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Sample records for mechanical folding kinetics

  1. Kinetic partitioning mechanism of HDV ribozyme folding

    SciTech Connect

    Chen, Jiawen; Gong, Sha; Wang, Yujie; Zhang, Wenbing

    2014-01-14

    RNA folding kinetics is directly tied to RNA biological functions. We introduce here a new approach for predicting the folding kinetics of RNA secondary structure with pseudoknots. This approach is based on our previous established helix-based method for predicting the folding kinetics of RNA secondary structure. In this approach, the transition rates for an elementary step: (1) formation, (2) disruption of a helix stem, and (3) helix formation with concomitant partial melting of an incompatible helix, are calculated with the free energy landscape. The folding kinetics of the Hepatitis delta virus (HDV) ribozyme and the mutated sequences are studied with this method. The folding pathways are identified by recursive searching the states with high net flux-in(out) population starting from the native state. The theory results are in good agreement with that of the experiments. The results indicate that the bi-phasic folding kinetics for the wt HDV sequence is ascribed to the kinetic partitioning mechanism: Part of the population will quickly fold to the native state along the fast pathway, while another part of the population will fold along the slow pathway, in which the population is trapped in a non-native state. Single mutation not only changes the folding rate but also the folding pathway.

  2. Kinetic partitioning mechanism of HDV ribozyme folding.

    PubMed

    Chen, Jiawen; Gong, Sha; Wang, Yujie; Zhang, Wenbing

    2014-01-14

    RNA folding kinetics is directly tied to RNA biological functions. We introduce here a new approach for predicting the folding kinetics of RNA secondary structure with pseudoknots. This approach is based on our previous established helix-based method for predicting the folding kinetics of RNA secondary structure. In this approach, the transition rates for an elementary step: (1) formation, (2) disruption of a helix stem, and (3) helix formation with concomitant partial melting of an incompatible helix, are calculated with the free energy landscape. The folding kinetics of the Hepatitis delta virus (HDV) ribozyme and the mutated sequences are studied with this method. The folding pathways are identified by recursive searching the states with high net flux-in(out) population starting from the native state. The theory results are in good agreement with that of the experiments. The results indicate that the bi-phasic folding kinetics for the wt HDV sequence is ascribed to the kinetic partitioning mechanism: Part of the population will quickly fold to the native state along the fast pathway, while another part of the population will fold along the slow pathway, in which the population is trapped in a non-native state. Single mutation not only changes the folding rate but also the folding pathway.

  3. Kinetic partitioning mechanism of HDV ribozyme folding

    NASA Astrophysics Data System (ADS)

    Chen, Jiawen; Gong, Sha; Wang, Yujie; Zhang, Wenbing

    2014-01-01

    RNA folding kinetics is directly tied to RNA biological functions. We introduce here a new approach for predicting the folding kinetics of RNA secondary structure with pseudoknots. This approach is based on our previous established helix-based method for predicting the folding kinetics of RNA secondary structure. In this approach, the transition rates for an elementary step: (1) formation, (2) disruption of a helix stem, and (3) helix formation with concomitant partial melting of an incompatible helix, are calculated with the free energy landscape. The folding kinetics of the Hepatitis delta virus (HDV) ribozyme and the mutated sequences are studied with this method. The folding pathways are identified by recursive searching the states with high net flux-in(out) population starting from the native state. The theory results are in good agreement with that of the experiments. The results indicate that the bi-phasic folding kinetics for the wt HDV sequence is ascribed to the kinetic partitioning mechanism: Part of the population will quickly fold to the native state along the fast pathway, while another part of the population will fold along the slow pathway, in which the population is trapped in a non-native state. Single mutation not only changes the folding rate but also the folding pathway.

  4. Energy landscapes, folding mechanisms, and kinetics of RNA tetraloop hairpins.

    PubMed

    Chakraborty, Debayan; Collepardo-Guevara, Rosana; Wales, David J

    2014-12-31

    RNA hairpins play a pivotal role in a diverse range of cellular functions, and are integral components of ribozymes, mRNA, and riboswitches. However, the mechanistic and kinetic details of RNA hairpin folding, which are key determinants of most of its biological functions, are poorly understood. In this work, we use the discrete path sampling (DPS) approach to explore the energy landscapes of two RNA tetraloop hairpins, and provide insights into their folding mechanisms and kinetics in atomistic detail. Our results show that the potential energy landscapes have a distinct funnel-like bias toward the folded hairpin state, consistent with efficient structure-seeking properties. Mechanistic and kinetic information is analyzed in terms of kinetic transition networks. We find microsecond folding times, consistent with temperature jump experiments, for hairpin folding initiated from relatively compact unfolded states. This process is essentially driven by an initial collapse, followed by rapid zippering of the helix stem in the final phase. Much lower folding rates are predicted when the folding is initiated from extended chains, which undergo longer excursions on the energy landscape before nucleation events can occur. Our work therefore explains recent experiments and coarse-grained simulations, where the folding kinetics exhibit precisely this dependency on the initial conditions.

  5. Insights into Coupled Folding and Binding Mechanisms from Kinetic Studies*

    PubMed Central

    Crabtree, Michael D.; Dahal, Liza; Wicky, Basile I. M.; Clarke, Jane

    2016-01-01

    Intrinsically disordered proteins (IDPs) are characterized by a lack of persistent structure. Since their identification more than a decade ago, many questions regarding their functional relevance and interaction mechanisms remain unanswered. Although most experiments have taken equilibrium and structural perspectives, fewer studies have investigated the kinetics of their interactions. Here we review and highlight the type of information that can be gained from kinetic studies. In particular, we show how kinetic studies of coupled folding and binding reactions, an important class of signaling event, are needed to determine mechanisms. PMID:26851275

  6. Kinetic Intermediates in RNA Folding

    NASA Astrophysics Data System (ADS)

    Zarrinkar, Patrick P.; Williamson, James R.

    1994-08-01

    The folding pathways of large, highly structured RNA molecules are largely unexplored. Insight into both the kinetics of folding and the presence of intermediates was provided in a study of the Mg2+-induced folding of the Tetrahymena ribozyme by hybridization of complementary oligodeoxynucleotide probes. This RNA folds via a complex mechanism involving both Mg2+-dependent and Mg2+-independent steps. A hierarchical model for the folding pathway is proposed in which formation of one helical domain (P4-P6) precedes that of a second helical domain (P3-P7). The overall rate-limiting step is formation of P3-P7, and takes place with an observed rate constant of 0.72 ± 0.14 minute-1. The folding mechanism of large RNAs appears similar to that of many multidomain proteins in that formation of independently stable substructures precedes their association into the final conformation.

  7. Fast protein folding kinetics.

    PubMed

    Gelman, Hannah; Gruebele, Martin

    2014-05-01

    Fast-folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast-folding proteins has provided insight into the mechanisms, which allow some proteins to find their native conformation well <1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even 'slow' folding processes: fast folders are small; relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast-folding proteins and provides an overview of the major findings of fast-folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general, as well as some work that is left to do.

  8. Fast protein folding kinetics

    PubMed Central

    Gelman, Hannah; Gruebele, Martin

    2014-01-01

    Fast folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast folding proteins has provided insight into the mechanisms which allow some proteins to find their native conformation well less than 1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even “slow” folding processes: fast folders are small, relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast folding proteins and provides an overview of the major findings of fast folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general as well as some work that is left to do. PMID:24641816

  9. Pressure-Jump-Induced Kinetics Reveals a Hydration Dependent Folding/Unfolding Mechanism of Ribonuclease A

    PubMed Central

    Font, J.; Torrent, J.; Ribó, M.; Laurents, D. V.; Balny, C.; Vilanova, M.; Lange, R.

    2006-01-01

    Pressure-jump (p-jump)-induced relaxation kinetics was used to explore the energy landscape of protein folding/unfolding of Y115W, a fluorescent variant of ribonuclease A. Pressure-jumps of 40 MPa amplitude (5 ms dead-time) were conducted both to higher (unfolding) and to lower (folding) pressure, in the range from 100 to 500 MPa, between 30 and 50°C. Significant deviations from the expected symmetrical protein relaxation kinetics were observed. Whereas downward p-jumps resulted always in single exponential kinetics, the kinetics induced by upward p-jumps were biphasic in the low pressure range and monophasic at higher pressures. The relative amplitude of the slow phase decreased as a function of both pressure and temperature. At 50°C, only the fast phase remained. These results can be interpreted within the framework of a two-dimensional energy surface containing a pressure- and temperature-dependent barrier between two unfolded states differing in the isomeric state of the Asn-113–Pro-114 bond. Analysis of the activation volume of the fast kinetic phase revealed a temperature-dependent shift of the unfolding transition state to a larger volume. The observed compensation of this effect by glycerol offers an explanation for its protein stabilizing effect. PMID:16798802

  10. Understanding the mechanical properties of DNA origami tiles and controlling the kinetics of their folding and unfolding reconfiguration.

    PubMed

    Chen, Haorong; Weng, Te-Wei; Riccitelli, Molly M; Cui, Yi; Irudayaraj, Joseph; Choi, Jong Hyun

    2014-05-14

    DNA origami represents a class of highly programmable macromolecules that can go through conformational changes in response to external signals. Here we show that a two-dimensional origami rectangle can be effectively folded into a short, cylindrical tube by connecting the two opposite edges through the hybridization of linker strands and that this process can be efficiently reversed via toehold-mediated strand displacement. The reconfiguration kinetics was experimentally studied as a function of incubation temperature, initial origami concentration, missing staples, and origami geometry. A kinetic model was developed by introducing the j factor to describe the reaction rates in the cyclization process. We found that the cyclization efficiency (j factor) increases sharply with temperature and depends strongly on the structural flexibility and geometry. A simple mechanical model was used to correlate the observed cyclization efficiency with origami structure details. The mechanical analysis suggests two sources of the energy barrier for DNA origami folding: overcoming global twisting and bending the structure into a circular conformation. It also provides the first semiquantitative estimation of the rigidity of DNA interhelix crossovers, an essential element in structural DNA nanotechnology. This work demonstrates efficient DNA origami reconfiguration, advances our understanding of the dynamics and mechanical properties of self-assembled DNA structures, and should be valuable to the field of DNA nanotechnology.

  11. Mechanics of Curved Folds

    NASA Astrophysics Data System (ADS)

    Dias, Marcelo A.; Santangelo, Christian D.

    2011-03-01

    Despite an almost two thousand year history, origami, the art of folding paper, remains a challenge both artistically and scientifically. Traditionally, origami is practiced by folding along straight creases. A whole new set of shapes can be explored, however, if, instead of straight creases, one folds along arbitrary curves. We present a mechanical model for curved fold origami in which the energy of a plastically-deformed crease is balanced by the bending energy of developable regions on either side of the crease. Though geometry requires that a sheet buckle when folded along a closed curve, its shape depends on the elasticity of the sheet. NSF DMR-0846582.

  12. Simple Model of Protein Folding Kinetics

    NASA Astrophysics Data System (ADS)

    Zwanzig, Robert

    1995-10-01

    A simple model of the kinetics of protein folding is presented. The reaction coordinate is the "correctness" of a configuration compared with the native state. The model has a gap in the energy spectrum, a large configurational entropy, a free energy barrier between folded and partially folded states, and a good thermodynamic folding transition. Folding kinetics is described by a master equation. The folding time is estimated by means of a local thermodynamic equilibrium assumption and then is calculated both numerically and analytically by solving the master equation. The folding time has a maximum near the folding transition temperature and can have a minimum at a lower temperature.

  13. RNA folding: structure prediction, folding kinetics and ion electrostatics.

    PubMed

    Tan, Zhijie; Zhang, Wenbing; Shi, Yazhou; Wang, Fenghua

    2015-01-01

    Beyond the "traditional" functions such as gene storage, transport and protein synthesis, recent discoveries reveal that RNAs have important "new" biological functions including the RNA silence and gene regulation of riboswitch. Such functions of noncoding RNAs are strongly coupled to the RNA structures and proper structure change, which naturally leads to the RNA folding problem including structure prediction and folding kinetics. Due to the polyanionic nature of RNAs, RNA folding structure, stability and kinetics are strongly coupled to the ion condition of solution. The main focus of this chapter is to review the recent progress in the three major aspects in RNA folding problem: structure prediction, folding kinetics and ion electrostatics. This chapter will introduce both the recent experimental and theoretical progress, while emphasize the theoretical modelling on the three aspects in RNA folding.

  14. A New Folding Kinetic Mechanism for Human Transthyretin and the Influence of the Amyloidogenic V30M Mutation

    PubMed Central

    Jesus, Catarina S. H.; Almeida, Zaida L.; Vaz, Daniela C.; Faria, Tiago Q.; Brito, Rui M. M.

    2016-01-01

    Protein aggregation into insoluble amyloid fibrils is the hallmark of several neurodegenerative diseases, chief among them Alzheimer’s and Parkinson’s. Although caused by different proteins, these pathologies share some basic molecular mechanisms with familial amyloidotic polyneuropathy (FAP), a rare hereditary neuropathy caused by amyloid formation and deposition by transthyretin (TTR) in the peripheral and autonomic nervous systems. Among the amyloidogenic TTR mutations known, V30M-TTR is the most common in FAP. TTR amyloidogenesis (ATTR) is triggered by tetramer dissociation, followed by partial unfolding and aggregation of the low conformational stability monomers formed. Thus, tetramer dissociation kinetics, monomer conformational stability and competition between refolding and aggregation pathways do play a critical role in ATTR. Here, we propose a new model to analyze the refolding kinetics of WT-TTR and V30M-TTR, showing that at pH and protein concentrations close to physiological, a two-step mechanism with a unimolecular first step followed by a second-order second step adjusts well to the experimental data. Interestingly, although sharing the same kinetic mechanism, V30M-TTR refolds at a much slower rate than WT-TTR, a feature that may favor the formation of transient species leading to kinetic partition into amyloidogenic pathways and, thus, significantly increasing the probability of amyloid formation in vivo. PMID:27589730

  15. The early folding kinetics of apomyoglobin.

    PubMed Central

    Pappu, R. V.; Weaver, D. L.

    1998-01-01

    The folding pathway of apomyoglobin has been experimentally shown to have early kinetic intermediates involving the A, B, G, and H helices. The earliest detected kinetic events occur on a ns to micros time scale. We show that the early folding kinetics of apomyoglobin may be understood as the association of nascent helices through a network of diffusion-collision-coalescence steps G + H <--> GH + A <--> AGH + B <--> ABGH obtained by solving the diffusion-collision model in a chemical kinetics approximation. Our reproduction of the experimental results indicates that the model is a useful way to analyze folding data. One prediction from our fit is that the nascent A and H helices should be relatively more helix-like before coalescence than the other apomyoglobin helices. PMID:9521125

  16. Biphasic folding kinetics of RNA pseudoknots and telomerase RNA activity

    PubMed Central

    Cao, Song; Chen, Shi-Jie

    2007-01-01

    Using a combined master equation and kinetic cluster approach, we investigate RNA pseudoknot folding and unfolding kinetics. The energetic parameters are computed from a recently developed Vfold model for RNA secondary structure and pseudoknot folding thermodynamics. The folding kinetics theory is based on the complete conformational ensemble, including all the native-like and non-native states. The predicted folding and unfolding pathways, activation barriers, Arrhenius plots, and rate-limiting steps lead to several findings. First, for the PK5 pseudoknot, a misfolded 5′ hairpin emerges as a stable kinetic trap in the folding process, and the detrapping from this misfolded state is the rate-limiting step for the overall folding process. The calculated rate constant and activation barrier agree well with the experimental data. Second, as an application of the model, we investigate the kinetic folding pathways for hTR (human Telomerase RNA) pseudoknot. The predicted folding and unfolding pathways not only support the proposed role of conformational switch between hairpin and pseudoknot in hTR activity, but also reveal molecular mechanism for the conformational switch. Furthermore, for an experimentally studied hTR mutation, whose hairpin intermediate is destabilized, the model predicts a long-lived transient hairpin structure, and the switch between the transient hairpin intermediate and the native pseudoknot may be responsible for the observed hTR activity. Such finding would help resolve the apparent contradiction between the observed hTR activity and the absence of a stable hairpin. PMID:17276459

  17. Modelling RNA folding under mechanical tension

    PubMed Central

    VIEREGG, JEFFREY R.; TINOCO, IGNACIO

    2006-01-01

    We investigate the thermodynamics and kinetics of RNA unfolding and refolding under mechanical tension. The hierarchical nature of RNA structure and the existence of thermodynamic parameters for base pair formation based on nearest-neighbour interactions allows modelling of sequence-dependent folding dynamics for any secondary structure. We calculate experimental observables such as the transition force for unfolding, the end-to-end distribution function and its variance, as well as kinetic information, for a representative RNA sequence and for a sequence containing two homopolymer segments: A.U and G.C. PMID:16969426

  18. Protein folding and misfolding: mechanism and principles.

    PubMed

    Englander, S Walter; Mayne, Leland; Krishna, Mallela M G

    2007-11-01

    Two fundamentally different views of how proteins fold are now being debated. Do proteins fold through multiple unpredictable routes directed only by the energetically downhill nature of the folding landscape or do they fold through specific intermediates in a defined pathway that systematically puts predetermined pieces of the target native protein into place? It has now become possible to determine the structure of protein folding intermediates, evaluate their equilibrium and kinetic parameters, and establish their pathway relationships. Results obtained for many proteins have serendipitously revealed a new dimension of protein structure. Cooperative structural units of the native protein, called foldons, unfold and refold repeatedly even under native conditions. Much evidence obtained by hydrogen exchange and other methods now indicates that cooperative foldon units and not individual amino acids account for the unit steps in protein folding pathways. The formation of foldons and their ordered pathway assembly systematically puts native-like foldon building blocks into place, guided by a sequential stabilization mechanism in which prior native-like structure templates the formation of incoming foldons with complementary structure. Thus the same propensities and interactions that specify the final native state, encoded in the amino-acid sequence of every protein, determine the pathway for getting there. Experimental observations that have been interpreted differently, in terms of multiple independent pathways, appear to be due to chance misfolding errors that cause different population fractions to block at different pathway points, populate different pathway intermediates, and fold at different rates. This paper summarizes the experimental basis for these three determining principles and their consequences. Cooperative native-like foldon units and the sequential stabilization process together generate predetermined stepwise pathways. Optional misfolding errors

  19. Thermodynamics and kinetics of protein folding: an evolutionary perspective.

    PubMed

    Demetrius, Lloyd

    2002-08-07

    This article appeals to an evolutionary model which postulates that primordial proteins were described by small polypeptide chains which (i) lack disulfide bridges, and (ii) display slow folding rates with multi-state kinetics, to determine relations between structural properties of proteins and their folding kinetics. We parameterize the energy landscape of proteins in terms of thermodynamic activation variables. The model studies evolutionary changes in these thermodynamic parameters, and we invoke relations between these activation variables and structural properties of the protein to predict the following correspondence between protein structure and folding kinetics. 1. Proteins with inter- and intra-chain disulfide bridges: large variability in both folding rates and stability of intermediates, multi-state kinetics. 2. Proteins which lack inter and intra-chain disulfide bridges. 2.1 Single-domain chains: fast folding rates; unstable intermediates; two-state kinetics. 2.2 Multi-domain monomers: intermediate rates; metastable intermediates; multi-state kinetics. 2.3 Multi-domain oligomers: slow rates; metastable intermediates; multi-state kinetics. The evolutionary model thus provides a kinetic characterization of one important subfamily of proteins which we describe by the following properties: Folding dynamics of single-domain proteins which lack disulfide bridges are described by two-state kinetics. Folding rate of this class of proteins is positively correlated with the thermodynamic stability of the folded state.

