Kinetic Bottlenecks Research Articles

Direct Electrochemical Regeneration of Monooxygenase Subunits for Biocatalytic Asymmetric Epoxidation

We report the first example of direct electrochemical regeneration of a flavin-dependent monooxygenase for asymmetric epoxidation catalysis. It is shown that electrochemical regeneration of the oxygenase subunit of the multicomponent styrene monooxygenase is sufficient to perform enantiospecific S-epoxidation of various styrene derivatives. Kinetic bottlenecks of the novel electroenzymatic reaction are identified. This work opens up new alternatives for asymmetric oxyfunctionalization catalysis.

Histone-DNA binding free energy cannot be measured in dilution-driven dissociation experiments.

Despite decades of study on nucleosomes, there has been no experimental determination of the free energy of association between histones and DNA. Instead, only the relative free energy of association of the histone octamer for differing DNA sequences has been available. Recently, a method was developed based on quantitative analysis of nucleosome dissociation in dilution experiments that provides a simple practical measure of nucleosome stability. Solution conditions were found in which nucleosome dissociation driven by dilution fit well to a simple model involving a noncooperative nucleosome assembly/disassembly equilibrium, suggesting that this approach might allow absolute equilibrium affinity of the histone octamer for DNA to be measured. Here, we show that the nucleosome assembly/disassembly process is not strictly reversible in these solution conditions, implying that equilibrium affinities cannot be obtained from these measurements. Increases in [NaCl] or temperature, commonly employed to suppress kinetic bottlenecks in nucleosome assembly, lead to cooperative behavior that cannot be interpreted with the simple assembly/disassembly equilibrium model. We conclude that the dilution experiments provide useful measures of kinetic but not equilibrium stability. Kinetic stability is of practical importance: it may govern nucleosome function in vivo, and it may (but need not) parallel absolute thermodynamic stability.

Folding a protein in a computer: An atomic description of the folding/unfolding of protein A

We study the folding mechanism of a three-helix bundle protein at atomic resolution, including effects of explicit water. Using replica exchange molecular dynamics we perform enough sampling over a wide range of temperatures to obtain the free energy, entropy, and enthalpy surfaces as a function of structural reaction coordinates. Simulations were started from different configurations covering the folded and unfolded states. Because many transitions between all minima at the free energy surface are observed, a quantitative determination of the free energy barriers and the ensemble of configurations associated with them is now possible. The kinetic bottlenecks for folding can be determined from the thermal ensembles of structures on the free energy barriers, provided the kinetically determined transition-state ensembles are similar to those determined from free energy barriers. A mechanism incorporating the interplay among backbone ordering, sidechain packing, and desolvation arises from these calculations. Large Phi values arise not only from native contacts, which mostly form at the transition state, but also from contacts already present in the unfolded state that are partially destroyed at the transition.

Fluorine incorporation into gate stacks of advanced silicon memory technologies: Simulation, depth distribution, and reliability

Fluorine segregation influences the intrinsic reliability of thin gate oxides in poly-Si/SiO2/Si structures. We analyze diffusion and segregation kinetics of F in gate stacks with 5 nm gate oxides using secondary ion mass spectroscopy. Well defined doses of F were introduced by ion implantation. We find that F segregation at interfaces to the gate oxide is diffusion limited with an effective activation energy of 1.4 eV. For F concentrations lower than 5×1015 cm−2, F segregation is beneficial improving, for example, the tails of the Weibull plots and slightly increasing the breakdown field. For F doses higher than 1×1016 cm−2, detrimental consequences were found, degrading the charge to breakdown values by about a factor of 5 after long-term thermal treatments. From ab initio density functional theory pseudopotential calculations for atomic structures and total energies of segregated F, we conclude that the segregation to the interface is driven mainly by the energy difference between Si–O and Si–Si bonds in the interfacial region, by oxygen assisted strain relaxation around Si atoms attacked by F and, possibly, by kinetic bottlenecks in diffusion of network oxygen through SiO2.

