer network computed for an aspartate-water cluster shows the pre-orientation of water molecules and aspartate side chains to be a pre-requisite for the subsequent concerted proton transfer to the product state. [ABSTRACT FROM AUTHOR]

We study the exchange processes ofthe metabolite creatine, whichis present in both tumorous and normal tissues and has NH2and NH groups that can transfer protons to water. Creatine produceschemical exchange saturation transfer (CEST) contrast in magneticresonance imaging (MRI). The proton transfer pathway from zwitterioniccreatine to water is examined using a kinetic transition network constructedfrom the discrete path sampling approach and an approximate quantum-chemicalenergy function, employing the self-consistent-charge density-functionaltight-binding (SCC-DFTB) method. The resulting potential energy surfaceis visualized by constructing disconnectivity graphs. The energy landscapeconsists of two distinct regions corresponding to the zwitterioniccreatine structures and deprotonated creatine. The activation energythat characterizes the proton transfer from the creatine NH2group to water was determined from an Arrhenius fit of rate constantsas a function of temperature, obtained from harmonic transition statetheory. The result is in reasonable agreement with values obtainedin water exchange spectroscopy (WEX) experiments. [ABSTRACT FROM AUTHOR]

The Diels-Alderase ribozyme is an in vitro-evolved ribonucleic acid enzyme that catalyzes a [4 + 2] cycloaddition reaction between an anthracene diene and a maleimide dienophile. The ribozyme can in principle be used to selectively synthesize only one product enantiomer, depending on which of the two entrances to the catalytic pocket, 'front' or 'back', the substrate is permitted to use. Here, we investigate stereoselection and substrate recognition in the ribozyme by means of multiple molecular dynamics simulations, performed on each of the two substrates individually in the pocket, on the reactant state, and on the product state. The results are consistent with a binding mechanism in which the maleimide likely binds first followed by the anthracene, which enters preferentially through the front door. The free energy profiles for anthracene binding indicate that the pre-( R, R)-enantiomer conformation is slightly preferred, in agreement with the experimentally observed small enantiomeric excess of the ( R, R)-enantiomer of the product. The reactant state is stabilized by the simultaneous presence of both substrates bound to their binding sites in the hydrophobic pocket as well as by stacking interactions between them. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: EcoRV, a restriction enzyme in Escherichia coli, destroys invading foreign DNA by cleaving it at the center step of a GATATC sequence. In the EcoRV–cognate DNA crystallographic complex, a sharp kink of 50° has been found at the center base-pair step (TA). Here, we examine the interplay between the intrinsic propensity of the cognate sequence to kink and the induction by the enzyme by performing all-atom molecular dynamics simulations of EcoRV unbound and interacting with three DNA sequences: the cognate sequence, GATATC (TA); the non-cognate sequence, GAATTC (AT); and with the cognate sequence methylated on the first adenine GACH3 TATC (TA-CH3). In the unbound EcoRV, the cleft between the two C-terminal subdomains is found to be open. Binding to AT narrows the cleft and forms a partially bound state. However, the intrinsic bending propensity of AT is insufficient to allow tight binding. In contrast, the cognate TA sequence is easier to bend, allowing specific, high-occupancy hydrogen bonds to form in the complex. The absence of cleavage for this methylated sequence is found to arise from the loss of specific hydrogen bonds between the first adenine of the recognition sequence and Asn185. On the basis of the results, we suggest a three-step recognition mechanism. In the first step, EcoRV, in an open conformation, binds to the DNA at a random sequence and slides along it. In the second step, when the two outer base pairs, GAxxTC, are recognized, the R loops of the protein become more ordered, forming strong hydrogen-bonding interactions, resulting in a partially bound EcoRV–DNA complex. In the third step, the flexibility of the center base pair is probed, and in the case of the full cognate sequence the DNA bends, the complex strengthens and the protein and DNA interact more closely, allowing cleavage. [ABSTRACT FROM AUTHOR]

DNA repair proteins are able to discriminate DNA lesions among an abundance of intact DNA with high selectivity. To investigate detectable characteristics of one specific lesion, we compare statistical results from molecular dynamics simulations of two different DNA in water, one with an intact C:G pair and one that contains a U:G mispair, and perform a comparative analysis of the water dynamics around the two. Our data show that in addition to the local DNA conformation, also the surrounding water shell exhibits significant differences that may help mispair discrimination. The chemical groups which account for a U:G mispair to exhibit a wobble conformation instead of the 'proper' Watson-Crick pairing of a C:G pair, that is an oxygen atom (in uracil) instead of an amino group (in cytosine), also order the water molecules around the bases such that they act predominantly as hydrogen-bond donor or acceptor to the uracil or cytosine base, respectively. These changes in water conformation stretch into the second solvation shell, which may be exploited by repair enzymes to achieve lesion detection with high efficiency. [Display omitted] • DNA locally deformed at U:G mispair • Water orientates according to chemical groups of C or U • Water is slowed down between U:G [ABSTRACT FROM AUTHOR]

Homeothermic organisms maintain their core body temperature in a narrow, tightly controlled range. Whether and how subtle circadian oscillations or disease-associated changes in core body temperature are sensed and integrated in gene expression programs remain elusive. Furthermore, a thermo-sensor capable of sensing the small temperature differentials leading to temperature-dependent sex determination (TSD) in poikilothermic reptiles has not been identified. Here, we show that the activity of CDC-like kinases (CLKs) is highly responsive to physiological temperature changes, which is conferred by structural rearrangements within the kinase activation segment. Lower body temperature activates CLKs resulting in strongly increased phosphorylation of SR proteins in vitro and in vivo. This globally controls temperature-dependent alternative splicing and gene expression, with wide implications in circadian, tissue-specific, and disease-associated settings. This temperature sensor is conserved across evolution and adapted to growth temperatures of diverse poikilotherms. The dynamic temperature range of reptilian CLK homologs suggests a role in TSD. • CLK kinases are thermo-sensors reactive to subtle body-temperature changes • Body temperature globally controls splicing and gene expression through CLKs • CLK homologs are evolutionarily adapted to diverse body/growth temperatures • Wide functionality of CLKs: from TSD in reptiles to circadian biology in mammals Haltenhof et al. show that the activity of CLK kinases is extremely sensitive to physiological temperature changes. This cellular thermometer is based on minor structural rearrangements of the kinase activation segment, representing a new concept in enzymology. CLKs globally control alternative splicing and gene expression in a (body) temperature-dependent manner, with wide implications ranging from circadian biology to disease conditions. This thermometer is evolutionarily adapted to the growth temperature of diverse organisms with implications for reptilian temperature dependent sex determination. [ABSTRACT FROM AUTHOR]