  20. Sampling Kinetic Protein Folding Pathways using All-Atom Models

    NASA Astrophysics Data System (ADS)

    Bolhuis, P. G.

    This chapter summarizes several computational strategies to study the kinetics of two-state protein folding using all atom models. After explaining the background of two state folding using energy landscapes I introduce common protein models and computational tools to study folding thermodynamics and kinetics. Free energy landscapes are able to capture the thermodynamics of two-state protein folding, and several methods for efficient sampling of these landscapes are presented. An accurate estimate of folding kinetics, the main topic of this chapter, is more difficult to achieve. I argue that path sampling methods are well suited to overcome the problems connected to the sampling of folding kinetics. Some of the major issues are illustrated in the case study on the folding of the GB1 hairpin.

  1. Osmolyte-induced folding of an intrinsically disordered protein: folding mechanism in the absence of ligand.

    PubMed

    Chang, Yu-Chu; Oas, Terrence G

    2010-06-29

    Understanding the interconversion between thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of the bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in this study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations exhibited multiphasic kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea during the titration to produce a urea-TMAO titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated, and the folding rate constants are relatively slow compared to those of intrinsically folded proteins similar in size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.

  2. Cotranscriptional folding kinetics of ribonucleic acid secondary structures

    NASA Astrophysics Data System (ADS)

    Zhao, Peinan; Zhang, Wenbing; Chen, Shi-Jie

    2011-12-01

    We develop a systematic helix-based computational method to predict RNA folding kinetics during transcription. In our method, the transcription is modeled as stepwise process, where each step is the transcription of a nucleotide. For each step, the kinetics algorithm predicts the population kinetics, transition pathways, folding intermediates, and the transcriptional folding products. The folding pathways, rate constants, and the conformational populations for cotranscription folding show contrastingly different features than the refolding kinetics for a fully transcribed chain. The competition between the transcription speed and rate constants for the transitions between the different nascent structures determines the RNA folding pathway and the end product of folding. For example, fast transcription favors the formation of branch-like structures than rod-like structures and chain elongation in the folding process may reduce the probability of the formation of misfolded structures. Furthermore, good theory-experiment agreements suggest that our method may provide a reliable tool for quantitative prediction for cotranscriptional RNA folding, including the kinetics for the population distribution for the whole conformational ensemble.

  3. Predicting Secondary Structural Folding Kinetics for Nucleic Acids

    PubMed Central

    Zhao, Peinan; Zhang, Wen-Bing; Chen, Shi-Jie

    2010-01-01

    Abstract We report a new computational approach to the prediction of RNA secondary structure folding kinetics. In this approach, each elementary kinetic step is represented as the transformation between two secondary structures that differ by a helix. Based on the free energy landscape analysis, we identify three types of dominant pathways and the rate constants for the kinetic steps: 1), formation; 2), disruption of a helix stem; and 3), helix formation with concomitant partial melting of a competing (incompatible) helix. The third pathway, termed the tunneling pathway, is the low-barrier dominant pathway for the conversion between two incompatible helices. Comparisons with experimental data indicate that this new method is quite reliable in predicting the kinetics for RNA secondary structural folding and structural rearrangements. The approach presented here may provide a robust first step for further systematic development of a predictive theory for the folding kinetics for large RNAs. PMID:20409482

  4. Ligand-Promoted Protein Folding by Biased Kinetic Partitioning

    PubMed Central

    Hingorani, Karan S.; Metcalf, Matthew C.; Deming, Derrick T.; Garman, Scott C.; Powers, Evan T.; Gierasch, Lila M.

    2017-01-01

    Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems. PMID:28218913

  5. A kinetic folding intermediate probed by native state hydrogen exchange.

    PubMed

    Parker, M J; Marqusee, S

    2001-01-19

    Stopped-flow fluorescence studies on the N-terminal domain of rat CD2 (CD2.d1) have demonstrated that folding from the fully denatured state (U) proceeds via the transient accumulation of an apparent intermediate (I) in a so-called burst phase that precedes the rate-limiting transition leading to the native state (N). A previous pH-dependent equilibrium hydrogen exchange (HX) study identified a subset of amides in CD2.d1 which, under EX2 conditions, exchange from N with free energies greater than or equal to the free energy difference between the N and I states calculated from the stopped-flow data. Under EX1 conditions the rates of HX for these amides tend towards an asymptote that matches the global unfolding rate calculated from the stopped-flow data, suggesting that exchange for these amides requires traversing the N-to-I transition state barrier. Exchange for these amides presumably occurs from exchange-competent forms comprising the kinetic burst phase therefore. To explore this idea further, native state HX (NHX) data have been collected for CD2.d1 under EX2 conditions using denaturant concentrations which span either side of the denaturant concentration where, according to the stopped-flow data, the apparent U and I states are iso-energetic. The data fit to a two-component, sub-global (sg)/global (g) NHX mechanism, yielding Delta G and m value parameters (where the m value is a measure of hydrocarbon solvation). Regression analysis demonstrates that the (m(sg), Delta G(sg)) and (m(g), Delta G(g)) values calculated for this subset of amides correspond with those describing the kinetic burst phase transition. This result confirms the ability of the NHX technique to explore the structural and energetic properties of kinetic folding intermediates. Copyright 2001 Academic Press.

  6. Enhanced protein folding by removal of kinetic traps

    NASA Astrophysics Data System (ADS)

    Liu, Yanxin; Chapagain, Prem; Parra, Jose; Gerstman, Bernard

    2007-03-01

    The presence of non-native kinetic traps along the free energy landscape of a protein may significantly lengthen the overall folding time so that the folding process becomes unreliable. We used a computational 3-D lattice model to investigate the free energy landscape of a model alpha helical hairpin peptide. We used two slightly different sequences and show that strategic substitutions of only a few amino acid residues greatly enhance the folding process. These strategic substitutions prevent the formation of long-lived misfolded configurations which not only lengthen the folding time but also may cause unwanted aggregation. Detailed kinetic and thermodynamic analysis was carried out for the folding of these two sequences and the results are consistent with the experimental and molecular dynamics simulations of small helical bundle proteins.

  7. Effects of monovalent cations on folding kinetics of G-quadruplexes.

    PubMed

    You, Jing; Li, Hui; Lu, Xi-Ming; Li, Wei; Wang, Peng-Ye; Dou, Shuo-Xing; Xi, Xu-Guang

    2017-08-31

    G-quadruplexes are special structures existing at the ends of human telomeres, the folding kinetics of which are essential for their functions, such as in the maintenance of genome stability and the protection of chromosome ends. In the present study, we investigated the folding kinetics of G-quadruplex in different monovalent cation environments and determined the detailed kinetic parameters for Na(+)- and K(+)-induced G-quadruplex folding, and for its structural transition from the basket-type Na(+) form to the hybrid-type K(+) form. More interestingly, although Li(+) was often used in previous studies of G-quadruplex folding as a control ion supposed to have no effect, we have found that Li(+) can actually influence the folding kinetics of both Na(+)- and K(+)-induced G-quadruplexes significantly and in different ways, by changing the folding fraction of Na(+)-induced G-quadruplexes and greatly increasing the folding rates of K(+)-induced G-quadruplexes. The present study may shed new light on the roles of monovalent cations in G-quadruplex folding and should be useful for further studies of the underlying folding mechanism. © 2017 The Author(s).

  8. Effects of monovalent cations on folding kinetics of G-quadruplexes

    PubMed Central

    You, Jing; Lu, Xi-Ming; Li, Wei; Wang, Peng-Ye; Xi, Xu-Guang

    2017-01-01

    G-quadruplexes are special structures existing at the ends of human telomeres, the folding kinetics of which are essential for their functions, such as in the maintenance of genome stability and the protection of chromosome ends. In the present study, we investigated the folding kinetics of G-quadruplex in different monovalent cation environments and determined the detailed kinetic parameters for Na+- and K+-induced G-quadruplex folding, and for its structural transition from the basket-type Na+ form to the hybrid-type K+ form. More interestingly, although Li+ was often used in previous studies of G-quadruplex folding as a control ion supposed to have no effect, we have found that Li+ can actually influence the folding kinetics of both Na+- and K+-induced G-quadruplexes significantly and in different ways, by changing the folding fraction of Na+-induced G-quadruplexes and greatly increasing the folding rates of K+-induced G-quadruplexes. The present study may shed new light on the roles of monovalent cations in G-quadruplex folding and should be useful for further studies of the underlying folding mechanism. PMID:28588052

  9. Oxidative folding of peptides with cystine-knot architectures: kinetic studies and optimization of folding conditions.

    PubMed

    Reinwarth, Michael; Glotzbach, Bernhard; Tomaszowski, Michael; Fabritz, Sebastian; Avrutina, Olga; Kolmar, Harald

    2013-01-02

    Bioactive peptides often contain several disulfide bonds that provide the main contribution to conformational rigidity and structural, thermal, or biological stability. Among them, cystine-knot peptides-commonly named "knottins"-make up a subclass with several thousand natural members. Hence, they are considered promising frameworks for peptide-based pharmaceuticals. Although cystine-knot peptides are available through chemical and recombinant synthetic routes, oxidative folding to afford the bioactive isomers still remains a crucial step. We therefore investigated the oxidative folding of ten protease-inhibiting peptides from two knottin families, as well as that of an HIV entry inhibitor and of aprotinin, under two conventional sets of folding conditions and by a newly developed procedure. Kinetic studies identified folding conditions that resulted in correctly folded miniproteins with high rates of conversion even for highly hydrophobic and aggregation-prone peptides in concentrated solutions.

  10. Decoding the folding of Burkholderia glumae lipase: folding intermediates en route to kinetic stability.

    PubMed

    Pauwels, Kris; Sanchez del Pino, Manuel M; Feller, Georges; Van Gelder, Patrick

    2012-01-01

    The lipase produced by Burkholderia glumae folds spontaneously into an inactive near-native state and requires a periplasmic chaperone to reach its final active and secretion-competent fold. The B. glumae lipase-specific foldase (Lif) is classified as a member of the steric-chaperone family of which the propeptides of α-lytic protease and subtilisin are the best known representatives. Steric chaperones play a key role in conferring kinetic stability to proteins. However, until present there was no solid experimental evidence that Lif-dependent lipases are kinetically trapped enzymes. By combining thermal denaturation studies with proteolytic resistance experiments and the description of distinct folding intermediates, we demonstrate that the native lipase has a kinetically stable conformation. We show that a newly discovered molten globule-like conformation has distinct properties that clearly differ from those of the near-native intermediate state. The folding fingerprint of Lif-dependent lipases is put in the context of the protease-prodomain system and the comparison reveals clear differences that render the lipase-Lif systems unique. Limited proteolysis unveils structural differences between the near-native intermediate and the native conformation and sets the stage to shed light onto the nature of the kinetic barrier.

  11. Decoding the Folding of Burkholderia glumae Lipase: Folding Intermediates En Route to Kinetic Stability

    PubMed Central

    Pauwels, Kris; Sanchez del Pino, Manuel M.; Feller, Georges; Van Gelder, Patrick

    2012-01-01

    The lipase produced by Burkholderia glumae folds spontaneously into an inactive near-native state and requires a periplasmic chaperone to reach its final active and secretion-competent fold. The B. glumae lipase-specific foldase (Lif) is classified as a member of the steric-chaperone family of which the propeptides of α-lytic protease and subtilisin are the best known representatives. Steric chaperones play a key role in conferring kinetic stability to proteins. However, until present there was no solid experimental evidence that Lif-dependent lipases are kinetically trapped enzymes. By combining thermal denaturation studies with proteolytic resistance experiments and the description of distinct folding intermediates, we demonstrate that the native lipase has a kinetically stable conformation. We show that a newly discovered molten globule-like conformation has distinct properties that clearly differ from those of the near-native intermediate state. The folding fingerprint of Lif-dependent lipases is put in the context of the protease-prodomain system and the comparison reveals clear differences that render the lipase-Lif systems unique. Limited proteolysis unveils structural differences between the near-native intermediate and the native conformation and sets the stage to shed light onto the nature of the kinetic barrier. PMID:22615867

  12. The folding thermodynamics and kinetics of crambin using an all-atom Monte Carlo simulation.

    PubMed

    Shimada, J; Kussell, E L; Shakhnovich, E I

    2001-04-20

    We present a novel Monte Carlo simulation of protein folding, in which all heavy atoms are represented as interacting hard spheres. This model includes all degrees of freedom relevant to folding, all side-chain and backbone torsions, and uses a Go potential. In this study, we focus on the 46 residue alpha/beta protein crambin and two of its structural components, the helix and helix hairpin. For a wide range of temperatures, we recorded multiple folding events of these three structures from random coils to native conformations that differ by less than 1 A C(alpha) dRMS from their crystal structure coordinates. The thermodynamics and kinetic mechanism of the helix-coil transition obtained from our simulation shows excellent agreement with currently available experimental and molecular dynamics data. Based on insights obtained from folding its smaller structural components, a possible folding mechanism for crambin is proposed. We observed that the folding occurs via a cooperative, first order-like process, and that many folding pathways to the native state exist. One particular sequence of events constitutes a "fast-folding" pathway where kinetic traps are avoided. At very low temperatures, a kinetic trap arising from the incorrect packing of side-chains was observed. These results demonstrate that folding to the native state can be observed in a reasonable amount of time on desktop computers even when an all-atom representation is used, provided the energetics sufficiently stabilize the native state.

  13. Ion specificity in α-helical folding kinetics.

    PubMed

    von Hansen, Yann; Kalcher, Immanuel; Dzubiella, Joachim

    2010-11-04

    The influence of the salts KCl, NaCl, and NaI at molar concentrations on the α-helical folding kinetics of the alanine-based oligopeptide Ace-AEAAAKEAAAKA-Nme is investigated by means of (explicit-water) molecular dynamics simulations and a diffusional analysis. The mean first passage times for folding and unfolding are found to be highly salt-specific. In particular, the folding times increase about 1 order of magnitude for the sodium salts. The drastic slowing can be traced to long-lived, compact configurations of the partially folded peptide, in which sodium ions are tightly bound by several carbonyl and carboxylate groups. This multiple trapping leads to a nonexponential residence time distribution of the cations in the first solvation shell of the peptide. The analysis of α-helical folding in the framework of diffusion in a reduced (one-dimensional) free energy landscape further shows that the salt not only specifically modifies equilibrium properties but also induces kinetic barriers due to individual ion binding. In the sodium salts, for instance, the peptide's configurational mobility (or "diffusivity") can decrease about 1 order of magnitude. This study demonstrates the highly specific action of ions and highlights the intimate coupling of intramolecular friction and solvent effects in protein folding.

