Characterization by tandem mass spectrometry of stable cysteine sulfenic acid in a cysteine switch peptide of matrix metalloproteinases

Cysteine sulfenic acid (Cys-SOH) is an elusive intermediate in reactive oxygen species-induced oxidation reactions of many proteins such as peroxiredoxins and tyrosine phosphatases. Cys-SOH is proposed to play a vital role in catalytic and signaling functions. The formation of cysteine sulfinic acid (Cys-SO(2)H) and cysteine sulfonic acid (Cys-SO(3)H) has been implicated in the activation of matrix metalloproteinase-7 (MMP-7) and oxidation of thiol to cysteine sulfinic acid has been associated with the autolytic cleavage of MMP-7. We have examined the formation of cysteine sulfenic acid in a synthetic peptide PRCGVPDVA, which is a cysteine switch domain of MMP-7 and other matrix metalloproteases. We have prepared the cysteine sulfenic acid containing peptide, PRC(SOH)GVPDVA, by reaction with hydroxyl radicals generated by the Fenton reaction (Fe(+2)/H(2)O(2)). We characterized this modified peptide by tandem mass spectrometry and accurate mass measurement experiments. In addition, we used 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl) reagent to form an adduct with PRC(SOH)GVPDVA to provide additional evidence for the viability of PRC(SOH)GVPDVA in solution. We also characterized an intramolecular cysteine sulfinamide cross-link product PRC[S(O)N]GVPDVA based on tandem mass spectrometry and accurate mass measurement experiments. These results contribute to the understanding of a proteolytic cleavage mechanism that is traditionally associated with MMP activation.     

Scope and mechanism of the C-H bond activation reactivity within a supramolecular host by an iridium guest: a stepwise ion pair guest dissociation mechanism

A chiral self-assembled supramolecular M(4)L(6) assembly has been shown to be a suitable host for a series of reactive monocationic half-sandwich iridium guests 1, 3, and 4 that are capable of activating C-H bonds. Upon encapsulation, selective C-H bond activation of organic substrates occurs. Precise size and shape selectivity are observed in the C-H bond activation of aldehydes and ether substrates. The reactions exhibit significant kinetic diastereoselectivities. Thermodynamic studies have shown that the iridium starting materials and products are bound strongly by the host assembly. The encapsulation process is largely entropy-driven. Kinetic investigations with water-soluble phosphine traps and added salts have provided evidence for a unique stepwise mechanism of guest dissociation for [4 subset Ga(4)L(6)]. Iridium guest 4 first dissociates from the host cavity to form an ion pair with the host exterior. This species then fully dissociates from the host exterior into the bulk solution. Model ion pair intermediates were characterized directly with (1)H NMR NOESY techniques. The rate of iridium guest dissociation is slower than the rate observed for the C-H bond activation processes, indicating that the selective C-H bond activation reactivity occurs within the cavity of the supramolecular host.     

Revisiting the dispersion mechanism of grafted nanoparticles in polymer matrix: a detailed molecular dynamics simulation

By focusing on the grafted nanoparticles (NPs) embedded in polymer melts, a detailed coarse-grained molecular dynamics simulation is adopted to investigate the effects of the grafting density, the length of the matrix and grafted chains on the dispersion of the NPs. We have employed visualization snapshots, radial distribution functions (RDFs), the interaction energy between NPs, the number of neighbor NPs, and the conformation of the brush chains to clearly analyze the dispersion state of the grafted NPs. Our simulated results generally indicate that the dispersion of the NPs is controlled by both the excluded volume of the grafted NPs and the interface between the brushes and the matrix. It is found that increasing grafting density or grafted chain length leads to better dispersion, owing to larger excluded volume; however, increasing the length of the matrix chains leads to aggregation of NPs, attributed to both a progressive loss of the interface between the brushes and the matrix and the overlap between brushes of different NPs, intrinsically driven by entropy. Meanwhile, it is found that there exists an optimum grafting density (σ(c)) for the dispersion of the NPs, which roughly obeys the following mathematical relation: σ(c) is proportional to N(m)(K)/N(g)(L), where K, L > 0 and N(m) and N(g) represent the length of the matrix and grafted chain length, respectively. Considering the practical situation that the grafted brushes and the matrix polymer are mostly not chemically identical, we also studied the effect of the compatibility between the brushes and the matrix polymer by taking into account the attraction between the grafted chains and the matrix chains. In general, our comprehensive simulation results are believed to guide the design and preparation of high-performance polymer nanocomposites with good or even tailored dispersion of NPs.     