Isotopic Tracer Studies of Thiophene Desulfurization Reactions Using Hydrogen from Alkanes on H-ZSM5 and Co/H-ZSM5

Reaction pathways for the desulfurization of thiophene using alkanes as hydrogen sources were probed by measuring the chemical and isotopic composition of products formed from 13C-labeled C3H8 and unlabeled C4H4S mixtures on H-ZSM5 and Co/H-ZSM5. Aliphatic hydrocarbons formed only from propane carbon atoms while aromatic molecules contained carbon atoms from both propane and thiophene. Hydrogen-deficient thiophene and thiophene-derived species react with surface hydrogen and alkenes formed from propane and desorb as unreactive aromatics, in steps that lead to the irreversible exit of alkenes from oligomerization-cracking cycles. These steps remove kinetic bottlenecks in thiophene desulfurization resulting from the irreversible formation of strongly adsorbed unsaturated species, which cannot desorb without reactions with hydrogen or hydrogen-rich surface species. This kinetic coupling between alkane and thiophene reactions leads to the observed concurrent increase in the rates of both propane aromatization and thiophene desulfurization compared with those achieved with each pure reactant. The scavenging of unsaturated intermediates formed via thiophene decomposition using hydrogen or hydrogen-rich intermediates formed from propane decreases the rate of bimolecular Diels–Alder reactions, which lead to larger organosulfur compounds and to low H2S selectivity. As a result, H2S selectivities are higher and deactivation rates are lower when propane is present as a co-reactant during reactions of thiophene.

A mechanistic investigation of gelation. The sol-gel polymerization of precursors to bridged polysilsesquioxanes.

The study of a homologous series of precursors to bridged polysilsesquioxanes has uncovered striking discontinuities in gelation behavior. An investigation of the chemistry during the early stages of the polymerization has provided a molecular basis for these observations. Monomers containing from one to four bridging carbon atoms exhibit a pronounced tendency to undergo rapid intra- or bimolecular cyclization. The cyclic and bicyclic intermediates have been characterized by (29)Si NMR spectroscopy, chemical ionization mass spectrometry, and isolation from the reaction solution. These carbosiloxanes are local thermodynamic sinks that produce kinetic bottlenecks in the production of high-molecular-weight silsesquioxanes. The formation of cyclic carbosiloxanes results in slowing, or in some cases completely shutting down, gelation. An additional finding is that the cyclic structures are incorporated intact into the final xerogel.

Folding a protein by discretizing its backbone torsional dynamics.

The aim of this work is to provide a coarse codification of local conformational constraints associated with each folding motif of a peptide chain in order to obtain a rough solution to the protein folding problem. This is accomplished by implementing a discretized version of the soft-mode dynamics on a personal computer (PC). Our algorithm mimics a parallel process as it evaluates concurrent folding possibilities by pattern recognition. It may be implemented in a PC as a sequence of perturbation-translation-renormalization (p-t-r) cycles performed on a matrix of local topological constraints (LTM). This requires suitable representational tools and a periodic quenching of the dynamics required for renormalization. We introduce a description of the peptide chain based on a local discrete variable the values of which label the basins of attraction of the Ramachandran map for each residue. Thus, the local variable indicates the basin in which the torsional coordinates of each residue lie at a given time. In addition, a coding of local topological constraints associated with each secondary and tertiary structural motif is introduced. Our treatment enables us to adopt a computation time step of 81 ps, a value far larger than hydrodynamic drag time scales. Folding pathways are resolved as transitions between patterns of locally encoded structural signals that change within the 10 micros-100 ms time scale range. These coarse folding pathways are generated by the periodic search for structural patterns in the time-evolving LTM. Each pattern is recorded as a contact matrix, an operation subject to a renormalization feedback loop. The validity of our approach is tested vis-a-vis experimentally-probed folding pathways eventually generating tertiary interactions in proteins which recover their active structure under in vitro renaturation conditions. As an illustration, we focus on determining significant folding intermediates and late kinetic bottlenecks that occur within the first 10 ms of the bovine pancreatic trypsin inhibitor renaturation process. The probed cooperativity and nucleation effects, as well as diffusion-collision stabilization of secondary structure are shown to result from the persistence of relatively stable patterns through successive (p-t-r) cycles, thus acting as seeding patterns for further growth or hierarchical development.

