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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.

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.

Non

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.

Experimental and theoretical methods were used to study two mixtures, 1-hexanol with n-hexane and cis-3-hexen-1-ol with n-hexane. The experimental technics were used to determine excess molar volume and viscosity deviations in the temperature range from 288.15K to 318.15K. The data of excess molar volume indicate similar behavior of the two alcohols, 1-hexanol and cis-3-hexen-1-ol in mixtures with n-hexane. The experimental data are in accordance with molecular dynamics simulation of these mixtures, showing similar number of hydrogen bonds in the two mixtures. The experimental data of viscosity deviations show difference of the two mixtures, where the deviation is significantly larger for cis-3-hexen-1-ol and n-hexane mixture, indicating stronger interactions within cis-3-hexen-1-ol and n-hexane binary mixture. These experimental data are in accordance with quantum chemical calculations on interactions energies. Namely, calculated interaction energies show that interaction between single and double bonds are stronger, than interactions between two single bonds. Eksperimentalne i teorijske metode korišćene su za proučavanje dve smeše, 1-heksanola sa n-heksanom i cis-3-heksen-1-ola sa n-heksanom. Eksperimentalne tehnike su korišćene za određivanje dopunskih zapremina i promene viskoznosti za temperaturni opseg od 288.15K do 318.15K. Podaci o dopunskoj molarnoj zapremini ukazuje na slično ponašanje dva alkohola, 1-heksanola i cis-3-heksen-1-ola u smešama sa n-heksanom. Eksperimentalni podaci su u skladu sa simulacijom molekularne dinamike ovih smeša, pokazujući sličan broj vodoničnih veza u dve smeše. Eksperimentalni podaci o promeni viskoznosti pokazuju značajnu razliku između dve smeše, odstupanje je značajno veće za smešu cis-3-heksen-1-ol i n-heksan, što ukazuje na jaču interakciju u binarnoj smeši cis-3-heksen-1-ola i nheksana. Ovi eksperimentalni podaci su u skladu sa kvantno-hemijskim proračunima energija interakcija na molekulskom nivou. Naime, izračunate energije interakcije pokazuju da je interakcija između jednostrukih i dvostrukih veza jača, nego interakcija između dve jednostruke veze. Related to: [http://cer.ihtm.bg.ac.rs/handle/123456789/3291]

Experimental and theoretical methods were used to study two mixtures, 1-hexanol with nhexane and cis-3-hexen-1-ol with n-hexane. The experimental technics were used to determine excess molar volume and viscosity deviations in the temperature range from 288.15K to 318.15K. The data of excess molar volume indicate similar behavior of the two alcohols, 1-hexanol and cis-3-hexen-1-ol in mixtures with n-hexane. The experimental data are in accordance with molecular dynamics simulation of these mixtures, showing similar number of hydrogen bonds in the two mixtures. The experimental data of viscosity deviations show difference of the two mixtures, where the deviation is significantly larger for cis-3- hexen-1-ol and n-hexane mixture, indicating stronger interactions within cis-3-hexen-1-ol and n-hexane binary mixture. These experimental data are in accordance with quantum chemical calculations on interactions energies. Namely, calculated interaction energies show that interaction between single and double bonds are stronger, than interactions between two single bonds. Eksperimentalne i teorijske metode korišćene su za proučavanje dve smeše, 1-heksanola sa n-heksanom i cis-3-heksen-1-ola sa n-heksanom. Eksperimentalne tehnike su korišćene za određivanje dopunskih zapremina i promene viskoznosti za temperaturni opseg od 288.15K do 318.15K. Podaci o dopunskoj molarnoj zapremini ukazuje na slično ponašanje dva alkohola, 1-heksanola i cis-3-heksen-1-ola u smešama sa n-heksanom. Eksperimentalni podaci su u skladu sa simulacijom molekularne dinamike ovih smeša, pokazujući sličan broj vodoničnih veza u dve smeše. Eksperimentalni podaci o promeni viskoznosti pokazuju značajnu razliku između dve smeše, odstupanje je značajno veće za smešu cis-3-heksen-1- ol i n-heksan, što ukazuje na jaču interakciju u binarnoj smeši cis-3-heksen-1-ola i nheksana. Ovi eksperimentalni podaci su u skladu sa kvantno-hemijskim proračunima energija interakcija na molekulskom nivou. Naime, izračunate energije interakcije pokazuju da je interakcija između jednostrukih i dvostrukih veza jača, nego interakcija između dve jednostruke veze. Related to: [http://cer.ihtm.bg.ac.rs/handle/123456789/3290]

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.