  14. Mapping the kinetic barriers of a Large RNA molecule's folding landscape.

    PubMed

    Schlatterer, Jörg C; Martin, Joshua S; Laederach, Alain; Brenowitz, Michael

    2014-01-01

    The folding of linear polymers into discrete three-dimensional structures is often required for biological function. The formation of long-lived intermediates is a hallmark of the folding of large RNA molecules due to the ruggedness of their energy landscapes. The precise thermodynamic nature of the barriers (whether enthalpic or entropic) that leads to intermediate formation is still poorly characterized in large structured RNA molecules. A classic approach to analyzing kinetic barriers are temperature dependent studies analyzed with Eyring's transition state theory. We applied Eyring's theory to time-resolved hydroxyl radical (•OH) footprinting kinetics progress curves collected at eight temperature from 21.5 °C to 51 °C to characterize the thermodynamic nature of folding intermediate formation for the Mg(2+)-mediated folding of the Tetrahymena thermophila group I ribozyme. A common kinetic model configuration describes this RNA folding reaction over the entire temperature range studied consisting of primary (fast) transitions to misfolded intermediates followed by much slower secondary transitions, consistent with previous studies. Eyring analysis reveals that the primary transitions are moderate in magnitude and primarily enthalpic in nature. In contrast, the secondary transitions are daunting in magnitude and entropic in nature. The entropic character of the secondary transitions is consistent with structural rearrangement of the intermediate species to the final folded form. This segregation of kinetic control reveals distinctly different molecular mechanisms during the two stages of RNA folding and documents the importance of entropic barriers to defining rugged RNA folding landscapes.

  15. Mapping the Kinetic Barriers of a Large RNA Molecule's Folding Landscape

    PubMed Central

    Schlatterer, Jörg C.; Martin, Joshua S.; Laederach, Alain; Brenowitz, Michael

    2014-01-01

    The folding of linear polymers into discrete three-dimensional structures is often required for biological function. The formation of long-lived intermediates is a hallmark of the folding of large RNA molecules due to the ruggedness of their energy landscapes. The precise thermodynamic nature of the barriers (whether enthalpic or entropic) that leads to intermediate formation is still poorly characterized in large structured RNA molecules. A classic approach to analyzing kinetic barriers are temperature dependent studies analyzed with Eyring's transition state theory. We applied Eyring's theory to time-resolved hydroxyl radical (•OH) footprinting kinetics progress curves collected at eight temperature from 21.5°C to 51°C to characterize the thermodynamic nature of folding intermediate formation for the Mg2+-mediated folding of the Tetrahymena thermophila group I ribozyme. A common kinetic model configuration describes this RNA folding reaction over the entire temperature range studied consisting of primary (fast) transitions to misfolded intermediates followed by much slower secondary transitions, consistent with previous studies. Eyring analysis reveals that the primary transitions are moderate in magnitude and primarily enthalpic in nature. In contrast, the secondary transitions are daunting in magnitude and entropic in nature. The entropic character of the secondary transitions is consistent with structural rearrangement of the intermediate species to the final folded form. This segregation of kinetic control reveals distinctly different molecular mechanisms during the two stages of RNA folding and documents the importance of entropic barriers to defining rugged RNA folding landscapes. PMID:24586236

  16. Protein Folding and Mechanisms of Proteostasis

    PubMed Central

    Díaz-Villanueva, José Fernando; Díaz-Molina, Raúl; García-González, Victor

    2015-01-01

    Highly sophisticated mechanisms that modulate protein structure and function, which involve synthesis and degradation, have evolved to maintain cellular homeostasis. Perturbations in these mechanisms can lead to protein dysfunction as well as deleterious cell processes. Therefore in recent years the etiology of a great number of diseases has been attributed to failures in mechanisms that modulate protein structure. Interconnections among metabolic and cell signaling pathways are critical for homeostasis to converge on mechanisms associated with protein folding as well as for the preservation of the native structure of proteins. For instance, imbalances in secretory protein synthesis pathways lead to a condition known as endoplasmic reticulum (ER) stress which elicits the adaptive unfolded protein response (UPR). Therefore, taking this into consideration, a key part of this paper is developed around the protein folding phenomenon, and cellular mechanisms which support this pivotal condition. We provide an overview of chaperone protein function, UPR via, spatial compartmentalization of protein folding, proteasome role, autophagy, as well as the intertwining between these processes. Several diseases are known to have a molecular etiology in the malfunction of mechanisms responsible for protein folding and in the shielding of native structure, phenomena which ultimately lead to misfolded protein accumulation. This review centers on our current knowledge about pathways that modulate protein folding, and cell responses involved in protein homeostasis. PMID:26225966

  17. Periodic and stochastic thermal modulation of protein folding kinetics

    SciTech Connect

    Platkov, Max; Gruebele, Martin

    2014-07-21

    Chemical reactions are usually observed either by relaxation of a bulk sample after applying a sudden external perturbation, or by intrinsic fluctuations of a few molecules. Here we show that the two ideas can be combined to measure protein folding kinetics, either by periodic thermal modulation, or by creating artificial thermal noise that greatly exceeds natural thermal fluctuations. We study the folding reaction of the enzyme phosphoglycerate kinase driven by periodic temperature waveforms. As the temperature waveform unfolds and refolds the protein, its fluorescence color changes due to FRET (Förster resonant Energy Transfer) of two donor/acceptor fluorophores labeling the protein. We adapt a simple model of periodically driven kinetics that nicely fits the data at all temperatures and driving frequencies: The phase shifts of the periodic donor and acceptor fluorescence signals as a function of driving frequency reveal reaction rates. We also drive the reaction with stochastic temperature waveforms that produce thermal fluctuations much greater than natural fluctuations in the bulk. Such artificial thermal noise allows the recovery of weak underlying signals due to protein folding kinetics. This opens up the possibility for future detection of a stochastic resonance for protein folding subject to noise with controllable amplitude.

  18. Periodic and stochastic thermal modulation of protein folding kinetics.

    PubMed

    Platkov, Max; Gruebele, Martin

    2014-07-21

    Chemical reactions are usually observed either by relaxation of a bulk sample after applying a sudden external perturbation, or by intrinsic fluctuations of a few molecules. Here we show that the two ideas can be combined to measure protein folding kinetics, either by periodic thermal modulation, or by creating artificial thermal noise that greatly exceeds natural thermal fluctuations. We study the folding reaction of the enzyme phosphoglycerate kinase driven by periodic temperature waveforms. As the temperature waveform unfolds and refolds the protein, its fluorescence color changes due to FRET (Förster resonant Energy Transfer) of two donor/acceptor fluorophores labeling the protein. We adapt a simple model of periodically driven kinetics that nicely fits the data at all temperatures and driving frequencies: The phase shifts of the periodic donor and acceptor fluorescence signals as a function of driving frequency reveal reaction rates. We also drive the reaction with stochastic temperature waveforms that produce thermal fluctuations much greater than natural fluctuations in the bulk. Such artificial thermal noise allows the recovery of weak underlying signals due to protein folding kinetics. This opens up the possibility for future detection of a stochastic resonance for protein folding subject to noise with controllable amplitude.

  19. Mechanical Models of Fault-Related Folding

    SciTech Connect

    Johnson, A. M.

    2003-01-09

    The subject of the proposed research is fault-related folding and ground deformation. The results are relevant to oil-producing structures throughout the world, to understanding of damage that has been observed along and near earthquake ruptures, and to earthquake-producing structures in California and other tectonically-active areas. The objectives of the proposed research were to provide both a unified, mechanical infrastructure for studies of fault-related foldings and to present the results in computer programs that have graphical users interfaces (GUIs) so that structural geologists and geophysicists can model a wide variety of fault-related folds (FaRFs).

  20. Kinetic Monte Carlo method applied to nucleic acid hairpin folding.

    PubMed

    Sauerwine, Ben; Widom, Michael

    2011-12-01

    Kinetic Monte Carlo on coarse-grained systems, such as nucleic acid secondary structure, is advantageous for being able to access behavior at long time scales, even minutes or hours. Transition rates between coarse-grained states depend upon intermediate barriers, which are not directly simulated. We propose an Arrhenius rate model and an intermediate energy model that incorporates the effects of the barrier between simulated states without enlarging the state space itself. Applying our Arrhenius rate model to DNA hairpin folding, we demonstrate improved agreement with experiment compared to the usual kinetic Monte Carlo model. Further improvement results from including rigidity of single-stranded stacking.

  1. Antibody-detected folding: kinetics of surface epitope formation are distinct from other folding phases.

    PubMed Central

    Raman, C. S.; Jemmerson, R.; Nall, B. T.

    2000-01-01

    The rate of macromolecular surface formation in yeast iso-2 cytochrome c and its site-specific mutant, N52I iso-2, has been studied using a monoclonal antibody that recognizes a tertiary epitope including K58 and H39. The results indicate that epitope refolding occurs after fast folding but prior to slow folding, in contrast to horse cytochrome c where surface formation occurs early. The antibody-detected (ad) kinetic phase accompanying epitope formation has k(ad) = 0.2 s(-1) and is approximately 40-fold slower than the fastest detectable event in the folding of yeast iso-2 cytochrome c (k2f approximately 8 s(-1)), but occurs prior to the absorbance- and fluorescence-detected slow folding steps (k1a approximately 0.06 s(-1); k1b approximately 0.09 s(-1)). N5I iso-2 cytochrome c exhibits similar kinetic behavior with respect to epitope formation. A detailed dissection of the mechanistic differences between the folding pathways of horse and yeast cytochromes c identifies possible reasons for the slow surface formation in the latter. Our results suggest that non-native ligation involving H33 or H39 during refolding may slow down the formation of the tertiary epitope in iso-2 cytochrome c. This study illustrates that surface formation can be coupled to early events in protein folding. Thus, the rate of macromolecular surface formation is fine tuned by the residues that make up the surface and the interactions they entertain during refolding. PMID:10739255

  2. Extreme Mechanics: Self-Folding Origami

    NASA Astrophysics Data System (ADS)

    Santangelo, Christian D.

    2017-03-01

    Origami has emerged as a tool for designing three-dimensional structures from flat films. Because they can be fabricated by lithographic or roll-to-roll processing techniques, they have great potential for the manufacture of complicated geometries and devices. This article discusses the mechanics of origami and kirigami with a view toward understanding how to design self-folding origami structures. Whether an origami structure can be made to fold autonomously depends strongly on the geometry and kinematics of the origami fold pattern. This article collects some of the results on origami rigidity into a single framework, and discusses how these aspects affect the foldability of origami. Despite recent progress, most problems in origami and origami design remain completely open.

  3. Kinetics of chain motions within a protein-folding intermediate

    PubMed Central

    Neuweiler, Hannes; Banachewicz, Wiktor; Fersht, Alan R.

    2010-01-01

    Small proteins can fold remarkably rapidly, even in μs. What limits their rate of folding? The Engrailed homeodomain is a particularly well-characterized example, which folds ultrafast via an intermediate, I, of solved structure. It is a puzzle that the helix2-turn-helix3 motif of the 3-helix bundle forms in approximately 2 μs, but the final docking of preformed helix1 in I requires approximately 20 μs. Simulation and structural data suggest that nonnative interactions may slow down helix docking. Here we report the direct measurement of chain motions in I by using photoinduced electron transfer fluorescence-quenching correlation spectroscopy (PET-FCS). We use a mutant that traps I at physiological ionic strength but refolds at higher ionic strength. A single Trp in helix3 quenches the fluorescence of an extrinsic label on contact with it. We placed the label along the sequence to probe segmental chain motions. At high ionic strength, we found two relaxations for all probed positions on the 2- and 20-μs time scale, corresponding to the known folding processes, and a 200-ns phase attributable to loop closure kinetics in the unfolded state. At low ionic strength, we found only the 2-μs and 200-ns phase for labels in the helix2-turn-helix3 motif of I, because the native state is not significantly populated. But for labels in helix1 we observed an additional approximately 10-μs phase showing that it was moving slowly, with a rate constant similar to that for overall folding under native conditions. Folding was rate-limited by chain motions on a rough energy surface where nonnative interactions constrain motion. PMID:21135210

  4. Femtomole Mixer for Microsecond Kinetic Studies of Protein Folding

    PubMed Central

    Hertzog, David E.; Michalet, Xavier; Jäger, Marcus; Kong, Xiangxu; Santiago, Juan G.; Weiss, Shimon; Bakajin, Olgica

    2005-01-01

    We have developed a microfluidic mixer for studying protein folding and other reactions with a mixing time of 8 μs and sample consumption of femtomoles. This device enables us to access conformational changes under conditions far from equilibrium and at previously inaccessible time scales. In this paper, we discuss the design and optimization of the mixer using modeling of convective diffusion phenomena and a characterization of the mixer performance using microparticle image velocimetry, dye quenching, and Förster resonance energy-transfer (FRET) measurements of single-stranded DNA. We also demonstrate the feasibility of measuring fast protein folding kinetics using FRET with acyl-CoA binding protein. PMID:15595857

  5. Microsecond Microfluidic Mixing for Investigation of Protein Folding Kinetics

    SciTech Connect

    Hertzog, D E; Santiago, J G; Bakajin, O

    2005-02-10

    We have developed and characterized a mixer to study the reaction kinetics of protein folding on a microsecond timescale. The mixer uses hydrodynamic focusing of pressure-driven flow in a microfluidic channel to reduce diffusion times as first demonstrated by Knight et al.[1]. Features of the mixer include 1 {micro}s mixing times, sample consumptions of order 1 nl/s, loading sample volumes on the order of microliters, and the ability to manufacture in fused silica for compatibility with most spectroscopic methods.

  6. Microsecond Microfluidic Mixing for Investigation of Protein Folding Kinetics

    SciTech Connect

    Hertzog, D E; Santiago, J G; Bakajin, O

    2003-06-25

    We have developed and characterized a mixer to study the reaction kinetics of protein folding on a microsecond timescale. The mixer uses hydrodynamic focusing of pressure-driven flow in a microfluidic channel to reduce diffusion times as first demonstrated by Knight et al.[1]. Features of the mixer include 1 {micro}s mixing times, sample consumptions of order 1 nl/s, loading sample volumes on the order of microliters, and the ability to manufacture in fused silica for compatibility with most spectroscopic methods.

  7. Folded membrane dialyzer with mechanically sealed edges

    DOEpatents

    Markley, Finley W.

    1976-01-01

    A semipermeable membrane is folded in accordion fashion to form a stack of pleats and the edges are sealed so as to isolate the opposite surfaces of the membrane. The stack is contained within a case that provides ports for flow of blood in contact with one surface of the membrane through channels formed by the pleats and also provides ports for flow of a dialysate through channels formed by the pleats in contact with the other surface of the membrane. The serpentine side edges of the membrane are sealed by a solidified plastic material, whereas effective mechanical means are provided to seal the end edges of the folded membrane. The mechanical means include a clamping strip which biases case sealing flanges into a sealed relationship with end portions of the membrane near the end edges, which portions extend from the stack and between the sealing flanges.

  8. Folding of a model three-helix bundle protein: a thermodynamic and kinetic analysis.

    PubMed

    Zhou, Y; Karplus, M

    1999-11-05

    The kinetics and thermodynamics of an off-lattice model for a three-helix bundle protein are investigated as a function of a bias gap parameter that determines the energy difference between native and non-native contacts. A simple dihedral potential is used to introduce the tendency to form right-handed helices. For each value of the bias parameter, 100 trajectories of up to one microsecond are performed. Such statistically valid sampling of the kinetics is made possible by the use of the discrete molecular dynamics method with square-well interactions. This permits much faster simulations for off-lattice models than do continuous potentials. It is found that major folding pathways can be defined, although ensembles with considerable structural variation are involved. The large gap models generally fold faster than those with a smaller gap. For the large gap models, the kinetic intermediates are non-obligatory, while both obligatory and non-obligatory intermediates are present for small gap models. Certain large gap intermediates have a two-helix microdomain with one helix extended outward (as in domain-swapped dimers); the small gap intermediates have more diverse structures. The importance of studying the kinetic, as well as the thermodynamics, of folding for an understanding of the mechanism is discussed and the relation between kinetic and equilibrium intermediates is examined. It is found that the behavior of this model system has aspects that encompass both the "new" view and the "old" view of protein folding.

  9. Kinetic role of helix caps in protein folding is context-dependent.

    PubMed

    Kapp, Gregory T; Richardson, Jane S; Oas, Terrence G

    2004-04-06

    Secondary structure punctuation through specific backbone and side chain interactions at the beginning and end of alpha-helices has been proposed to play a key role in hierarchical protein folding mechanisms [Baldwin, R. L., and Rose, G. D. (1999) Trends Biochem. Sci. 24, 26-33; Presta, L. G., and Rose, G. D. (1988) Science 240, 1632-1641]. We have made site-specific substitutions in the N- and C-cap motifs of the 5-helix protein monomeric lambda repressor (lambda(6-85)) and have measured the rate constants for folding and unfolding of each variant. The consequences of C-cap changes are strongly context-dependent. When the C-cap was located at the chain terminus, changes had little energetic and no kinetic effect. However, substitutions in a C-cap at the boundary between helix 4 and the subsequent interhelical loop resulted in large changes to the stability and rate constants of the variant, showing a substantial kinetic role for this interior C-cap and suggesting a general kinetic role for interior helix C-caps. Statistical preferences tabulated separately for internal and terminal C-caps also show only weak residue preferences in terminal C-caps. This kinetic distinction between interior and terminal C-caps can explain the discrepancy between the near-absence of stability and kinetic effects seen for C-caps of isolated peptides versus the very strong C-cap effects seen for proteins in statistical sequence preferences and mutational energetics. Introduction of consensus, in-register N-capping motifs resulted in increased stability, accelerated folding, and slower unfolding. The kinetic measurements indicate that some of the new native-state capping interactions remain unformed in the transition state. The accelerated folding rates could result from helix stabilization without invoking a specific role for N-caps in the folding reaction.