Coarsely defined solution to the protein folding problem

In this work we introduce a least-action formulation of the protein folding problem casted within a coarse description of the peptide chain dynamics. Thus, a binary coding of local topological costraints associated to each structural motif is introduced to computationally mimick the long-time dynamics. Folding pathways are resolved as transitions between patterns of locally encoded structural signals. Our variational approach is aimed at identifying the most economic pathway with respect to the stepwise cost in conformational freedom. Our treatment allows us to account for the expediency of the process in proteins effectively capable of in vitro renaturation. We identify the dominant pathway by introducing a coarse version of Lagrangian microscopic dynamics. The coarse folding pathways are generated by a parallel search for structural patterns in a matrix of local topological constraints (LTM) of the chain. Each local topological constraint represents a coarse description of a local torsional state and each pattern is evaluated, translated and finally recorded as a contact matrix (CM), an operation which is subject to a renormalization feedback loop. The renormalization operation periodically introduces long-range correlations on the LTM according to the latest CM generated by translation. Local topological constraints may form consensus regions in portions of the chain which translate as secondary structure motifs or tertiary interactions. Nucleation steps and cooperative effects are accounted for by means of the renormalization operation which warrants the persistence of seeding patterns upon successive LTM evaluations. Relevant folding timescales beyond the realm of molecular-dynamics simulations become accessible through the coarsely codified representation of local torsional constraints. The validity of our approach is tested vis-a-vis experimentally probed folding pathways generating tertiary interactions in proteins which may recover their active structure under in vitro renaturation conditions. We focus on determining significant folding intermediates and the late kinetic bottlenecks which occur within the first 1/100 s of the renaturation process.

Microscopic dynamics from a coarsely defined solution to the protein folding problem

In this work we introduce a least-action formulation of the protein folding problem casted within a coarse description of the soft mode dynamics of the peptide chain. Ultimately, we show that this coarse variational approach can be lifted to yield the microscopic long-time torsional dynamics responsible for the actual folding process. As a first step, a binary coding of local topological constraints associated to each structural motif is introduced to coarsely mimic the long-time dynamics. Folding pathways are initially resolved as transitions between patterns of locally encoded structural signals. Our variational approach is aimed at identifying the most economic pathway with respect to the stepwise cost in conformational freedom. Our treatment allows us to account for the expediency of the process in proteins effectively capable of in vitro renaturation. We identify the dominant pathway by introducing a coarse version of Lagrangian microscopic dynamics. The coarse folding pathways are generated by a parallel search for structural patterns in a matrix of local topological constraints (LTM) of the chain. Each local topological constraint represents a coarse description of a local torsional state and each pattern is evaluated, translated, and finally recorded as a contact matrix (CM), an operation that is subject to a renormalization feedback loop. The renormalization operation periodically introduces long-range correlations on the LTM according to the latest CM generated by translation. Local topological constraints may form consensus regions in portions of the chain that translate as secondary structure motifs or tertiary interactions. Nucleation steps and cooperative effects are accounted for by means of the renormalization operation, which warrants the persistence of seeding patterns upon successive LTM evaluations. Relevant folding time scales beyond the realm of molecular dynamics simulations become accessible through the coarsely codified representation of local torsional constraints. The validity of our approach is tested vis à vis experimentally probed folding pathways generating tertiary interactions in proteins that may recover their active structure under in vitro renaturation conditions. We focus on determining significant folding intermediates and the late kinetic bottlenecks that occur within the first 1100 s of the renaturation process. After the computational accessibility of this coarse solution of the folding problem becomes apparent, we show how to lift our variational problem to microscopic dynamics of the peptide chain. The consistency of our approach is revealed by an actual generation of Newton’s laws at the microscopic level through an inverse projection of the coarse dynamics originally generated through the pattern recognition computation.