Phytochromes have an interesting ability to photoconvert between red-absorbing (Pr) and far-red-absorbing (Pfr) states. However, this photoconversion process depends on key interactions between a bilin chromophore and protein matrix, thus, the identification of the chemical nature and quantification of chromophore-protein interactions constitute an essential step for understanding the photoconversion process, as well as the proton transfer events taking place during the photocycle. In this work, we presented a powerful and straightforward approach based on the fragment molecular orbital (FMO) method to identify the nature and quantify the strength of the noncovalent interactions at a fully quantum mechanical level between the biliverdin (BV) chromophore and protein of the Deinococcus radiodurans phytochrome (DrBphP) in the Pr state. One of the most interesting features of the FMO method is the pair interaction energy decomposition analysis (PIEDA) approach, the application of PIEDA led to the identification of the pyrrole water, Asp207, and Glu27 as key residues for the stabilization of the pyrrole rings of the BV chromophore through the formation of six H-bonds. Furthermore, the conserved Arg254 and His260 were also identified as essential residues in the conformational stability of both propionic side chains B and C. Interestingly, new interactions were identified in the chromophore-binding pocket, two nonclassical H-bonds (CH/O interactions) between Asp207 and Tyr263, and an OH/�� interaction between Tyr263 and ring D of the BV chromophore, which might have photochemical relevance. Since the photoconversion reaction of phytochromes is achieved in part by protonation state changes of the chromophore and key residues of the protein matrix. The accurate determination of the protonation states of the chromophore and titratable amino acids constitutes an essential step in order to understand the proton transfer process between the BV and protein matrix. To this end, two different approaches were applied to the Agp2 phytochrome in the Pfr and Meta-F states. The first approach is based on the combination of electrostatic energy computations by solving the Poisson Boltzmann equation (PBE) with classical molecular dynamics (MD) simulations. The second one is the constant pH molecular dynamics (CpHMD) method, which has been used for studying several important biological processes caused by changes in solution pH. One of the main advantages of the CpHMD approaches is that the protonation states may change during the conformational dynamics. Thus, the dynamical processes coupled to a change in protonation states can be directly studied. The results obtained with the first approach led to a propionic side chain of ring C deprotonated in both Pfr and Meta-F states. However, these results are in contradiction with the experimental Raman spectra of Agp2 phytochrome. In contrast, the application of the CpHMD method to the Agp2 phytochrome led to a propionic side chain of ring C protonated in both Pfr and Meta-F states as well as the identification of the His278 as a potential proton site acceptor. Thus, the pKa values obtained with the CpHMD method are in good agreement with the experimental results. Furthermore, this approach identified the His278 as a potential proton site acceptor, since the protonation state of pscC and His278 are highly correlated. In summary, by using the CpHMD approach, the dynamical processes coupled to a change in protonation states of phytochromes structures can be directly studied, whilst, by using classical MD simulations, the protonation states are fixed during the conformational dynamic, which leads to an unrealistic sampling. Interestingly, the conclusions derived for the Agp2 phytochrome can be extended to all bathy phytochromes, since the residues located in the chromophore binding pocket are highly conserved, furthermore, these results, not only reveal the role of His278 in the photocycle but also the importance of carrying out a better sampling of the His residues that are in direct contact with the chromophore molecule., Phytochrome haben die interessante F��higkeit, zwischen rot-absorbierenden (Pr) und fern-rotabsorbierenden (Pfr) Zust��nden zu photokonvertieren. Dieser Photokonversionsprozess h��ngt jedoch von Schl��sselinteraktionen