  10. Folding thermodynamics and kinetics of imprinted renaturable heteropolymers

    NASA Astrophysics Data System (ADS)

    Pande, V. S.; Grosberg, A. Yu.; Tanaka, T.

    1994-11-01

    Recently, a procedure was suggested to synthesize polymers with characteristics similar to those observed in globular proteins: renaturability and the existence of an ``active site'' capable of specifically recognizing a given target molecule. This procedure was originally studied using a computer simulation of the thermodynamics of lattice 27-mers. This analysis is extended to the thermodynamic study of longer chains (36-mers) and different types of short range interactions. We found, in the best conditions, a 50% success rate of creating renaturable heteropolymers, thus confirming the original results. Folding kinetics as examined by Monte Carlo simulation show that the imprinted sequences can reach the ground state reliably and quickly. Finally, we compare the correlations found in the imprinted sequences with those found in natural proteins. We interpret these results as the confirmation of the efficacy of the polymerization procedure.

  11. Dynamics of protein folding: probing the kinetic network of folding-unfolding transitions with experiment and theory.

    PubMed

    Buchner, Ginka S; Murphy, Ronan D; Buchete, Nicolae-Viorel; Kubelka, Jan

    2011-08-01

    The problem of spontaneous folding of amino acid chains into highly organized, biologically functional three-dimensional protein structures continues to challenge the modern science. Understanding how proteins fold requires characterization of the underlying energy landscapes as well as the dynamics of the polypeptide chains in all stages of the folding process. In recent years, important advances toward these goals have been achieved owing to the rapidly growing interdisciplinary interest and significant progress in both experimental techniques and theoretical methods. Improvements in the experimental time resolution led to determination of the timescales of the important elementary events in folding, such as formation of secondary structure and tertiary contacts. Sensitive single molecule methods made possible probing the distributions of the unfolded and folded states and following the folding reaction of individual protein molecules. Discovery of proteins that fold in microseconds opened the possibility of atomic-level theoretical simulations of folding and their direct comparisons with experimental data, as well as of direct experimental observation of the barrier-less folding transition. The ultra-fast folding also brought new questions, concerning the intrinsic limits of the folding rates and experimental signatures of barrier-less "downhill" folding. These problems will require novel approaches for even more detailed experimental investigations of the folding dynamics as well as for the analysis of the folding kinetic data. For theoretical simulations of folding, a main challenge is how to extract the relevant information from overwhelmingly detailed atomistic trajectories. New theoretical methods have been devised to allow a systematic approach towards a quantitative analysis of the kinetic network of folding-unfolding transitions between various configuration states of a protein, revealing the transition states and the associated folding pathways at

  12. Evolution of Enzyme Kinetic Mechanisms.

    PubMed

    Ulusu, Nuriye Nuray

    2015-06-01

    This review paper discusses the reciprocal kinetic behaviours of enzymes and the evolution of structure-function dichotomy. Kinetic mechanisms have evolved in response to alterations in ecological and metabolic conditions. The kinetic mechanisms of single-substrate mono-substrate enzyme reactions are easier to understand and much simpler than those of bi-bi substrate enzyme reactions. The increasing complexities of kinetic mechanisms, as well as the increasing number of enzyme subunits, can be used to shed light on the evolution of kinetic mechanisms. Enzymes with heterogeneous kinetic mechanisms attempt to achieve specific products to subsist. In many organisms, kinetic mechanisms have evolved to aid survival in response to changing environmental factors. Enzyme promiscuity is defined as adaptation to changing environmental conditions, such as the introduction of a toxin or a new carbon source. Enzyme promiscuity is defined as adaptation to changing environmental conditions, such as the introduction of a toxin or a new carbon source. Enzymes with broad substrate specificity and promiscuous properties are believed to be more evolved than single-substrate enzymes. This group of enzymes can adapt to changing environmental substrate conditions and adjust catalysing mechanisms according to the substrate's properties, and their kinetic mechanisms have evolved in response to substrate variability.

  13. RNA folding pathways and kinetics using 2D energy landscapes.

    PubMed

    Senter, Evan; Dotu, Ivan; Clote, Peter

    2015-01-01

    RNA folding pathways play an important role in various biological processes, such as (i) the hok/sok (host-killing/suppression of killing) system in E. coli to check for sufficient plasmid copy number, (ii) the conformational switch in spliced leader (SL) RNA from Leptomonas collosoma, which controls trans splicing of a portion of the '5 exon, and (iii) riboswitches--portions of the 5' untranslated region of messenger RNA that regulate genes by allostery. Since RNA folding pathways are determined by the energy landscape, we describe a novel algorithm, FFTbor2D, which computes the 2D projection of the energy landscape for a given RNA sequence. Given two metastable secondary structures A, B for a given RNA sequence, FFTbor2D computes the Boltzmann probability p(x, y) = Z(x,y)/Z that a secondary structure has base pair distance x from A and distance y from B. Using polynomial interpolationwith the fast Fourier transform,we compute p(x, y) in O(n(5)) time and O(n(2)) space, which is an improvement over an earlier method, which runs in O(n(7)) time and O(n(4)) space. FFTbor2D has potential applications in synthetic biology, where one might wish to design bistable switches having target metastable structures A, B with favorable pathway kinetics. By inverting the transition probability matrix determined from FFTbor2D output, we show that L. collosoma spliced leader RNA has larger mean first passage time from A to B on the 2D energy landscape, than 97.145% of 20,000 sequences, each having metastable structures A, B. Source code and binaries are freely available for download at http://bioinformatics.bc.edu/clotelab/FFTbor2D. The program FFTbor2D is implemented in C++, with optional OpenMP parallelization primitives.

  14. When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches

    PubMed Central

    Muñoz, Victor; Cerminara, Michele

    2016-01-01

    Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico. All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats. PMID:27574021

  15. When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches.

    PubMed

    Muñoz, Victor; Cerminara, Michele

    2016-09-01

    Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats.

  16. Perturbations of the denatured state ensemble: modeling their effects on protein stability and folding kinetics.

    PubMed Central

    Wrabl, J. O.; Shortle, D.

    1996-01-01

    By considering the denatured state of a protein as an ensemble of conformations with varying numbers of sequence-specific interactions, the effects on stability, folding kinetics, and aggregation of perturbing these interactions can be predicted from changes in the molecular partition function. From general considerations, the following conclusions are drawn: (1) A perturbation that enhances a native interaction in denatured state conformations always increases the stability of the native state. (2) A perturbation that promotes a non-native interaction in the denatured state always decreases the stability of the native state. (3) A change in the denatured state ensemble can alter the kinetics of aggregation and folding. (4) The loss (or increase) in stability accompanying two mutations, each of which lowers (or raises) the free energy of the denatured state, will be less than the sum of the effects of the single mutations, except in cases where both mutations affect the same set of partially folded conformations. By modeling the denatured state as the ensemble of all non-native conformations of hydrophobic-polar (HP) chains configured on a square lattice, it can be shown that the stabilization obtained from enhancement of native interactions derives in large measure from the avoidance of non-native interactions in the D state. In addition, the kinetic effects of fixing single native contacts in the denatured state or imposing linear gradients in the HH contact probabilities are found, for some sequences, to significantly enhance the efficiency of folding by a simple hydrophobic zippering algorithm. Again, the dominant mechanism appears to be avoidance of non-native interactions. These results suggest stabilization of native interactions and imposition of gradients in the stability of local structure are two plausible mechanisms involving the denatured state that could play a role in the evolution of protein folding and stability. PMID:8931153

  17. Thermal and mechanical multistate folding of ribonuclease H.

    PubMed

    Schmitt, Terry J; Clark, Jonathan E; Knotts, Thomas A

    2009-12-21

    Two different classes of experimental techniques exist by which protein folding mechanisms are ascertained. The first class, of which circular dichroism is an example, probes thermally-induced folding. The second class, which includes atomic force microscopy and optical tweezers, measures mechanically-induced folding. In this article, we investigate if proteins fold/unfold via the same mechanisms both thermally and mechanically. We do so using Ribonuclease H, a protein that has been shown to fold through a three-state mechanism using both types of experimental techniques. A detailed, molecular-level description of the states involved in thermal and mechanical folding shows that mechanisms for both types are globally similar, but small difference exist in the most unfolded conformations. Comparison to previous work suggests a universal folding behavior for proteins with a core helical bundle.

  18. Thermal and mechanical multistate folding of ribonuclease H

    NASA Astrophysics Data System (ADS)

    Schmitt, Terry J.; Clark, Jonathan E.; Knotts, Thomas A.

    2009-12-01

    Two different classes of experimental techniques exist by which protein folding mechanisms are ascertained. The first class, of which circular dichroism is an example, probes thermally-induced folding. The second class, which includes atomic force microscopy and optical tweezers, measures mechanically-induced folding. In this article, we investigate if proteins fold/unfold via the same mechanisms both thermally and mechanically. We do so using Ribonuclease H, a protein that has been shown to fold through a three-state mechanism using both types of experimental techniques. A detailed, molecular-level description of the states involved in thermal and mechanical folding shows that mechanisms for both types are globally similar, but small difference exist in the most unfolded conformations. Comparison to previous work suggests a universal folding behavior for proteins with a core helical bundle.

  19. Coupled folding and binding kinetics in the intrinsically disordered peptide IA3

    NASA Astrophysics Data System (ADS)

    Narayanan, Ranjani; Ganesh, Omjoy; Edison, Arthur; Hagen, Stephen

    2008-03-01

    IA3 is an intrinsically disordered 68 residue peptide and is an endogenous inhibitor of yeast proteinase A (YPrA). X-ray crystallography of the IA3.YPrA complex [Li et al, Nat. Struct. Biol. (7), 113-117 (2000)] indicates that the N-terminus of IA3 adopts an alpha-helical fold when it is bound to the YPrA active site. We have used equilibrium circular dichroism and multi-wavelength, nanosecond time-resolved laser temperature-jump spectroscopy to study the coupled folding and binding interaction of IA3 with YPrA. Our initial measurements of the rate of helix formation in free IA3 indicate mono-exponential folding kinetics that extrapolate to kF˜ 10^5/s at room temperature in aqueous solutions. By comparing this rate to the kinetics we observe for IA3 interacting with YPrA, we can assess possible mechanisms for the coupled folding and binding of IA3.

  20. Kinetic evidence of an on-pathway intermediate in the folding of lysozyme.

    PubMed Central

    Bai, Y.

    2000-01-01

    By means of a kinetic test, it was demonstrated that one of the folding intermediates (Ialpha) of hen lysozyme with alpha-domain folded and beta-domain unfolded is on the folding pathway under the classical definition. Ialpha folds to the native (N) state directly (unfolded (U) <==> Ialpha <==> N) without having to unfold to U and then refold to N through alternative folding pathways as in Ialpha <==> U <==> N. PMID:10739262

  1. Protein folding kinetics: barrier effects in chemical and thermal denaturation experiments

    PubMed Central

    Naganathan, Athi N.; Doshi, Urmi; Muñoz, Victor

    2008-01-01

    Recent experimental work on fast protein folding brings about an intriguing paradox. Microsecond-folding proteins are supposed to fold near or at the folding speed limit (downhill folding), but yet their folding behavior seems to comply with classical two-state analyses, which imply the crossing of high free energy barriers. However, close inspection of chemical and thermal denaturation kinetic experiments in fast-folding proteins reveals systematic deviations from two-state behavior. Using a simple one-dimensional free energy surface approach we find that such deviations are indeed diagnostic of marginal folding barriers. Furthermore, the quantitative analysis of available fast-kinetic data indicates that many microsecond-folding proteins fold downhill in native conditions. All of these proteins are then promising candidates for an atom-by-atom analysis of protein folding using nuclear magnetic resonance1. We also find that the diffusion coefficient for protein folding is strongly temperature dependent, corresponding to an activation energy of ~1 kJ.mol−1 per protein residue. As a consequence, the folding speed limit at room temperature is about an order of magnitude slower than the ~ 1μs estimates from high temperature T-jump experiments. Our analysis is quantitatively consistent with the available thermodynamic and kinetic data on two-state folding proteins, and provides a straightforward explanation for the apparent fast-folding paradox. PMID:17419630

  2. Folding mechanism of a multiple independently-folding domain protein: double B domain of protein A.

    PubMed

    Arora, Pooja; Hammes, Gordon G; Oas, Terrence G

    2006-10-10

    The antibody binding properties of staphylococcal protein A (SpA) can be attributed to the presence of five highly homologous domains (E, D, A, B, and C). Although the folding of the B domain of protein A (BdpA) is well-characterized, the folding behavior of this domain in the context of full-length SpA in the cell remains unexplored. The sequence of the B domain is 89 and 91% identical to those of domains A and C, respectively. We have fused B domain sequences (BBdpA) as a close approximation of the A-B or B-C portion of SpA. Circular dichroism and fluorescence-detected denaturation curves of BBdpA are experimentally indistinguishable from those of BdpA. The rate constants for folding and unfolding from NMR line shape analysis for the single- and double-domain proteins are the same within experimental uncertainties (+/-20%). These results support the designation of SpA as a multiple independently-folding domain (MIFD) protein. We develop a mathematical model that describes the folding thermodynamics and kinetics of MIFD proteins. The model depicts MIFD protein folding and unfolding as a parallel network and explicitly calculates the flux through all parallel pathways. These fluxes are combined to give a complete description of the global thermodynamics and kinetics of the folding and unfolding of MIFD proteins. The global rates for complete folding and unfolding of a MIFD protein and those of the individual domains depend on the stability of the protein. We show that the global unfolding rate of a MIFD protein may be many orders of magnitude slower than that of the constituent domains.

  3. Monitoring the folding kinetics of a β-hairpin by time-resolved IR spectroscopy in silico.

    PubMed

    Daidone, Isabella; Thukral, Lipi; Smith, Jeremy C; Amadei, Andrea

    2015-04-09

    Protein folding is one of the most fundamental problems in modern biochemistry. Time-resolved infrared (IR) spectroscopy in the amide I region is commonly used to monitor folding kinetics. However, associated atomic detail information on the folding mechanism requires simulations. In atomistic simulations structural order parameters are typically used to follow the folding process along the simulated trajectories. However, a rigorous test of the reliability of the mechanisms found in the simulations requires calculation of the time-dependent experimental observable, i.e., in the present case the IR signal in the amide I region. Here, we combine molecular dynamics simulation with a mixed quantum mechanics/molecular mechanics theoretical methodology, the Perturbed Matrix Method, in order to characterize the folding of a β-hairpin peptide, through modeling the time-dependence of the amide I IR signal. The kinetic and thermodynamic data (folding and unfolding rate constants, and equilibrium folded- and unfolded-state probabilities) obtained from the fit of the calculated signal are in good agreement with the available experimental data [Xu et al. J. Am. Chem. Soc. 2003, 125, 15388-15394]. To the best of our knowledge, this is the first report of the simulation of the time-resolved IR signal of a complex process occurring on a long (microsecond) time scale.

  4. Protein folding funnels: a kinetic approach to the sequence-structure relationship.

    PubMed

    Leopold, P E; Montal, M; Onuchic, J N

    1992-09-15

    A lattice model of protein folding is developed to distinguish between amino acid sequences that do and do not fold into unique conformations. Although Monte Carlo simulations provide insights into the long-time processes involved in protein folding, these simulations cannot systematically chart the conformational energy surface that enables folding. By assuming that protein folding occurs after chain collapse, a kinetic map of important pathways on this surface is constructed through the use of an analytical theory of probability flow. Convergent kinetic pathways, or "folding funnels," guide folding to a unique, stable, native conformation. Solution of the probability flow equations is facilitated by limiting treatment to diffusion between geometrically similar collapsed conformers. Similarity is measured in terms of a reconfigurational distance. Two specific amino acid sequences are deemed foldable and nonfoldable because one gives rise to a single, large folding funnel leading to a native conformation and the other has multiple pathways leading to several stable conformers. Monte Carlo simulations demonstrate that folding funnel calculations accurately predict the fact of and the pathways involved in folding-specific sequences. The existence of folding funnels for specific sequences suggests that geometrically related families of stable, collapsed conformers fulfill kinetic and thermodynamic requirements of protein folding.