熔融ZnCl2结构的分子动力学模拟研究

熔融ZnCl2作为一种离子性共价性参半的典型熔盐, 其近邻结构在实验测量和分子动力学模拟方面均作过一些研究。本文依据新近EXAFS实验结果, 比较了不同的有效势下模拟得到的径向分布函数,表明KDR势可作为一种实用势。并进一步在KDR势模拟产生的瞬态构型基础上, 使用键序参数方法研究了晶态和熔融态ZnCl2中的近邻结构。结果表明, 和晶态ZnCl2一样, 在熔融ZnCl2中存在稳定的Zn/Cl正四面体结构, 但熔态和晶态Zn/Cl近邻结构热波方差σ不同。计算表明300K晶态σ=5.0℃, 613K熔融态σ=12.2℃。也对熔融ZnCl2的网络状结构和宏观输运性质进行了讨论。     

Theoretical study on the mechanism of the oxygen activation process in cysteine dioxygenase enzymes

Cysteine dioxygenase (CDO) is a vital enzyme for human health involved in the biodegradation of toxic cysteine and thereby regulation of the cysteine concentration in the body. The enzyme belongs to the group of nonheme iron dioxygenases and utilizes molecular oxygen to transfer two oxygen atoms to cysteinate to form cysteine sulfinic acid products. The mechanism for this reaction is currently disputed, with crystallographic studies implicating a persulfenate intermediate in the catalytic cycle. To resolve the dispute we have performed quantum mechanics/molecular mechanics (QM/MM) calculations on substrate activation by CDO enzymes using an enzyme monomer and a large QM active region. We find a stepwise mechanism, whereby the distal oxygen atom of the iron(II)-superoxo complex attacks the sulfur atom of cysteinate to form a ring structure, followed by dioxygen bond breaking and the formation of a sulfoxide bound to an iron(IV)-oxo complex. A sulfoxide rotation precedes the second oxygen atom transfer to the substrate to give cysteine sulfinic acid products. The reaction takes place on several low-lying spin-state surfaces via multistate reactivity patterns. It starts in the singlet ground state of the iron(II)-superoxo reactant and then proceeds mainly on the quintet and triplet surfaces. The initial and rate-determining attack of the superoxo group on the cysteinate sulfur atom involves a spin-state crossing from singlet to quintet. We have also investigated an alternative mechanism via a persulfenate intermediate, with a realignment of hydrogen bonding interactions in the substrate binding pocket. However, this alternative mechanism of proximal oxygen atom attack on the sulfur atom of cysteinate is computed to be a high-energy pathway, and therefore, the persulfenate intermediate is unlikely to participate in the catalytic cycle of CDO enzymes.     

Molecular dynamics simulations of the catalytic pathway of a cysteine protease: a combined QM/MM study of human cathepsin K

Molecular dynamics simulations using a combined QM/MM potential have been performed to study the catalytic mechanism of human cathepsin K, a member of the papain family of cysteine proteases. We have determined the two-dimensional free energy surfaces of both acylation and deacylation steps to characterize the reaction mechanism. These free energy profiles show that the acylation step is rate limiting with a barrier height of 19.8 kcal/mol in human cathepsin K and of 29.3 kcal/mol in aqueous solution. The free energy of activation for the deacylation step is 16.7 kcal/mol in cathepsin K and 17.8 kcal/mol in aqueous solution. The reduction of free energy barrier is achieved by stabilization of the oxyanion in the transition state. Interestingly, although the "oxyanion hole" has been formed in the Michaelis complex, the amide units do not donate hydrogen bonds directly to the carbonyl oxygen of the substrate, but they stabilize the thiolate anion nucleophile. Hydrogen-bonding interactions are induced as the substrate amide group approaches the nucleophile, moving more than 2 A and placing the oxyanion in contact with Gln19 and the backbone amide of Cys25. The hydrolysis of peptide substrate shares a common mechanism both for the catalyzed reaction in human cathepsin K and for the uncatalyzed reaction in water. Overall, the nucleophilic attack by Cys25 thiolate and the proton-transfer reaction from His162 to the amide nitrogen are highly coupled, whereas a tetrahedral intermediate is formed along the nucleophilic reaction pathway.     