Direct electrochemistry of Megasphaera elsdenii iron hydrogenase. Definition of the enzyme's catalytic operating potential and quantitation of the catalytic behaviour over a continuous potential range.

The Fe-hydrogenase from Megasphaera elsdenii undergoes direct electron exchange with glassy carbon electrodes. Cyclic voltammetry defines the catalytic-performance of the enzyme over a continuous but precisely defined range of potentials. In the presence of H2 and protons the bias of the enzyme towards H2 production is readily visualised. Variation of the response with pH indicates that protein ionisations with pK of approximately 6.7 and 8.3 regulate the catalytic activity. Possible origins for these observations in the chemistry of the H2-activating site are discussed. The mid-wave potential of the catalytic response, Emid, is defined as the catalytic operating potential of the enzyme. Under an atmosphere of hydrogen Emid = -421 +/- 10 mV, pH 7 with a variation of -21 +/- 4 mV pH-1, 22 degrees C. Deviation of Emid from the thermodynamic potential of the hydrogen/proton couple reflects the enzyme's influence over the catalysed reaction. Emid is the reduction potential of the H2-activating centre (H-cluster) in the absence of kinetic bottle-necks at other steps in the reaction mechanism.

Cationic polymerization within clusters composed of unsaturated molecules

Abstract Electron impact ionization of large neutral clusters composed of simple olefinic molecules (ethene, 1,1-difluoroethene and propene) display highly anomalous ion intensity distributions. These cluster ion distributions typically exhibit a magic number at n = 4, for cluster ions of the type {M}+, where M is an olefinic molecule. The explanation which best explains this observation is that these small ‘cluster’ ions are in actuality covalently bound molecular ions formed by sequential ion-molecule association (polymerization) reactions which have occurred within the cluster ion. This size dependent cluster chemistry is accounted for by kinetic bottlenecks which occur in the ionic chain growth reactions and which terminate with the formation of cyclic molecular ions (i.e. cyclopentanes and cyclohexanes ions). The condensed phase counterparts of these intracluster reactions are the free cationic polymerization reactions initiated by radiolysis of bulk samples of the olefins. In addition to reviewing our own efforts in this field, we will discuss previous work on cationic polymerization which is relevant to the new chemistry we have observed within clusters of olefinic molecules. We also will present work concerning acetylene and methylacetylene cluster ions where we also observe similar intracluster ion-molecule reactions giving rise to what are most likely benzene and trimethylbenzene ions. Lastly, we will show how mixed cluster of acetylene-acetone also are capable of producing covalently bonded cyclic molecular ions again via intracluster ion-molecule polymerization reactions. We therefore feel that these new cluster condensation reactions constitutes a more general family of chemical reactions for all cluster ions containing unsaturated molecules.

Two-dimensional melting revisited

The two-dimensional melting behaviour of the inverse-twelfth-power system and its dependence on boundary and initial conditions has been re-examined with long molecular dynamics simulation runs. The system consists of 1225 particles, which in a first series of runs is being simulated under standard periodic boundary conditions. It is feasible to reconcile some central simulation results with the experimental data of Murray et al., 1987, Phys. Rev. Lett., 58, 1200 for possible two-stage melting of submicron-sized charged colloidal spheres in liquid water. In another series of simulations, the particles are confined to a roughly rectangular cell with irregularly shaped walls. The energy as a function of density is found to be very sensitive to the choice of boundary conditions. The dependence on the initial conditions is studied by setting up the latter simulations with different sets of starting coordinates—some of them probably those of a hexatic phase—which were directly obtained from Murray et al.'s experimental data for colloidal suspensions. Using the short ranged r -12 potential, molecular dynamics simulations of these configurations were examined over five million time steps each. The initial order, even if experimentally found to be consistent with that of a hexatic phase, is not seen to undergo any rapid breakup. This suggests that kinetic bottlenecks limiting equilibration are still important, even for runs of this length.