  5. Folding kinetics of WW domains with the united residue force field for bridging microscopic motions and experimental measurements

    PubMed Central

    Zhou, Rui; Maisuradze, Gia G.; Suñol, David; Todorovski, Toni; Macias, Maria J.; Xiao, Yi; Scheraga, Harold A.; Czaplewski, Cezary; Liwo, Adam

    2014-01-01

    To demonstrate the utility of the coarse-grained united-residue (UNRES) force field to compare experimental and computed kinetic data for folding proteins, we have performed long-time millisecond-timescale canonical Langevin molecular dynamics simulations of the triple β-strand from the Formin binding protein 28 WW domain and six nonnatural variants, using UNRES. The results have been compared with available experimental data in both a qualitative and a quantitative manner. Complexities of the folding pathways, which cannot be determined experimentally, were revealed. The folding mechanisms obtained from the simulated folding kinetics are in agreement with experimental results, with a few discrepancies for which we have accounted. The origins of single- and double-exponential kinetics and their correlations with two- and three-state folding scenarios are shown to be related to the relative barrier heights between the various states. The rate constants obtained from time profiles of the fractions of the native, intermediate, and unfolded structures, and the kinetic equations fitted to them, correlate with the experimental values; however, they are about three orders of magnitude larger than the experimental ones for most of the systems. These differences are in agreement with the timescale extension derived by scaling down the friction of water and averaging out the fast degrees of freedom when passing from all-atom to a coarse-grained representation. Our results indicate that the UNRES force field can provide accurate predictions of folding kinetics of these WW domains, often used as models for the study of the mechanisms of proein folding. PMID:25489078

  6. Single-molecule kinetics under force: probing protein folding and enzymatic activity with optical tweezers

    NASA Astrophysics Data System (ADS)

    Wong, Wesley

    2010-03-01

    Weak non-covalent bonds between and within single molecules govern many aspects of biological structure and function (e.g. DNA base-paring, receptor-ligand binding, protein folding, etc.) In living systems, these interactions are often subject to mechanical forces, which can greatly alter their kinetics and activity. My group develops and applies novel single-molecule manipulation techniques to explore and quantify these force-dependent kinetics. Using optical tweezers, we have quantified the force-dependent unfolding and refolding kinetics of different proteins, including the cytoskeletal protein spectrin in collaboration with E. Evans's group [1], and the A2 domain of the von Willebrand factor blood clotting protein in collaboration with T. Springer's group [2]. Furthermore, we have studied the kinetics of the ADAMTS13 enzyme acting on a single A2 domain, and have shown that physiolgical forces in the circulation can act as a cofactor for enzymatic cleavage, regulating hemostatic activity [2]. References: 1. E. Evans, K. Halvorsen, K. Kinoshita, and W.P. Wong, Handbook of Single Molecule Biophysics, P. Hinterdorfer, ed., Springer (2009). 2. X. Zhang, K. Halvorsen, C.-Z. Zhang, W.P. Wong, and T.A. Springer, Science 324 (5932), 1330-1334 (2009).

  7. Folding Kinetics of Staphylococcal Nuclease Studied by Tryptophan Engineering and Rapid Mixing Methods

    PubMed Central

    Maki, Kosuke; Cheng, Hong; Dolgikh, Dimitry A.; Roder, Heinrich

    2007-01-01

    To monitor the development of tertiary structural contacts during folding, a unique tryptophan residue was introduced at seven partially buried locations (residues 15, 27, 61, 76, 91, 102 and 121) of a tryptophan-free variant of staphylococcal nuclease (P47G/P117G/H124L/W140H). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side chains that act as intramolecular quenchers. All other variants exhibit enhanced fluorescence under native conditions consistent with burial of the tryptophans in an apolar environment. The kinetics of folding was observed by continuous- and stopped-flow fluorescence measurements over refolding times ranging from 100 μs to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophans in the β-barrel and the N-terminal part of the α-helical domain become partially shielded from the solvent at an early stage (< 1 ms), indicating that this region of the protein undergoes a rapid specific collapse and remains uncoupled from the rest of the α-helical domain until the late stages of folding. For several variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophans reach their buried native environment only during the late stages of folding. Other variants show more complex behavior with a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate

  8. In vitro refolding of human proinsulin. Kinetic intermediates, putative disulfide-forming pathway folding initiation site, and potential role of C-peptide in folding process.

    PubMed

    Qiao, Zhi-Song; Min, Cheng-Yin; Hua, Qing-Xin; Weiss, Michael A; Feng, You-Min

    2003-05-16

    Human insulin is a double-chain peptide that is synthesized in vivo as a single-chain human proinsulin (HPI). We have investigated the disulfide-forming pathway of a single-chain porcine insulin precursor (PIP). Here we further studied the folding pathway of HPI in vitro. While the oxidized refolding process of HPI was quenched, four obvious intermediates (namely P1, P2, P3, and P4, respectively) with three disulfide bridges were isolated and characterized. Contrary to the folding pathway of PIP, no intermediates with one- or two-disulfide bonds could be captured under different refolding conditions. CD analysis showed that P1, P2, and P3 retained partially structural conformations, whereas P4 contained little secondary structure. Based on the time-dependent distribution, disulfide pair analysis, and disulfide-reshuffling process of the intermediates, we have proposed that the folding pathway of HPI is significantly different from that of PIP. These differences reveal that the C-peptide not only facilitates the folding of HPI but also governs its kinetic folding pathway of HPI. Detailed analysis of the molecular folding process reveals that there are some similar folding mechanisms between PIP and HPI. These similarities imply that the initiation site for the folding of PIP/HPI may reside in the central alpha-helix of the B-chain. The formation of disulfide A20-B19 may guide the transfer of the folding information from the B-chain template to the unstructured A-chain. Furthermore, the implications of this in vitro refolding study on the in vivo folding process of HPI have been discussed.

  9. Solvent-Exposed Salt Bridges Influence the Kinetics of α-Helix Folding and Unfolding.

    PubMed

    Meuzelaar, Heleen; Tros, Martijn; Huerta-Viga, Adriana; van Dijk, Chris N; Vreede, Jocelyne; Woutersen, Sander

    2014-03-06

    Salt bridges are known to play an essential role in the thermodynamic stability of the folded conformation of many proteins, but their influence on the kinetics of folding remains largely unknown. Here, we investigate the effect of Glu-Arg salt bridges on the kinetics of α-helix folding using temperature-jump transient-infrared spectroscopy and steady-state UV circular dichroism. We find that geometrically optimized salt bridges (Glu(-) and Arg(+) are spaced four peptide units apart, and the Glu/Arg order is such that the side-chain rotameric preferences favor salt-bridge formation) significantly speed up folding and slow down unfolding, whereas salt bridges with unfavorable geometry slow down folding and slightly speed up unfolding. Our observations suggest a possible explanation for the surprising fact that many biologically active proteins contain salt bridges that do not stabilize the native conformation: these salt bridges might have a kinetic rather than a thermodynamic function.

  10. Solvent-Exposed Salt Bridges Influence the Kinetics of α-Helix Folding and Unfolding

    PubMed Central

    2014-01-01

    Salt bridges are known to play an essential role in the thermodynamic stability of the folded conformation of many proteins, but their influence on the kinetics of folding remains largely unknown. Here, we investigate the effect of Glu-Arg salt bridges on the kinetics of α-helix folding using temperature-jump transient-infrared spectroscopy and steady-state UV circular dichroism. We find that geometrically optimized salt bridges (Glu– and Arg+ are spaced four peptide units apart, and the Glu/Arg order is such that the side-chain rotameric preferences favor salt-bridge formation) significantly speed up folding and slow down unfolding, whereas salt bridges with unfavorable geometry slow down folding and slightly speed up unfolding. Our observations suggest a possible explanation for the surprising fact that many biologically active proteins contain salt bridges that do not stabilize the native conformation: these salt bridges might have a kinetic rather than a thermodynamic function. PMID:24634715

  11. Insights into fold growth using fold-related joint patterns and mechanical stratigraphy

    NASA Astrophysics Data System (ADS)

    Savage, Heather M.; Ryan Shackleton, J.; Cooke, Michele L.; Riedel, Jeffrey J.

    2010-10-01

    Despite how common folds are as structural features, along-strike fold propagation has proven elusive to document. However, if a fold grows laterally along its axis, early-formed fold-related joints may differ significantly in orientation from joints that form later. In this paper, we integrate mechanical stratigraphy with joint pattern analysis to determine relative timing of jointing. Additionally, we demonstrate that joint patterns can be related to stresses on both the top and bottom of the bed during flexure. We present joint data from eight sedimentary beds on the fold terminus at Sheep Mountain Anticline, Wyoming, USA. The joint patterns around the terminus show two distinct patterns: joints in six of the beds show a radial pattern around the terminus whereas joint patterns in the two remaining beds differ from proximal units, despite being in the same structural position. Fracture resistance calculations confirm that the beds with mis-oriented fractures are less resistant to fracturing than other units in the study, and therefore would have fractured earlier in fold growth history. We present a plate bending model that illustrates potential joint patterns around a plunging fold nose from stresses along both the top and bottom of the bed. The joint strike predictions for the area in front of the inflection line on the fold nose match the orientations in our less resistant beds, which are now positioned behind the inflection line, suggesting that the fold grew laterally. The combined analysis of fracture pattern and mechanical stratigraphy provides a new way to investigate fold evolution.

  12. Modelling proteins: conformational sampling and reconstruction of folding kinetics.

    PubMed

    Klenin, Konstantin; Strodel, Birgit; Wales, David J; Wenzel, Wolfgang

    2011-08-01

    In the last decades biomolecular simulation has made tremendous inroads to help elucidate biomolecular processes in-silico. Despite enormous advances in molecular dynamics techniques and the available computational power, many problems involve long time scales and large-scale molecular rearrangements that are still difficult to sample adequately. In this review we therefore summarise recent efforts to fundamentally improve this situation by decoupling the sampling of the energy landscape from the description of the kinetics of the process. Recent years have seen the emergence of many advanced sampling techniques, which permit efficient characterisation of the relevant family of molecular conformations by dispensing with the details of the short-term kinetics of the process. Because these methods generate thermodynamic information at best, they must be complemented by techniques to reconstruct the kinetics of the process using the ensemble of relevant conformations. Here we review recent advances for both types of methods and discuss their perspectives to permit efficient and accurate modelling of large-scale conformational changes in biomolecules. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. Mechanical Folding and Unfolding of Protein Barnase at the Single-Molecule Level

    PubMed Central

    Alemany, Anna; Rey-Serra, Blanca; Frutos, Silvia; Cecconi, Ciro; Ritort, Felix

    2016-01-01

    The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, ΔG0. The estimated value of ΔG0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale. PMID:26745410

  14. Mechanical development of folded chert beds in Monterey Formation, California

    SciTech Connect

    Crowther, D.; Snyder, W.S.

    1988-03-01

    Small-scale folds in the upper siliceous facies of the Miocene Monterey Formation, at Lions Head, California (Santa Maria basin) are of tectonic origin. Folding is well developed in the chert-dominated zones and dies out rapidly in the adjacent siliceous mudstones. A tectonic origin is evidenced by the dominantly brittle deformation of the competent chert layers. Mechanically, the folds formed through a complex interrelationship between fracture and flexural slip. Opal-CT and quartz-chert layers display brittle fractures and rotated fracture blocks that responded to shortening. Thrusting of the chert layers is common in folds where fold propagation was impeded. Dilation breccia and void space occur in the hinges and reflect room problems during development of these disharmonic folds. Subsequent diagenesis has partially healed the fractures and slip surfaces, creating the erroneous appearance that ductile deformation was an important factor in the formation of the folds.

  15. The folding kinetics of the SDS-induced molten globule form of reduced cytochrome c.

    PubMed

    Chen, Eefei; Van Vranken, Vanessa; Kliger, David S

    2008-05-13

    The folding of reduced cytochrome c (redcyt c) is increasingly being recognized as undergoing a mechanism that deviates from a two-state process. In previous far-UV TRORD studies of redcyt c folding, a rapidly forming intermediate was attributed to the appearance of a molten-globule-like (MG) state [Chen, E., Goldbeck, R. A., and Kliger, D. S. (2003) J. Phys. Chem. A 107, 8149-8155]. A slow folding phase (>1 ms) was identified with the formation of native (N) secondary structure from that MG form. Here, using 0.65 mM SDS to induce the MG state in oxidized cytochrome c, folding of redcyt c was triggered with fast photoreduction and probed from early microseconds to milliseconds using far-UV TRORD spectroscopy. The kinetics of the reaction are described with a time constant of 50 +/- 16 ms, which corresponds to 1 +/- 0.6 ms upon extrapolation of the data to zero SDS concentration. The latter folding time is about 5 times faster than the calculated GuHCl-free time constant of 5.5 +/- 1.4 ms for slow-phase folding obtained in our previous study. This ratio of rates would be consistent with a scenario in which 20-30% MG that is suggested to form in the fast phase of redcyt c folding in GuHCl is an obligatory intermediate. The native state forms from this obligatory intermediate with an observed rate, k(f) = fk(G-->N) where f is the fractional population of MG and k(G-->N) is the microscopic rate for MG --> N. Calculation and comparison of the m(#)/m values show agreement within the uncertainties between the SDS ( approximately 0.5) and GuHCl ( approximately 0.3) based redcyt c folding experiments, suggesting that the two experiments report on comparable intermediates. The m values were obtained from far-UV CD SDS titration experiments, from which calculated thermodynamic parameters allowed estimation of the reduction potential for the MG state to be approximately 155 mV (-15 kJ/mol) vs NHE which, like the reduction potential for the native state, is more favorable than

  16. Buffed energy landscapes: another solution to the kinetic paradoxes of protein folding.

    PubMed

    Plotkin, Steven S; Wolynes, Peter G

    2003-04-15

    The energy landscapes of proteins have evolved to be different from most random heteropolymers. Many studies have concluded that evolutionary selection for rapid and reliable folding to a given structure that is stable at biological temperatures leads to energy landscapes having a single dominant basin and an overall funnel topography. We show here that, although such a landscape topography is indeed a sufficient condition for folding, another possibility also exists, giving a previously undescribed class of foldable sequences. These sequences have landscapes that are only weakly funneled in the conventional thermodynamic sense but have unusually low kinetic barriers for reconfigurational motion. Traps have been specifically removed by selection. Here we examine the possibility of folding on these "buffed" landscapes by mapping the determination of statistics of pathways for the heterogeneous nucleation processes involved in escaping from traps to the solution of an imaginary time Schroedinger equation. This equation is solved analytically in adiabatic and "soft-wall" approximations, and numerical results are shown for the general case. The fraction of funneled vs. buffed proteins in sequence space is estimated, suggesting the statistical dominance of the funneling mechanism for achieving foldability.

  17. Mechanics of large folds in thin interfacial films

    NASA Astrophysics Data System (ADS)

    Démery, Vincent; Davidovitch, Benny; Santangelo, Christian D.

    2014-10-01

    A thin film confined to a liquid interface responds to uniaxial compression by wrinkling, and then by folding, that has been solved exactly before self-contact. Here, we address the mechanics of large folds, i.e., folds that absorb a length much larger than the wrinkle wavelength. With scaling arguments and numerical simulations, we show that the antisymmetric fold is energetically favorable and can absorb any excess length at zero pressure. Then, motivated by puzzles arising in the comparison of this simple model to experiments on lipid monolayers or capillary rafts, we discuss how to incorporate film weight, self-adhesion, or energy dissipation.

  18. The folding of spectrin domains I: wild-type domains have the same stability but very different kinetic properties.

    PubMed

    Scott, Kathryn A; Batey, Sarah; Hooton, Karen A; Clarke, Jane

    2004-11-12

    The study of proteins with the same architecture, but different sequence has proven to be a valuable tool in the protein folding field. As a prelude to studies on the folding mechanism of spectrin domains we present the kinetic characterisation of the wild-type forms of the 15th, 16th, and 17th domains of chicken brain alpha-spectrin (referred to as R15, R16 and R17, respectively). We show that the proteins all behave in a two-state manner, with different kinetic properties. The folding rate varies remarkably between different members, with a 5000-fold variation in folding rate and 3000-fold variation in unfolding rate seen for proteins differing only 1 kcal mol(-1) in stability. We show clear evidence for significant complexity in the energy landscape of R16, which shows a change in amplitude outside the stopped-flow timescale and curvature in the unfolding arm of the chevron plot. The accompanying paper describes the characterisation of the folding pathway of this domain.

  19. DNA Origami Folding Pathways: Implications for Design, Thermodynamics, and Kinetics

    NASA Astrophysics Data System (ADS)

    Majikes, Jacob Michael

    DNA nanotechnology implements the predictable self-assembly rules of DNA, allowing the adaptation of DNA from a biological tool for storage of genetic information to a biomimetic structural nanomaterial. DNA has been employed to organize organic and inorganic materials, as well as to create both static and dynamic nanostructures. Aided by the low cost of arbitrary sequence DNA oligomer synthesis and robust conjugation chemistries, DNA has developed as a promising nanofabrication tool. While under biological conditions the formation and thermodynamics of DNA are well known, nanotechnology applications typically lie well outside of those conditions. This dissertation presents a new scaffold (miniM13) for DNA nanostructures and three new protocols to probe the folding and formation of DNA nanostructures. Development of these novel techniques improves the molecular assembly toolkit to enable new and exciting experimental systems. (Abstract shortened by ProQuest.).

  20. Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers.

    PubMed

    Zhang, Yongli

    2017-07-01

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers. We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain association initiates SNARE assembly; a middle domain suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain and the linker domain directly drive membrane fusion. In addition, the kinetics and pathway of the stagewise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins. © 2017 The Protein Society.