Molecular dynamics calculation of molecular volumes and volumes of activation

Experimentally, the effects of pressure on reaction rates are described by their pressure derivatives, known as volumes of activation. Transition state theory directly links activation volumes to partial molar volumes of reactants and transition states. We discuss a molecular dynamics method for the accurate calculation of molecular volumes, within which the volumes of molecular species are obtained as a difference between the volumes of pure solvent and solvent with a single molecule inserted. The volumes thus obtained depend on the molecular geometry, the strength and type of the solute-solvent interactions, as well as temperature and pressure. The partial molar volumes calculated using this approach agree well with experimental data. Since this method can also be applied to transition state species, it allows for quantitative analysis of experimental volumes of activation in terms of structural parameters of the corresponding transition states. The efficiency of the approach is illustrated by calculation of volumes of activation for three nonpolar reactions in nonpolar solvents. The results agree well with the experimental data.     

Molecular dynamics characterization of the structures and induction mechanisms of a reverse phenotype of the tetracycline receptor

Molecular-dynamics simulations have been used to investigate the mechanism of induction of a mutant (revTetR) of the tetracycline repressor protein (TetR) that shows the reverse phenotype (i.e., it is induced in the absence of tetracyclines and not in their presence). Low-frequency, normal-mode analyses demonstrate that the reverse phenotype is reproduced by the simulations on the basis of criteria established for wild-type TetR. The reverse phenotype is caused by the fact that the DNA-binding heads in revTetR are closer than the ideal distance needed for DNA-binding when no inducer is present. This distance increases on binding an inducer. Whereas this distance increase makes the interhead distance too large in wild-type TetR, it increases to the ideal value in revTetR. Thus, the mechanism of induction is the same for the two proteins, but the consequences are reversed because of the smaller interhead distance in revTetR when no inducer is present.     

Molecular dynamics modeling of the interface between surface functionalized graphitic structures and calcium-silicate-hydrate: interaction energies, structure, and dynamics

Molecular dynamics simulations were performed to study the molecular-scale energetic, structural, and dynamic properties of the interface between surface functionalized graphitic structures and calcium-silicate-hydrate (C-S-H). The 9 A tobermorite structure was used as a model for C-S-H, the main building block ("the glue") that hold a cementitious matrix together. Six types of carbon surface structures were investigated: a pristine graphite plane and five graphite planes functionalized with hydroxyl (OH), carboxyl (COOH), carboxylate (COO(-), deprotonated carboxyl), carbonyl (CO), and amine (NH(2)) groups. Results demonstrated the dominant role of electrostatic forces in the interfacial interactions and indicated that the polarity of the functional group can be used as an indicator of affinity to C-S-H. MD simulations revealed that an optimal number of polar oxygen containing groups may exist for efficient graphitic structure/cement interaction and emphasized the mediating role of Ca(2+) counterions in the interfacial interactions.     

Theoretical study of the neutral hydrolysis of methyl formate via a concerted and stepwise water-assisted mechanism using free-energy curves and molecular dynamics simulation

A procedure previously described by us is used for the theoretical study of chemical reactions in solution by means of molecular dynamics simulation, with solute–solvent interaction potentials LJ (12-6-1) derived from ab initio quantum calculations. We apply the procedure to the case of the neutral hydrolysis of methyl formate, HCOOCH₃ + 3H₂O → HCOOH + CH₃OH + 2H₂O in aqueous solution, via concerted and stepwise water-assisted mechanisms. We use the solvent as reaction coordinate, and the free-energy curves for the calculation of the activation energies. The theoretical calculation for the thermodynamics of this hydrolysis reaction in aqueous solution, assisted by three water molecules, is in agreement with the available experimental information. In particular our study gives values of ΔG ^≠ = 28.88 and 28.17 kcal/mol for the concerted and stepwise mechanisms, close to the experimental activation barrier of 28.8 kcal/mol, and a significant improvement over the values of 48.05 and 45.66 kcal/mol found in another similar study using the PCM model.     

Molecular dynamics prediction of the mechanism of ester hydrolysis in water

Hydrolysis reactions of the basic units of biological polymers with water, or the reverse reaction, the formation of ester, amide, ketal, or phosphate bonds, occur with very high activation barriers in the gas phase but occur much more rapidly in pure water. Car-Parrinello molecular dynamics simulations reported here show that the rate of hydrolysis of methyl formate in pure water is consistent with mechanisms involving cooperative catalysis by autoionization-generated hydroxide and hydronium, a process known to have an activation free energy of 23.8 kcal/mol. In this mechanism, autoionization is followed by rapid simultaneous acid-base catalysis.     

