Editorial : Computational and Experimental Insights in Redox-Coupled Proton Pumping in Proteins

Non peer reviewe

Phosphodiester hydrolysis computed for cluster models of enzymatic active sites

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

The Effect of a G:T Mispair on the Dynamics of DNA

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

Interplay of Hydration and Protonation Dynamics in the K-Channel of Cytochrome c Oxidase

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

Umbrella sampling of proton transfer in a creatine–water system

Proton transfer reactions are among the most common processes in chemistry and biology. Proton transfer between creatine and surrounding solvent water is underlying the chemical exchange saturation transfer used as a contrast in magnetic resonance imaging. The free energy barrier, determined by first- principles umbrella sampling simulations (View the MathML sourceEaDFT 3 kcal/mol) is in the same order of magnitude as the experimentally obtained activation energy. The underlying mechanism is a first proton transfer from the guanidinium group to the water pool, followed by a second transition where a proton is “transferred back” from the nearest water molecule to the deprotonated nitrogen atom of creatine

Effect of the Hydration Shell on the Carbonyl Vibration in the Ala-Leu-Ala-Leu Peptide

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

Base Flip in DNA Studied by Molecular Dynamics Simulations of Differently-Oxidized Forms of Methyl-Cytosine

Abstract: 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

Protonation Dynamics in the K-Channel of Cytochrome c Oxidase Estimated from Molecular Dynamics Simulations

Proton transfer reactions are one of the most fundamental processes in biochemistry. We present a simplistic approach for estimating proton transfer probabilities in a membrane protein, cytochrome c oxidase. We combine short molecular dynamics simulations at discrete protonation states with a Monte Carlo approach to exchange between those states. Requesting for a proton transfer the existence of a hydrogen-bonded connection between the two source and target residues of the exchange, restricts the acceptance of transfers to only those in which a proton-relay is possible. Together with an analysis of the hydrogen-bonded connectivity in one of the proton-conducting channels of cytochrome c oxidase, this approach gives insight into the protonation dynamics of the hydrogen-bonded networks. The connectivity and directionality of the networks are coupled to the conformation of an important protein residue in the channel, K362, rendering proton transfer in the entire channel feasible in only one of the two major conformations. Proton transport in the channel can thus be regulated by K362 not only through its possible role as a proton carrier itself, but also by allowing or preventing proton transport via water residues

Protonation-State Dependence of Hydration and Interactions in the Two Proton-Conducting Channels of Cytochrome c Oxidase

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

Mechanism of DNA recognition by the restriction enzyme EcoRV.

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 degrees 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 GA(CH(3))TATC (TA-CH(3)). 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 amore » 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.« les