  1. Using VIPT-Jump to Distinguish Between Different Folding Mechanisms: Application to BBL and a Trpzip

    PubMed Central

    Lin, Chun–Wei; Culik, Robert M.; Gai, Feng

    2013-01-01

    Protein folding involves a large number of sequential molecular steps or conformational substates. Thus, experimental characterization of the underlying folding energy landscape for any given protein is difficult. Herein, we present a new method that can be used to determine the major characteristics of the folding energy landscape in question, for example, to distinguish between activated and barrierless downhill folding scenarios. This method is based on the idea that the conformational relaxation kinetics of different folding mechanisms at a given final condition will show different dependences on the initial condition. We show, using both simulation and experiment, that it is possible to differentiate between disparate kinetic folding models by comparing temperature-jump (T-jump) relaxation traces obtained with a fixed final temperature and varied initial temperatures, which effectively varies the initial potential (VIP) of the system of interest. We apply this method (hereafter refer to as VIPT-jump) to two model systems, Trpzip-2c and BBL, and our results show that BBL exhibits characteristics of barrierless downhill folding, whereas Trpzip-2c folding encounters a free energy barrier. In addition, using the T-jump data of BBL we are able to provide, via Langevin Dynamics simulations, a realistic estimate of its conformational diffusion coefficient. PMID:23642153

  2. $\\cN$-FOLD SUPERSYMMETRY IN QUANTUM MECHANICAL MATRIX MODELS

    NASA Astrophysics Data System (ADS)

    Tanaka, Toshiaki

    2012-03-01

    We formulate Ņ-fold supersymmetry in quantum mechanical matrix models. As an example, we construct general two-by-two Hermitian matrix two-fold supersymmetric quantum mechanical systems. We find that there are two inequivalent such systems, both of which are characterized by two arbitrary scalar functions, and one of which does not reduce to the scalar system. The obtained systems are all weakly quasi-solvable.

  3. Isoprene: a photochemical kinetic mechanism

    SciTech Connect

    Killus, J.P.; Whitten, G.Z.

    1984-03-01

    A computer-modeling study has produced a photochemical kinetic mechanism for the atmospheric chemistry of isoprene, a naturally occurring common constituent of the troposphere. The kinetic mechnism is ready for use in atmospheric models because the reactions described are shown to adequately reproduce the results of a series of outdoor smog chamber experiments which encompass a wide range of precursor conditions of isoprene and NO/sub x/. Isoprene is a very reactive molecule that can contribute as much as 50% of the overall reactivity of rural air even though isoprene might be only 6% of the ambient hydrocarbon level. The major intermediate products of the atmospheric oxidation of isoprene, methyl vinyl ketone, methacrolein, methylglyoxal, and formaldehyde are also highly reactive. 25 references.

  4. Characterization of protein folding by a Φ-value calculation with a statistical-mechanical model

    PubMed Central

    Wako, Hiroshi; Abe, Haruo

    2016-01-01

    The Φ-value analysis approach provides information about transition-state structures along the folding pathway of a protein by measuring the effects of an amino acid mutation on folding kinetics. Here we compared the theoretically calculated Φ values of 27 proteins with their experimentally observed Φ values; the theoretical values were calculated using a simple statistical-mechanical model of protein folding. The theoretically calculated Φ values reflected the corresponding experimentally observed Φ values with reasonable accuracy for many of the proteins, but not for all. The correlation between the theoretically calculated and experimentally observed Φ values strongly depends on whether the protein-folding mechanism assumed in the model holds true in real proteins. In other words, the correlation coefficient can be expected to illuminate the folding mechanisms of proteins, providing the answer to the question of which model more accurately describes protein folding: the framework model or the nucleation-condensation model. In addition, we tried to characterize protein folding with respect to various properties of each protein apart from the size and fold class, such as the free-energy profile, contact-order profile, and sensitivity to the parameters used in the Φ-value calculation. The results showed that any one of these properties alone was not enough to explain protein folding, although each one played a significant role in it. We have confirmed the importance of characterizing protein folding from various perspectives. Our findings have also highlighted that protein folding is highly variable and unique across different proteins, and this should be considered while pursuing a unified theory of protein folding. PMID:28409079

  5. Evolutionary trend toward kinetic stability in the folding trajectory of RNases H

    PubMed Central

    Lim, Shion A.; Hart, Kathryn M.; Marqusee, Susan

    2016-01-01

    Proper folding of proteins is critical to producing the biological machinery essential for cellular function. The rates and energetics of a protein’s folding process, which is described by its energy landscape, are encoded in the amino acid sequence. Over the course of evolution, this landscape must be maintained such that the protein folds and remains folded over a biologically relevant time scale. How exactly a protein’s energy landscape is maintained or altered throughout evolution is unclear. To study how a protein’s energy landscape changed over time, we characterized the folding trajectories of ancestral proteins of the ribonuclease H (RNase H) family using ancestral sequence reconstruction to access the evolutionary history between RNases H from mesophilic and thermophilic bacteria. We found that despite large sequence divergence, the overall folding pathway is conserved over billions of years of evolution. There are robust trends in the rates of protein folding and unfolding; both modern RNases H evolved to be more kinetically stable than their most recent common ancestor. Finally, our study demonstrates how a partially folded intermediate provides a readily adaptable folding landscape by allowing the independent tuning of kinetics and thermodynamics. PMID:27799545

  6. Structure and function in bacteriorhodopsin: the effect of the interhelical loops on the protein folding kinetics.

    PubMed

    Allen, S J; Kim, J M; Khorana, H G; Lu, H; Booth, P J

    2001-04-27

    The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-alpha-1,2-dihexanoylphosphatidylcholine/l-alpha-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to

  7. Using Kinetic Network Models To Probe Non-Native Salt-Bridge Effects on α-Helix Folding.

    PubMed

    Zhou, Guangfeng; Voelz, Vincent A

    2016-02-11

    Salt-bridge interactions play an important role in stabilizing many protein structures, and have been shown to be designable features for protein design. In this work, we study the effects of non-native salt bridges on the folding of a soluble alanine-based peptide (Fs peptide) using extensive all-atom molecular dynamics simulations performed on the Folding@home distributed computing platform. Using Markov State Models, we show how non-native salt-bridges affect the folding kinetics of Fs peptide by perturbing specific conformational states. Furthermore, we present methods for the automatic detection and analysis of such states. These results provide insight into helix folding mechanisms and useful information to guide simulation-based computational protein design.

  8. Role of the Cys 2-Cys 10 disulfide bond for the structure, stability, and folding kinetics of ribonuclease T1.

    PubMed Central

    Mayr, L. M.; Willbold, D.; Landt, O.; Schmid, F. X.

    1994-01-01

    The Cys 2-Cys 10 disulfide bond in ribonuclease T1 was broken by substituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in ribonuclease F1. This C2S/C10N variant resembles the wild-type protein in structure and in catalytic activity. Minor structural changes were observed by 2-dimensional NMR in the local environment of the substituted amino acids only. The thermodynamic stability of ribonuclease T1 is strongly reduced by breaking the Cys 2-Cys 10 bond, and the free energy of denaturation is decreased by about 10 kJ/mol. The folding mechanism is not affected, and the trans to cis isomerizations of Pro 39 and Pro 55 are still the rate-limiting steps of the folding process. The differences in the time courses of unfolding and refolding are correlated with the decrease in stability: the folding kinetics of the wild-type protein and the C2S/C10N variant become indistinguishable when they are compared under conditions of identical stability. Apparently, the Cys 2-Cys 10 disulfide bond is important for the stability but not for the folding mechanism of ribonuclease T1. The breaking of this bond has the same effect on stability and folding kinetics as adding 1 M guanidinium chloride to the wild-type protein. PMID:8003959

  9. Folding mechanism of proteins and protein-like polymers

    NASA Astrophysics Data System (ADS)

    Pande, Vijay

    2000-03-01

    Proteins are amazing biomaterials: they both perform biological activity as well as assemble themselves. In order to understand how proteins fold and to design synthetic polymers with protein-like properties, we need to understand how these molecules assemble themselves. I will discuss results from recent simulations of proteins and protein-like polymers in order to examine which is common and potentially ``universal'' about the folding (self-assembly) mechanism. These results may shed light on protein and protein-like polymer design, experiments on folding, as well as areas in which misfolding may be important such as many neurodegenerative diseases.

  10. The prosegment catalyzes pepsin folding to a kinetically trapped native state.

    PubMed

    Dee, Derek R; Yada, Rickey Y

    2010-01-19

    Investigations of irreversible protein unfolding often assume that alterations to the unfolded state, rather than the nature of the native state itself, are the cause of the irreversibility. However, the present study describes a less common explanation for the irreversible denaturation of pepsin, a zymogen-derived aspartic peptidase. The presence of a large folding barrier combined with the thermodynamically metastable nature of the native state, the formation of which depends on a separate prosegment (PS) domain, is the source of the irreversibility. Pepsin is unable to refold to the native state upon return from denaturing conditions due to a large folding barrier (24.6 kcal/mol) and instead forms a thermodynamically stable, yet inactive, refolded state. The native state is kinetically stabilized by an unfolding activation energy of 24.5 kcal/mol, comparable to the folding barrier, indicating that native pepsin exists as a thermodynamically metastable state. However, in the presence of the PS, the native state becomes thermodynamically stable, and the PS catalyzes pepsin folding by stabilizing the folding transition state by 14.7 kcal/mol. Once folded, the PS is removed, and the native conformation exists as a kinetically trapped state. Thus, while PS-guided folding is thermodynamically driven, without the PS the pepsin energy landscape is dominated by kinetic barriers rather than by free energy differences between native and denatured states. As pepsin is the archetype of a broad class of aspartic peptidases of similar structure and function, and many require their PS for correct folding, these results suggest that the occurrence of native states optimized for kinetic rather than thermodynamic stability may be a common feature of protein design.

  11. Thermodynamics and kinetics of the hairpin ribozyme from atomistic folding/unfolding simulations

    PubMed Central

    Nivón, Lucas G.; Shakhnovich, Eugene I.

    2011-01-01

    We report a set of atomistic folding/unfolding simulations for the hairpin ribozyme using a monte carlo algorithm. The hairpin ribozyme folds in solution and catalyzes self-cleavage or ligation via a specific two-domain structure. The minimal active ribozyme has been studied extensively, showing stabilization of the active structure by cations and dynamic motion of the active structure. Here we introduce a simple model of tertiary structure formation that leads to a phase diagram for the RNA as a function of temperature and tertiary structure strength. We then employ this model to capture many folding/unfolding events and to examine the transition state ensemble (TSE) of the RNA during folding to its active “docked” conformation. The TSE is compact but with few tertiary interactions formed, in agreement with single-molecule dynamics experiments. To compare with experimental kinetic parameters we introduce a novel method to benchmark monte carlo kinetic parameters to docking/undocking rates collected over many single molecular trajectories. We find that topology alone, as encoded in a biased potential which discriminates between secondary and tertiary interactions, is sufficient to predict the thermodynamic behavior and kinetic folding pathway of the hairpin ribozyme. This method should be useful in predicting folding transition states for many natural or man-made RNA tertiary structures. PMID:21740912

  12. Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain.

    PubMed

    Gruber, Tobias; Balbach, Jochen

    2015-01-01

    The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state.

  13. Molecular-crowding effects on single-molecule RNA folding/unfolding thermodynamics and kinetics.

    PubMed

    Dupuis, Nicholas F; Holmstrom, Erik D; Nesbitt, David J

    2014-06-10

    The effects of "molecular crowding" on elementary biochemical processes due to high solute concentrations are poorly understood and yet clearly essential to the folding of nucleic acids and proteins into correct, native structures. The present work presents, to our knowledge, first results on the single-molecule kinetics of solute molecular crowding, specifically focusing on GAAA tetraloop-receptor folding to isolate a single RNA tertiary interaction using time-correlated single-photon counting and confocal single-molecule FRET microscopy. The impact of crowding by high-molecular-weight polyethylene glycol on the RNA folding thermodynamics is dramatic, with up to ΔΔG° ∼ -2.5 kcal/mol changes in free energy and thus >60-fold increase in the folding equilibrium constant (Keq) for excluded volume fractions of 15%. Most importantly, time-correlated single-molecule methods permit crowding effects on the kinetics of RNA folding/unfolding to be explored for the first time (to our knowledge), which reveal that this large jump in Keq is dominated by a 35-fold increase in tetraloop-receptor folding rate, with only a modest decrease in the corresponding unfolding rate. This is further explored with temperature-dependent single-molecule RNA folding measurements, which identify that crowding effects are dominated by entropic rather than enthalpic contributions to the overall free energy change. Finally, a simple "hard-sphere" treatment of the solute excluded volume is invoked to model the observed kinetic trends, and which predict ΔΔG° ∼ -5 kcal/mol free-energy stabilization at excluded volume fractions of 30%.

  14. PFD: a database for the investigation of protein folding kinetics and stability.

    PubMed

    Fulton, Kate F; Devlin, Glyn L; Jodun, Rachel A; Silvestri, Linda; Bottomley, Stephen P; Fersht, Alan R; Buckle, Ashley M

    2005-01-01

    We have developed a new database that collects all protein folding data into a single, easily accessible public resource. The Protein Folding Database (PFD) contains annotated structural, methodological, kinetic and thermodynamic data for more than 50 proteins, from 39 families. A user-friendly web interface has been developed that allows powerful searching, browsing and information retrieval, whilst providing links to other protein databases. The database structure allows visualization of folding data in a useful and novel way, with a long-term aim of facilitating data mining and bioinformatics approaches. PFD can be accessed freely at http://pfd.med.monash.edu.au.

  15. Kinetic potentials in quantum mechanics

    NASA Astrophysics Data System (ADS)

    Hall, Richard L.

    1984-09-01

    Suppose that the Hamiltonian H=-Δ+vf(r) represents the energy of a particle which moves in an attractive central potential and obeys nonrelativistic quantum mechanics. The discrete eigenvalues Enl=Fnl(v) of H may be expressed as a Legendre transformation Fnl(v)=mins≳0(s+vf¯nl(s)), n=1,2,3,..., l=0,1,2,..., where the ``kinetic potentials'' f¯nl(s) associated with f(r) are defined by f¯nl(s) =infDnl supψ∈Dnl, ∥ψ∥=1 ∫ ψ(r) f ([ψ,-Δψ)/s]1/2r)ψ(r)d3r, and Dnl is an n-dimensional subspace of L2(R3) labeled by Ylm(θ,φ), m=0, and contained in the domain D(H) of H. If the potential has the form f(r)=∑Ni=1 g(i)( f(i)(r)) then in many interesting cases it turns out that the corresponding kinetic potentials can be closely approximated by ∑Ni=1 g(i)( f¯nl(i)(s)). This nice behavior of the kinetic potentials leads to a constructive global approximation theory for Schrödinger eigenvalues. As an illustration, detailed recipes are provided for arbitrary linear combinations of power-law potentials and the log potential. For the linear plus Coulomb potential and the quartic anharmonic oscillator the approximate eigenvalues are compared to accurate values found by numerical integration.

  16. A comprehensive database of verified experimental data on protein folding kinetics

    PubMed Central

    Wagaman, Amy S; Coburn, Aaron; Brand-Thomas, Itai; Dash, Barnali; Jaswal, Sheila S

    2014-01-01

    Insights into protein folding rely increasingly on the synergy between experimental and theoretical approaches. Developing successful computational models requires access to experimental data of sufficient quantity and high quality. We compiled folding rate constants for what initially appeared to be 184 proteins from 15 published collections/web databases. To generate the highest confidence in the dataset, we verified the reported lnkf value and exact experimental construct and conditions from the original experimental report(s). The resulting comprehensive database of 126 verified entries, ACPro, will serve as a freely accessible resource (https://www.ats. amherst.edu/protein/) for the protein folding community to enable confident testing of predictive models. In addition, we provide a streamlined submission form for researchers to add new folding kinetics results, requiring specification of all the relevant experimental information according to the standards proposed in 2005 by the protein folding consortium organized by Plaxco. As the number and diversity of proteins whose folding kinetics are studied expands, our curated database will enable efficient and confident incorporation of new experimental results into a standardized collection. This database will support a more robust symbiosis between experiment and theory, leading ultimately to more rapid and accurate insights into protein folding, stability, and dynamics. PMID:25229122

  17. Prediction of folding pathway and kinetics among plant hemoglobins using an average distance map method.

    PubMed

    Nakajima, Shunsuke; Alvarez-Salgado, Emma; Kikuchi, Takeshi; Arredondo-Peter, Raúl

    2005-11-15

    Computational methods, such as the ADM (average distance map) method, have been developed to predict folding of homologous proteins. In this work we used the ADM method to predict the folding pathway and kinetics among selected plant nonsymbiotic (nsHb), symbiotic (Lb), and truncated (tHb) hemoglobins (Hbs). Results predicted that (1) folding of plant Hbs occurs throughout the formation of compact folding modules mostly formed by helices A, B, and C, and E, F, G, and H (folding modules A/C and E/H, respectively), and (2) primitive (moss) nsHbs fold in the C-->N direction, evolved (monocot and dicot) nsHbs fold either in the C-->N or N-->C direction, and Lbs and plant tHbs fold in the C-->N direction. We also predicted relative folding rates of plant Hbs from qualitative analyses of the stability of subdomains and classified plant Hbs into fast and moderate folding. ADM analysis of nsHbs predicted that prehelix A plays a role during folding of the N-terminal domain of Ceratodon nsHb, and that CD-loop plays a role in folding of primitive (Physcomitrella and Ceratodon) but not evolved nsHbs. Modeling of the rice Hb1 A/C and E/H modules showed that module E/H overlaps to the Mycobacterium tuberculosis HbO two-on-two folding. This observation suggests that module E/H is an ancient tertiary structure in plant Hbs.