Molecular dynamics simulations of a hard sphere crystal and reaction-like mechanism for homogeneous melting

Molecular dynamics simulations of a hard sphere crystal are performed for volume fractions ranging from solidification point to melting point. A local bond order parameter is chosen to assign a nature, liquid or solid, to a particle. The probability for a liquid or solid particle to change state presents a typical sigmoid shape as the nature of its neighbors changes. Using this property, I propose a reaction-like mechanism and introduce a small number of rate constants. A mean-field approach to melting and a kinetic Monte Carlo algorithm on a lattice are derived from these chemical processes. The results of these models successfully compare with molecular dynamics simulations, proving that the main properties of melting can be captured by a small number of dynamical parameters.     

Triple-helical transition state analogues: a new class of selective matrix metalloproteinase inhibitors

Alterations in activities of one family of proteases, the matrix metalloproteinases (MMPs), have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix (ECM) components, such as collagen and laminin. Since hydrolysis of the collagen triple-helix is one of the committed steps in ECM turnover, we envisioned modulation of collagenolytic activity as a strategy for creating selective MMP inhibitors. In the present study, a phosphinate transition state analogue has been incorporated within a triple-helical peptide template. The template sequence was based on the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly(439)-Val(440) bond selectively by MMP-2 and MMP-9. The phosphinate acts as a tetrahedral transition state analogue, which mimics the water-bound peptide bond of a protein substrate during hydrolysis. The phosphinate replaced the amide bond between Gly-Val in the P1-P1' subsites of the triple-helical peptide. Inhibition studies revealed Ki values in the low nanomolar range for MMP-2 and MMP-9 and low to middle micromolar range for MMP-8 and MMP-13. MMP-1, MMP-3, and MT1-MMP/MMP-14 were not inhibited effectively. Melting of the triple-helix resulted in a decrease in inhibitor affinity for MMP-2. The phosphinate triple-helical transition state analogue has high affinity and selectivity for the gelatinases (MMP-2 and MMP-9) and represents a new class of protease inhibitors that maximizes potential selectivity via interactions with both prime and nonprime active site subsites as well as with secondary binding sites (exosites).     

Molecular "light switch" for G-quadruplex DNA: cycling the switch on and off

We report a new G-quadruplex DNA "light switch", where the light switch can be cycled on and off through the successive introduction of G-quadruplex DNA and [Fe(CN)(6)](4-) ions.     

Molecular dynamics simulations on the structures and properties of ε-CL-20-based PBXs——Primary theoretical studies on HEDM formulation design

Five polymer bonded explosives (PBXs) with the base explosiveε-CL-20 (hexanitrohexaazaisowurtzitane), the most important high energy density compound (HEDC), and five polymer binders (Estane 5703, GAP, HTPB, PEG, and F2314) were constructed. Molecular dynamics (MD) method was employed to investigate their binding energies (Ebind), compatibility, safety, mechanical properties, and energetic properties. The information and rules were reported for choosing better binders and guiding formulation design of high energy density material (HEDM). According to the calculated binding energies, the ordering of compatibility and stability of the five PBXs was predicted as ε-CL-20/PEG ＞ ε-CL-20/ Estane5703 ≈ε-CL-20/GAP ＞ ε-CL-20/HTPB ＞ ε-CL-20/F2314. By pair correlation function g(r) analyses, hydrogen bonds and vdw are found to be the main interactions between the two components. The elasticity and isotropy of PBXs based ε-CL-20 can be obviously improved more than pure ε-CL-20 crystal. It is not by changing the molecular structures of ε-CL-20 for each binder to affect the sensitivity. The safety and energetic properties of these PBXs are mainly influenced by the thermal capability (C°p) and density (ρ) of binders, respectively.     