zwischen einem Bilin-Chromophor und der Proteinmatrix ab. Daher ist die Identifizierung der chemischen Natur und die Quantifizierung der Chromophor-Protein Interaktionen ein wesentlicher Schritt zum Verst��ndnis des Photokonversionsprozesses sowie der Protonentransfer-Ereignisse, die w��hrend des Photozyklus stattfinden. In dieser Arbeit pr��sentierten wir einen leistungsstarken und einfachen Ansatz, der auf der Fragment-Molekularorbital-Methode (FMO) basiert, um die Natur und die St��rke der nicht-kovalenten Wechselwirkungen zwischen dem Biliverdin (BV)-Chromophor und dem Protein des Deinococcus radiodurans Phytochroms (DrBphP) im Pr-Zustand zu identifizieren und zu quantifizieren. Eine der interessantesten Eigenschaften der FMO-Methode ist der Ansatz der ���pair interaction energy decomposition analysis��� (PIEDA). Die Anwendung von PIEDA f��hrte zur Identifizierung des Pyrrolwassers, Asp207 und Glu27 als Schl��sselreste f��r die Stabilisierung der Pyrrolringe des BV Chromophors durch die Bildung von sechs H-Bindungen. Dar��ber hinaus wurden die konservierten Arg254 und His260 als essentielle Reste f��r die Konformationsstabilit��t der beiden Propionseitenketten B und C identifiziert. Interessanterweise wurden neue Wechselwirkungen in der Chromophor-Bindungstasche identifiziert, zwei nicht-klassische H-Bindungen (CH/O Wechselwirkungen) zwischen Asp207 und Tyr263 sowie eine OH/��-Wechselwirkung zwischen Tyr263 und Ring D des BV-Chromophors, die photochemische Relevanz haben k��nnten. Denn die Photokonversionsreaktion von Phytochromen wird zum Teil durch Protonierungszustands��nderungen des Chromophors und von Schl��sselresten der Proteinmatrix erreicht. Die genaue Bestimmung der Protonierungszust��nde des Chromophors und der titrierbaren Aminos��uren ist ein wesentlicher Schritt, um den Protonentransferprozess zwischen BV und Proteinmatrix zu verstehen. Zu diesem Zweck wurden zwei verschiedene Ans��tze auf das Phytochrom Agp2 im Pfr- und Meta-F-Zustand angewendet. Der erste Ansatz basiert auf der Kombination von elektrostatischen Energieberechnungen durch L��sung der Poisson-Boltzmann-Gleichung (PBE) mit klassischen Molekulardynamik (MD)-Simulationen. Der zweite ist die Methode der konstanten pH Molekulardynamik (CpHMD), die f��r die Untersuchung mehrerer wichtiger biologischer Prozesse verwendet wurde, die durch ��nderungen des L��sungs-pH verursacht werden. Einer der Hauptvorteile der CpHMD-Ans��tze ist, dass sich die Protonierungszust��nde w��hrend der Konformationsdynamik ��ndern k��nnen. Somit k��nnen die dynamischen Prozesse, die mit einer ��nderung der Protonierungszust��nde gekoppelt sind, direkt untersucht werden. Die mit dem ersten Ansatz erhaltenen Ergebnisse f��hrten dazu, dass die Propionss��ureseitenkette von Ring C sowohl im Pfr- als auch im Meta-F-Zustand deprotoniert ist. Diese Ergebnisse stehen jedoch im Widerspruch zu den experimentellen Raman-Spektren vom Agp2 Phytochrom. Im Gegensatz dazu f��hrte die Anwendung der CpHMD-Methode auf das Agp2-Phytochrom zu einer sowohl im Pfr- als auch im Meta-F-Zustand protonierten Propions��ureseitenkette des Rings C sowie zur Identifizierung des His278 als potentieller Protonenakzeptor. Die mit der CpHMD-Methode erhaltenen pKa-Werte stehen somit in guter ��bereinstimmung mit den experimentellen Ergebnissen. Dar��ber hinaus identifizierte dieser Ansatz das His278 als potentiellen Protonenakzeptor, da der Protonierungszustand von pscC und His278 hoch korreliert sind. Zusammenfassend l��sst sich sagen, dass durch die Verwendung des CpHMD-Ansatzes die dynamischen Prozesse, die mit einer ��nderung der Protonierungszust��nde von Phytochromstrukturen gekoppelt sind, direkt untersucht werden k��nnen, w��hrend bei der Verwendung klassischer MD-Simulationen die Protonierungszust��nde w��hrend der Konformationsdynamik fixiert sind, was zu einem unrealistischen Sampling f��hrt. Interessanterweise k��nnen die f��r das Agp2-Phytochrom abgeleiteten Schlussfolgerungen auf alle Bathy-Phytochrome ausgedehnt werden, da die in der Chromophor-Bindungstasche befindlichen Reste hoch konserviert sind. Dar��ber hinaus zeigen diese Ergebnisse nicht nur die Rolle von His278 im Photozyklus, sondern auch die Wichtigkeit der Durchf��hrung eines besseren Samplings der His-Reste, die in direktem Kontakt mit dem Chromophor-Molek��l stehen.