  18. Macromolecular Crowding Modulates Folding Mechanism of α/β Protein Apoflavodoxin

    PubMed Central

    Homouz, Dirar; Stagg, Loren; Wittung-Stafshede, Pernilla; Cheung, Margaret S.

    2009-01-01

    Abstract Protein dynamics in cells may be different from those in dilute solutions in vitro, because the environment in cells is highly concentrated with other macromolecules. This volume exclusion because of macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, we investigated the folding energy landscape of an α/β protein, apoflavodoxin, in the presence of inert macromolecular crowding agents, using in silico and in vitro approaches. By means of coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fractions of crowding agents (ϕc) as well as of crowding agent geometry (sphere or spherocylinder) at high ϕc. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we identified the in silico crowding conditions that best enhance protein stability, and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either enhanced or relieved. Our test-tube experiments confirmed that apoflavodoxin's time-resolved folding path is modulated by crowding agent geometry. Macromolecular crowding effects may be a tool for the manipulation of protein-folding and function in living cells. PMID:19167312

  19. The X-38 V-201 Fin Fold Actuation Mechanism

    NASA Technical Reports Server (NTRS)

    Lupo, Christian; Robertson, Brandan; Gafka, George

    2004-01-01

    The X-38 Vehicle 201 (V-201) is a space flight prototype lifting body vehicle that was designed to launch to orbit in the Space Shuttle orbiter payload bay. Although the project was cancelled in May 2003, many of the systems were nearly complete. This paper will describe the fin folding actuation mechanism flight subsystems and development units as well as lessons learned in the design, assembly, development testing, and qualification testing. The two vertical tail fins must be stowed (folded inboard) to allow the orbiter payload bay doors to close. The fin folding actuation mechanism is a remotely or extravehicular activity (EVA) actuated single fault tolerant system consisting of seven subsystems capable of repeatedly deploying or stowing the fins.

  20. Kinetic and thermodynamic origins of osmolyte-influenced nucleic acid folding.

    PubMed

    Holmstrom, Erik D; Dupuis, Nicholas F; Nesbitt, David J

    2015-03-05

    The influential role of monovalent and divalent metal cations in facilitating conformational transitions in both RNA and DNA has been a target of intense biophysical research efforts. However, organic neutrally charged cosolutes can also significantly alter nucleic acid conformational transitions. For example, highly soluble small molecules such as trimethylamine N-oxide (TMAO) and urea are occasionally utilized by organisms to regulate cellular osmotic pressure. Ensemble studies have revealed that these so-called osmolytes can substantially influence the thermodynamics of nucleic acid conformational transitions. In the present work, we exploit single-molecule FRET (smFRET) techniques to measure, for first time, the kinetic origins of these osmolyte-induced changes to the folding free energy. In particular, we focus on smFRET RNA and DNA constructs designed as model systems for secondary and tertiary structure formation. These findings reveal that TMAO preferentially stabilizes both secondary and tertiary interactions by increasing kfold and decreasing kunfold, whereas urea destabilizes both conformational transitions, resulting in the exact opposite shift in kinetic rate constants (i.e., decreasing kfold and increasing kunfold). Complementary temperature-dependent smFRET experiments highlight a thermodynamic distinction between the two different mechanisms responsible for TMAO-facilitated conformational transitions, while only a single mechanism is seen for the destabilizing osmolyte urea. Finally, these results are interpreted in the context of preferential interactions between osmolytes, and the solvent accessible surface area (SASA) associated with the (i) nucleobase, (ii) sugar, and (iii) phosphate groups of nucleic acids in order to map out structural changes that occur during the conformational transitions.

  1. Microfluidic mixers for the investigation of rapid protein folding kinetics using synchrotron radiation circular dichroism spectroscopy.

    PubMed

    Kane, Avinash S; Hoffmann, Armin; Baumgärtel, Peter; Seckler, Robert; Reichardt, Gerd; Horsley, David A; Schuler, Benjamin; Bakajin, Olgica

    2008-12-15

    We have developed a microfluidic mixer optimized for rapid measurements of protein folding kinetics using synchrotron radiation circular dichroism (SRCD) spectroscopy. The combination of fabrication in fused silica and synchrotron radiation allows measurements at wavelengths below 220 nm, the typical limit of commercial instrumentation. At these wavelengths, the discrimination between the different types of protein secondary structure increases sharply. The device was optimized for rapid mixing at moderate sample consumption by employing a serpentine channel design, resulting in a dead time of less than 200 micros. Here, we discuss the design and fabrication of the mixer and quantify the mixing efficiency using wide-field and confocal epi-fluorescence microscopy. We demonstrate the performance of the device in SRCD measurements of the folding kinetics of cytochrome c, a small, fast-folding protein. Our results show that the combination of SRCD with microfluidic mixing opens new possibilities for investigating rapid conformational changes in biological macromolecules that have previously been inaccessible.

  2. Kinetic pathway for folding of the Tetrahymena ribozyme revealed by three UV-inducible crosslinks.

    PubMed Central

    Downs, W D; Cech, T R

    1996-01-01

    The kinetics of RNA folding were examined in the L-21 ribozyme, an RNA enzyme derived from the self-splicing Tetrahymena intron. Three UV-inducible crosslinks were mapped, characterized, and used as indicators for the folded state of the ribozyme. Together these data suggest that final structures are adopted first by the P4-P6 independently folding domain and only later in a region that positions the P1 helix (including the 5' splice site), a region whose folding is linked to that of a portion of the catalytic core. At intermediate times, a non-native structure forms in the region of the triple helical scaffold, which connects the major folding domains. At 30 degrees C, the unfolded ribozyme passes through these stages with a half-life of 2 min from the time magnesium cations are provided. At higher temperatures, the half-life is shortened but the order of events is unchanged. Thermal melting of the fully folded ribozyme also revealed a multi-stage process in which the steps of folding are reversed: the kinetically slowest structure is the least stable and melts first. These structures of the ribozyme also bind Mg2+ cooperatively and their relative affinity for binding seems to be a major determinant in the order of events during folding. Na+ can also substitute for Mg2+ to give rise to the same crosslinkable structures, but only at much higher concentrations. Specific binding sites for Mg2+ may make this cation particularly efficient at electrostatic stabilization during folding of these ribozyme structures. PMID:8756414

  3. Topography of funneled landscapes determines the thermodynamics and kinetics of protein folding

    PubMed Central

    Wang, Jin; Oliveira, Ronaldo J.; Chu, Xiakun; Whitford, Paul C.; Chahine, Jorge; Han, Wei; Wang, Erkang; Onuchic, José N.; Leite, Vitor B.P.

    2012-01-01

    The energy landscape approach has played a fundamental role in advancing our understanding of protein folding. Here, we quantify protein folding energy landscapes by exploring the underlying density of states. We identify three quantities essential for characterizing landscape topography: the stabilizing energy gap between the native and nonnative ensembles δE, the energetic roughness ΔE, and the scale of landscape measured by the entropy S. We show that the dimensionless ratio between the gap, roughness, and entropy of the system accurately predicts the thermodynamics, as well as the kinetics of folding. Large Λ implies that the energy gap (or landscape slope towards the native state) is dominant, leading to more funneled landscapes. We investigate the role of topological and energetic roughness for proteins of different sizes and for proteins of the same size, but with different structural topologies. The landscape topography ratio Λ is shown to be monotonically correlated with the thermodynamic stability against trapping, as characterized by the ratio of folding temperature versus trapping temperature. Furthermore, Λ also monotonically correlates with the folding kinetic rates. These results provide the quantitative bridge between the landscape topography and experimental folding measurements. PMID:23019359

  4. The energy landscape, folding pathways and the kinetics of a knotted protein.

    PubMed

    Prentiss, Michael C; Wales, David J; Wolynes, Peter G

    2010-07-01

    The folding pathway and rate coefficients of the folding of a knotted protein are calculated for a potential energy function with minimal energetic frustration. A kinetic transition network is constructed using the discrete path sampling approach, and the resulting potential energy surface is visualized by constructing disconnectivity graphs. Owing to topological constraints, the low-lying portion of the landscape consists of three distinct regions, corresponding to the native knotted state and to configurations where either the N or C terminus is not yet folded into the knot. The fastest folding pathways from denatured states exhibit early formation of the N terminus portion of the knot and a rate-determining step where the C terminus is incorporated. The low-lying minima with the N terminus knotted and the C terminus free therefore constitute an off-pathway intermediate for this model. The insertion of both the N and C termini into the knot occurs late in the folding process, creating large energy barriers that are the rate limiting steps in the folding process. When compared to other protein folding proteins of a similar length, this system folds over six orders of magnitude more slowly.

  5. Impact of Ion Binding on Poly-L-Lysine (Un)folding Energy Landscape and Kinetics

    PubMed Central

    Xiong, Kan; Asher, Sanford A.

    2012-01-01

    We utilize T-jump UV resonance Raman spectroscopy to study the impact of ion binding on the equilibrium energy landscape and on (un)folding kinetics of poly-L-lysine (PLL). We observe that the relaxation rates of the folded conformations (including π-helix (bulge), pure α-helix and turns) of PLL are slower than those of short alanine based peptides. The PLL pure α-helix folding time is similar to that of short alanine based peptides. We, for the first time have directly observed that turn conformations are α-helix and π-helix (bulge) unfolding intermediates. ClO4− binding to the lys side chain –NH3+ groups and the peptide backbone slows the α-helix unfolding rate compared to that in pure water, but little impacts the folding rate, resulting in an increased α-helix stability. ClO4− binding significantly increases the PLL unfolding activation barrier but little impacts the folding barrier. Thus, the PLL folding coordinate differs from the unfolding coordinate. The π-helix (bulge) unfolding and folding coordinates do not directly go through the α-helix energy well. Our results clearly demonstrate that PLL (un)folding is not a two-state process. PMID:22612556

  6. Asymmetric effect of domain interactions on the kinetics of folding in yeast phosphoglycerate kinase.

    PubMed

    Osváth, Szabolcs; Köhler, Gottfried; Závodszky, Péter; Fidy, Judit

    2005-06-01

    The aim of this work is to shed more light on the effect of domain-domain interactions on the kinetics and the pathway of protein folding. A model protein system consisting of several single-tryptophan variants of the two-domain yeast phosphoglycerate kinase (PGK) and its individual domains was studied. Refolding was initiated from the guanidine-unfolded state by stopped-flow or manual mixing and monitored by tryptophan fluorescence from 1 msec to 1000 sec. Denaturant titrations of both individual domains showed apparent two-state unfolding transitions. Refolding kinetics of the individual domains from different denaturant concentrations, however, revealed the presence of intermediate structures during titration for both domains. Refolding of the same domains within the complete protein showed that domain-domain interactions direct the folding of both domains, but in an asymmetric way. Folding of the N domain was already altered within 1 msec, while detectable changes in the folding of the C domain occurred only 60-100 msec after initiating refolding. All mutants showed a hyperfluorescent kinetic intermediate. Both the disappearance of this intermediate and the completion of the folding were significantly faster in the individual N domain than in the complete protein. On the contrary, folding of the individual C domain was slower than in the complete protein. The presence of the C domain directs the refolding of the N domain along a completely different pathway than that of the individual N domain, while folding of the individual C domain follows the same path as within the complete protein.

  7. Common folds and transport mechanisms of secondary active transporters.

    PubMed

    Shi, Yigong

    2013-01-01

    Secondary active transporters exploit the electrochemical potential of solutes to shuttle specific substrate molecules across biological membranes, usually against their concentration gradient. Transporters of different functional families with little sequence similarity have repeatedly been found to exhibit similar folds, exemplified by the MFS, LeuT, and NhaA folds. Observations of multiple conformational states of the same transporter, represented by the LeuT superfamily members Mhp1, AdiC, vSGLT, and LeuT, led to proposals that structural changes are associated with substrate binding and transport. Despite recent biochemical and structural advances, our understanding of substrate recognition and energy coupling is rather preliminary. This review focuses on the common folds and shared transport mechanisms of secondary active transporters. Available structural information generally supports the alternating access model for substrate transport, with variations and extensions made by emerging structural, biochemical, and computational evidence.

  8. Highly Anomalous Energetics of Protein Cold Denaturation Linked to Folding-Unfolding Kinetics

    PubMed Central

    Romero-Romero, M. Luisa; Inglés-Prieto, Alvaro; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

    2011-01-01

    Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a “mirror image” of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction) occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions. PMID:21829584

  9. Using enzyme folding to explore the mechanism of therapeutic touch: a feasibility study.

    PubMed

    Strickland, Mallory L; Boylan, Helen M

    2010-07-01

    The goal of this research is to design a novel model using protein folding to study Therapeutic Touch, a noncontact form of energy manipulation healing. Presented is a feasibility study suggesting that the denaturation path of ribonuclease A may be a useful model to study the energy exchange underlying therapeutic touch. The folding of ribonuclease A serves as a controlled energy-requiring system in which energy manipulation can be measured by the degree of folding achieved. A kinetic assay and fluorescence spectroscopy are used to assess the enzyme-folding state. The data suggest that the kinetic assay is a useful means of assessing the degree of refolding, and specifically, the enzyme function. However, fluorescence spectroscopy was not shown to be an effective measurement of enzyme structure for the purposes of this work. More research is needed to assess the underlying mechanism of therapeutic touch to complement the existing studies. An enzyme-folding model may provide a useful means of studying the energy exchange in therapeutic touch.

  10. Diffusion-collision of foldons elucidates the kinetic effects of point mutations and suggests control strategies of the folding process of helical proteins.

    PubMed

    Capriotti, Emidio; Compiani, Mario

    2006-07-01

    In this article we use mutation studies as a benchmark for a minimal model of the folding process of helical proteins. The model ascribes a pivotal role to the collisional dynamics of a few crucial residues (foldons) and predicts the folding rates by exploiting information drawn from the protein sequence. We show that our model rationalizes the effects of point mutations on the kinetics of folding. The folding times of two proteins and their mutants are predicted. Stability and location of foldons have a critical role as the determinants of protein folding. This allows us to elucidate two main mechanisms for the kinetic effects of mutations. First, it turns out that the mutations eliciting the most notable effects alter protein stability through stabilization or destabilization of the foldons. Secondly, the folding rate is affected via a modification of the foldon topology by those mutations that lead to the birth or death of foldons. The few mispredicted folding rates of some mutants hint at the limits of the current version of the folding model proposed in the present article. The performance of our folding model declines in case the mutated residues are subject to strong long-range forces. That foldons are the critical targets of mutation studies has notable implications for design strategies and is of particular interest to address the issue of the kinetic regulation of single proteins in the general context of the overall dynamics of the interactome.

  11. Statistical mechanics of simple models of protein folding and design.

    PubMed Central

    Pande, V S; Grosberg, A Y; Tanaka, T

    1997-01-01

    It is now believed that the primary equilibrium aspects of simple models of protein folding are understood theoretically. However, current theories often resort to rather heavy mathematics to overcome some technical difficulties inherent in the problem or start from a phenomenological model. To this end, we take a new approach in this pedagogical review of the statistical mechanics of protein folding. The benefit of our approach is a drastic mathematical simplification of the theory, without resort to any new approximations or phenomenological prescriptions. Indeed, the results we obtain agree precisely with previous calculations. Because of this simplification, we are able to present here a thorough and self contained treatment of the problem. Topics discussed include the statistical mechanics of the random energy model (REM), tests of the validity of REM as a model for heteropolymer freezing, freezing transition of random sequences, phase diagram of designed ("minimally frustrated") sequences, and the degree to which errors in the interactions employed in simulations of either folding and design can still lead to correct folding behavior. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 6 PMID:9414231

  12. Quantitative tests of a reconstitution model for RNA folding thermodynamics and kinetics.

    PubMed

    Bisaria, Namita; Greenfeld, Max; Limouse, Charles; Mabuchi, Hideo; Herschlag, Daniel

    2017-09-12

    Decades of study of the architecture and function of structured RNAs have led to the perspective that RNA tertiary structure is modular, made of locally stable domains that retain their structure across RNAs. We formalize a hypothesis inspired by this modularity-that RNA folding thermodynamics and kinetics can be quantitatively predicted from separable energetic contributions of the individual components of a complex RNA. This reconstitution hypothesis considers RNA tertiary folding in terms of ΔGalign, the probability of aligning tertiary contact partners, and ΔGtert, the favorable energetic contribution from the formation of tertiary contacts in an aligned state. This hypothesis predicts that changes in the alignment of tertiary contacts from different connecting helices and junctions (ΔGHJH) or from changes in the electrostatic environment (ΔG+/-) will not affect the energetic perturbation from a mutation in a tertiary contact (ΔΔGtert). Consistent with these predictions, single-molecule FRET measurements of folding of model RNAs revealed constant ΔΔGtert values for mutations in a tertiary contact embedded in different structural contexts and under different electrostatic conditions. The kinetic effects of these mutations provide further support for modular behavior of RNA elements and suggest that tertiary mutations may be used to identify rate-limiting steps and dissect folding and assembly pathways for complex RNAs. Overall, our model and results are foundational for a predictive understanding of RNA folding that will allow manipulation of RNA folding thermodynamics and kinetics. Conversely, the approaches herein can identify cases where an independent, additive model cannot be applied and so require additional investigation.

  13. The ensemble folding kinetics of protein G from an all-atom Monte Carlo simulation

    NASA Astrophysics Data System (ADS)

    Shimada, Jun; Shakhnovich, Eugene I.