Electroreduction of a series of alkylcobalamins: mechanism of stepwise reductive cleavage of the Co-C bond

The electrochemical (EC) reduction mechanism of methylcobalamin (Me-Cbl) in a mixed DMF/MeOH solvent in 0.2 M tetrabutylammonium fluoroborate electrolyte was studied as a function of temperature and solvent ratio vs a nonaqueous Ag/AgCl/Cl(-) reference electrode. Double-potential-step chronoamperometry allowed the rate constant of the subsequent homogeneous reaction to be measured over the temperature range from 0 to -80 degrees C in 40:60 and 50:50 DMF:MeOH ratios. Activation enthalpies are 5.8 +/- 0.5 and 7.6 +/- 0.3 kcal/mol in the 40:60 and 50:50 mixtures of DMF/MeOH, respectively. Digital simulation and curve-fitting for an EC mechanism using a predetermined homogeneous rate constant of 5.5 x 10(3) s(-1) give E degrees' = -1.466 V, k degrees = 0.016 cm/s, and alpha = 0.77 at 20 degrees C for a quasi-reversible electrode process. Digital simulation of the results of Lexa and Savéant (J. Am. Chem. Soc. 1978, 100, 3220-3222) shows that the mechanism is a series of stepwise homogeneous equilibrium processes with an irreversible step following the initial electron transfer (ET) and allows estimation of the equilibrium and rate constants of these reactions. An electron coupling matrix element of H(kA) = (4.7 +/- 1.1) x 10(-4) eV ( approximately 46 J/mol) is calculated for the nonadiabatic ET step for reduction to the radical anion. A reversible bond dissociation enthalpy for homolytic cleavage of Me-Cbl is calculated as 31 +/- 2 kcal/mol. The voltammetry of the ethyl-, n-propyl-, n-butyl-, isobutyl-, and adenosyl-substituted cobalamin was studied, and estimated reversible redox potentials were correlated with Co-C bond distances as determined by DFT (B3LYP/ LANL2DZ) calculations.     

Molecular dynamics: conductivity of a fused salt

The electrical conductivity of a model of liquid KCl was determined by molecular dynamics computations. In a constant temperature ensemble, the linear regime for Ohm's law was shown to extend up to at least 1.5 × 10⁷ V/cm. The results compared favorably with that calculated by monitoring the power dissipation and with the zero-field value obtained via the current autocorrelation function.     

分子动力学模拟研究康里新(Conessine)对MexB的抑制机理

通过分子动力学模拟结合自由能MM/PBSA方法,确定了康里新与MexB的结合位点与关键氨基酸.通过对康里新与内膜转运蛋白MexB的疏水作用和氢键的分析,发现康里新的结合口袋是一个"疏水陷阱",MexB中的"疏水陷阱"抑制了MexB蠕动外排作用.     

Molecular dynamics calculation of the activation volume for water exchange on Li+

The activation volume of water exchange around Li+ (aq) was determined from reactive flux calculations using molecular dynamics simulations with a classical force field. The barrier height for exchange decreases with pressure, giving a negative activation volume, in agreement with the current paradigm for inferring exchange mechanism from activation volume. However, it is also demonstrated that pressure-dependent transmission effects make a significant contribution to the overall activation volume. These calculations indicate that small activation volumes should not be regarded as mechanistically indicative because of the potential contributions from transmission effects.     

Excited states dynamics of DNA and RNA bases: characterization of a stepwise deactivation pathway in the gas phase

Radiationless deactivation pathways of excited gas phase nucleobases were investigated using mass-selected femtosecond resolved pump-probe resonant ionization. By comparison between nucleobases and methylated species, in which tautomerism cannot occur, we can access intrinsic mechanisms at a time resolution never reported so far (80 fs). At this time resolution, and using appropriate substitution, real nuclear motion corresponding to active vibrational modes along deactivation coordinates can actually be probed. We provide evidence for the existence of a two-step decay mechanism, following a 267 nm excitation of the nucleobases. The time resolution achieved together with a careful zero time-delay calibration between lasers allow us to show that the first step does correspond to intrinsic dynamics rather than to a laser cross correlation. For adenine and 9-methyladenine a first decay component of about 100 fs has been measured. This first step is radically increased to 200 fs when the amino group hydrogen atoms of adenine are substituted by methyl groups. Our results could be rationalized according to the effect of the highly localized nature of the excitation combined to the presence of efficient deactivation pathway along both pyrimidine ring and amino group out-of-plane vibrational modes. These nuclear motions play a key role in the vibronic coupling between the initially excited pipi* and the dark npi* states. This seems to be the common mechanism that opens up the earlier phase of the internal conversion pathway which then, in consideration of the rather fast relaxation times observed, would probably proceed via conical intersection between the npi* relay state and high vibrational levels of the ground state.