    2002-08-01

    Protein G is folded with an all-atom Monte Carlo simulation by using a G potential. When folding is monitored by using burial of the lone tryptophan in protein G as the reaction coordinate, the ensemble kinetics is single exponential. Other experimental observations, such as the burst phase and mutational data, are also reproduced. However, more detailed analysis reveals that folding occurs over three distinct, three-state pathways. We show that, because of this tryptophan's asymmetric location in the tertiary fold, its burial (i) does not detect certain intermediates and (ii) may not correspond to the folding event. This finding demonstrates that ensemble averaging can disguise the presence of multiple pathways and intermediates when a non-ideal reaction coordinate is used. Finally, all observed folding pathways eventually converge to a common rate-limiting step, which is the formation of a specific nucleus involving hydrophobic core residues. These residues are conserved in the ubiquitin superfamily and in a phage display experiment, suggesting that fold topology is a strong determinant of the transition state.

  14. Analyzing complicated protein folding kinetics rapidly by analytical Laplace inversion using a Tikhonov regularization variant.

    PubMed

    Mulligan, Vikram Khipple; Hadley, Kevin Charles; Chakrabartty, Avijit

    2012-02-01

    Kinetic experiments provide much information about protein folding mechanisms. Time-resolved signals are often best described by expressions with many exponential terms, but this hinders the extraction of rate constants by nonlinear least squares (NLS) fitting. Numerical inverse Laplace transformation, which converts a time-resolved dataset into a spectrum of amplitudes as a function of rate constant, allows easy estimation of the rate constants, amplitudes, and number of processes underlying the data. Here, we present a Tikhonov regularization-based method that converts a dataset into a rate spectrum, subject to regularization constraints, without requiring an iterative search of parameter space. This allows more rapid generation of rate spectra as well as analysis of datasets too noisy to process by existing iterative search algorithms. This method's simplicity also permits highly objective, largely automatic analysis with minimal human guidance. We show that this regularization method reproduces results previously obtained by NLS fitting and that it is effective for analyzing datasets too complex for traditional fitting methods. This method's reliability and speed, as well as its potential for objective, model-free analysis, make it extremely useful as a first step in analysis of complicated noisy datasets and an excellent guide for subsequent NLS analysis. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Kinetic folding design of aptazyme-regulated expression devices as riboswitches for metabolic engineering.

    PubMed

    Sparkman-Yager, David; Correa-Rojas, Rodrigo A; Carothers, James M

    2015-01-01

    Recent developments in the fields of synthetic biology and metabolic engineering have opened the doors for the microbial production of biofuels and other valuable organic compounds. There remain, however, significant metabolic hurdles to the production of these compounds in cost-effective quantities. This is due, in part, to mismatches between the metabolic engineer's desire for high yields and the microbe's desire to survive. Many valuable compounds, or the intermediates necessary for their biosynthesis, prove deleterious at the desired production concentrations. One potential solution to these toxicity-related issues is the implementation of nonnative dynamic genetic control mechanisms that sense excessively high concentrations of metabolic intermediates and respond accordingly to alleviate their impact. One potential class of dynamic regulator is the riboswitch: cis-acting RNA elements that regulate the expression of downstream genes based on the presence of an effector molecule. Here, we present combined methods for constructing aptazyme-regulated expression devices (aREDs) through computational cotranscriptional kinetic folding design and experimental validation. These approaches can be used to engineer aREDs within novel genetic contexts for the predictable, dynamic regulation of gene expression in vivo. © 2015 Elsevier Inc. All rights reserved.

  16. Mechanisms and Kinetics of Catalytic Reactions

    DTIC Science & Technology

    1990-08-01

    CHEMICAL RESEARCH, r- DEVELOPMENT 5 N ENGINEERING CRDE-R-084 "" CENTER CENER(GC-TR-1728-008) ’ 04 N MECHANISMS AND KINETICS OF CATALYTIC REACTIONS Q...and Kinetics of Catalytic Reactions &AUTHOR(S) Garlick, Stephanie M. 7. PERFORMING ORGANIZATION NAME(S) AND ADORESS(ES) . PERFORMING ORGANIZATION...Tables........................87 vi MECHANISMS AND KINETICS OF CATALYTIC REACTIONS 1. INTRODUCTION The hydrolysis of phosphate esters in microemulsion

  17. Kinetically coupled folding of a single HIV-1 glycoprotein 41 complex in viral membrane fusion and inhibition.

    PubMed

    Jiao, Junyi; Rebane, Aleksander A; Ma, Lu; Gao, Ying; Zhang, Yongli

    2015-06-02

    HIV-1 glycoprotein 41 (gp41) mediates viral entry into host cells by coupling its folding energy to membrane fusion. Gp41 folding is blocked by fusion inhibitors, including the commercial drug T20, to treat HIV/AIDS. However, gp41 folding intermediates, energy, and kinetics are poorly understood. Here, we identified the folding intermediates of a single gp41 trimer-of-hairpins and measured their associated energy and kinetics using high-resolution optical tweezers. We found that folding of gp41 hairpins was energetically independent but kinetically coupled: Each hairpin contributed a folding energy of ∼-23 kBT, but folding of one hairpin successively accelerated the folding rate of the next one by ∼20-fold. Membrane-mimicking micelles slowed down gp41 folding and reduced the stability of the six-helix bundle. However, the stability was restored by cooperative folding of the membrane-proximal external region. Surprisingly, T20 strongly inhibited gp41 folding by actively displacing the C-terminal hairpin strand in a force-dependent manner. The inhibition was abolished by a T20-resistant gp41 mutation. The energetics and kinetics of gp41 folding established by us provides a basis to understand viral membrane fusion, infection, and therapeutic intervention.

  18. TOPICAL REVIEW: The nucleation mechanism of protein folding: a survey of computer simulation studies

    NASA Astrophysics Data System (ADS)

    Faísca, Patrícia F. N.

    2009-09-01

    The nucleation mechanism of protein folding, originally proposed by Baldwin in the early 1970s, was firstly observed by Shakhnovich and co-workers two decades later in the context of Monte Carlo simulations of a simple lattice model. At about the same time the extensive use of phi-value analysis provided the first experimental evidence that the folding of Chymotrypsin-inhibitor 2, a small single-domain protein, which folds with two-state kinetics, is also driven by a nucleation mechanism. Since then, the nucleation mechanism is generally considered the most common form of folding mechanism amongst two-state proteins. However, recent experimental data has put forward the idea that this may not necessarily be so, since the accuracy of the experimentally determined phi values, which are used to identify the critical (i.e. nucleating) residues, is typically poor. Here, we provide a survey of in silico results on the nucleation mechanism, ranging from simple lattice Monte Carlo to more sophisticated off-lattice molecular dynamics simulations, and discuss them in light of experimental data.

  19. Demonstration of a folding after binding mechanism in the recognition between the measles virus NTAIL and X domains.

    PubMed

    Dosnon, Marion; Bonetti, Daniela; Morrone, Angela; Erales, Jenny; di Silvio, Eva; Longhi, Sonia; Gianni, Stefano

    2015-03-20

    In the past decade, a wealth of experimental data has demonstrated that a large fraction of proteins, while functional, are intrinsically disordered at physiological conditions. Many intrinsically disordered proteins (IDPs) undergo a disorder-to-order transition upon binding to their biological targets, a phenomenon known as induced folding. Induced folding may occur through two extreme mechanisms, namely conformational selection and folding after binding. Although the pre-existence of ordered structures in IDPs is a prerequisite for conformational selection, it does not necessarily commit to this latter mechanism, and kinetic studies are needed to discriminate between the two possible scenarios. So far, relatively few studies have addressed this issue from an experimental perspective. Here, we analyze the interaction kinetics between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein. Data reveal that NTAIL recognizes XD by first forming a weak encounter complex in a disordered conformation, which is subsequently locked-in by a folding step; i.e., binding precedes folding. The implications of our kinetic results, in the context of previously reported equilibrium data, are discussed. These results contribute to enhancing our understanding of the molecular mechanisms by which IDPs recognize their partners and represent a paradigmatic example of the need of kinetic methods to discriminate between reaction mechanisms.

  20. Thermal stability and folding kinetics analysis of intrinsically disordered protein, securin

    NASA Astrophysics Data System (ADS)

    Chang, Chia-Ching; Chu, Hsueh-Liang; Ho, Li-Ping

    2014-03-01

    Lacking a stable tertiary structure, intrinsically disordered proteins (IDPs) possess particular functions in cell regulation, signaling, and controlling pathways. The study of their unique structure features, thermal stabilities, and folding kinetics is intriguing. In this study, an identified IDP, securin, was used as a model protein. By using a quasi-static five-step (on-path) folding process, the function of securin was restored and analyzed by isothermal titration calorimetry. Fluorescence spectroscopy and particle size analysis indicated that securin possessed a compact hydrophobic core and particle size. The glass transition of securin was characterized using differential scanning microcalorimetry. Furthermore, the folding/unfolding rates (kobs) of securin were undetectable, implying that the folding/unfolding rate is very fast and that the conformation of securin is sensitive to solvent environment change. Therefore, securin may fold properly under specific physiological conditions. In summary, the thermal glass transition behavior and undetectable kobs of folding/unfolding reactions may be two of the indices of IDP. This study was supported in part by grants NSC 97-2112-M-009-009-YM3 and NSC 100-2112-M-009-004-MY3, Taiwan, R.O.C.

  1. The precursor of beta-lactamase: purification, properties and folding kinetics.

    PubMed

    Laminet, A A; Plückthun, A

    1989-05-01

    The precursor of Escherichia coli RTEM beta-lactamase was purified to homogeneity on a milligram scale by a procedure independent of the binding properties of the protein and refolded to an active, reduced form. For comparing the folding kinetics, the wild-type enzyme was reduced and a mutant was constructed, in which the two cysteines that form a very stable disulfide bond in the RTEM enzyme were both changed into alanines. The rate of folding was determined by directly measuring the increase in enzymatic activity. The reduced precursor folds at least 15 times more slowly than either the reduced mature enzyme or the mature Cys----Ala double mutant under identical conditions. The wild-type enzyme, the Cys----Ala double mutant and the precursor protein all had similar KM values, demonstrating a very similar native state. The slow folding of the precursor compared with the mature form may be an essential and general feature to secure a transport competent conformation necessary for the translocation through a membrane in protein export. This folding assay of a precursor by directly following its enzymatic activity may facilitate the characterization of putative folding modulators in bacterial membrane transport.

  2. Universality and diversity of folding mechanics for three-helix bundle proteins.

    PubMed

    Yang, Jae Shick; Wallin, Stefan; Shakhnovich, Eugene I

    2008-01-22

    In this study we evaluate, at full atomic detail, the folding processes of two small helical proteins, the B domain of protein A and the Villin headpiece. Folding kinetics are studied by performing a large number of ab initio Monte Carlo folding simulations using a single transferable all-atom potential. Using these trajectories, we examine the relaxation behavior, secondary structure formation, and transition-state ensembles (TSEs) of the two proteins and compare our results with experimental data and previous computational studies. To obtain a detailed structural information on the folding dynamics viewed as an ensemble process, we perform a clustering analysis procedure based on graph theory. Moreover, rigorous p(fold) analysis is used to obtain representative samples of the TSEs and a good quantitative agreement between experimental and simulated Phi values is obtained for protein A. Phi values for Villin also are obtained and left as predictions to be tested by future experiments. Our analysis shows that the two-helix hairpin is a common partially stable structural motif that gets formed before entering the TSE in the studied proteins. These results together with our earlier study of Engrailed Homeodomain and recent experimental studies provide a comprehensive, atomic-level picture of folding mechanics of three-helix bundle proteins.

  3. Navigating ligand protein binding free energy landscapes: universality and diversity of protein folding and molecular recognition mechanisms

    NASA Astrophysics Data System (ADS)

    Verkhivker, Gennady M.; Rejto, Paul A.; Bouzida, Djamal; Arthurs, Sandra; Colson, Anthony B.; Freer, Stephan T.; Gehlhaar, Daniel K.; Larson, Veda; Luty, Brock A.; Marrone, Tami; Rose, Peter W.

    2001-03-01

    Thermodynamic and kinetic aspects of ligand-protein binding are studied for the methotrexate-dihydrofolate reductase system from the binding free energy profile constructed as a function of the order parameter. Thermodynamic stability of the native complex and a cooperative transition to the unique native structure suggest the nucleation kinetic mechanism at the equilibrium transition temperature. Structural properties of the transition state ensemble and the ensemble of nucleation conformations are determined by kinetic simulations of the transmission coefficient and ligand-protein association pathways. Structural analysis of the transition states and the nucleation conformations reconciles different views on the nucleation mechanism in protein folding.

  4. The shape and mechanics of curved-fold origami structures

    NASA Astrophysics Data System (ADS)

    Dias, Marcelo A.; Santangelo, Christian D.

    2012-12-01

    We develop recursion equations to describe the three-dimensional shape of a sheet upon which a series of concentric curved folds have been inscribed. In the case of no stretching outside the fold, the three-dimensional shape of a single fold prescribes the shape of the entire origami structure. To better explore these structures, we derive continuum equations, valid in the limit of vanishing spacing between folds, to describe the smooth surface intersecting all the mountain folds. We find that this surface has negative Gaussian curvature with magnitude equal to the square of the fold's torsion. A series of open folds with constant fold angle generate a helicoid.

  5. Kinetic barriers to the folding of horse cytochrome C in the reduced state.

    PubMed

    Bhuyan, Abani K; Kumar, Rajesh

    2002-10-22

    To determine the kinetic barrier in the folding of horse cytochrome c, a CO-liganded derivative of cytochrome c, called carbonmonoxycytochrome c, has been prepared by exploiting the thermodynamic reversibility of ferrocytochrome c unfolding induced by guanidinium hydrochloride (GdnHCl), pH 7. The CO binding properties of unfolded ferrocytochrome c, studied by 13C NMR and optical spectroscopy, are remarkably similar to those of native myoglobin and isolated chains of human hemoglobin. Equilibrium unfolding transitions of ferrocytochrome c in the presence and the absence of CO observed by both excitation energy transfer from the lone tryptophan to the ferrous heme and far-UV circular dichroism (CD) indicate no accumulation of structural intermediates to a detectable level. Values of thermodynamic parameters obtained by two-state analysis of fluorescence transitions are DeltaG(H2O) = 11.65(+/-1.13) kcal x mol(-1) and C(m) = 3.9(+/-0.1) M GdnHCl in the presence of CO, and DeltaG(H2O)=19.3(+/-0.5) kcal x mol(-1) and C(m) = 5.1(+/-0.1) M GdnHCl in the absence of CO, indicating destabilization of ferrocytochrome c by approximately 7.65 kcal x mol(-1) due to CO binding. The native states of ferrocytochrome c and carbonmonoxycytochrome c are nearly identical in terms of structure and conformation except for the Fe2+-M80 --> Fe2+-CO replacement. Folding and unfolding kinetics as a function of GdnHCl, studied by stopped-flow fluorescence, are significantly different for the two proteins. Both refold fast, but carbonmonoxycytochrome c refolds 2-fold faster (tau = 1092 micros at 10 degrees C) than ferrocytochrome c. Linear extrapolation of the folding rates to the ordinate of the chevron plot projects this value of tau to 407 micros. The unfolding rate of the former in water, estimated by extrapolation, is faster by more than 10 orders of magnitude. Significant differences are also observed in rate-denaturant gradients in the chevron. Formation and disruption of the Fe2+-M80

  6. Kinetics and Mechanism--A Games Approach.

    ERIC Educational Resources Information Center

    Harsch, Gunther

    1984-01-01

    Proposes an approach to chemical kinetics and mechanism using statistical games, illustrating its use in monomolecular, catalytic, autocatalytic, consecutive, and equilibrium reactions. Major features of the games are also outlined and discussed. (JN)

  7. Role of mechanical factors in cortical folding development

    NASA Astrophysics Data System (ADS)

    Razavi, Mir Jalil; Zhang, Tuo; Li, Xiao; Liu, Tianming; Wang, Xianqiao

    2015-09-01

    Deciphering mysteries of the structure-function relationship in cortical folding has emerged as the cynosure of recent research on brain. Understanding the mechanism of convolution patterns can provide useful insight into the normal and pathological brain function. However, despite decades of speculation and endeavors the underlying mechanism of the brain folding process remains poorly understood. This paper focuses on the three-dimensional morphological patterns of a developing brain under different tissue specification assumptions via theoretical analyses, computational modeling, and experiment verifications. The living human brain is modeled with a soft structure having outer cortex and inner core to investigate the brain development. Analytical interpretations of differential growth of the brain model provide preliminary insight into the critical growth ratio for instability and crease formation of the developing brain followed by computational modeling as a way to offer clues for brain's postbuckling morphology. Especially, tissue geometry, growth ratio, and material properties of the cortex are explored as the most determinant parameters to control the morphogenesis of a growing brain model. As indicated in results, compressive residual stresses caused by the sufficient growth trigger instability and the brain forms highly convoluted patterns wherein its gyrification degree is specified with the cortex thickness. Morphological patterns of the developing brain predicted from the computational modeling are consistent with our neuroimaging observations, thereby clarifying, in part, the reason of some classical malformation in a developing brain.

  8. Complex RNA Folding Kinetics Revealed by Single-Molecule FRET and Hidden Markov Models

    PubMed Central

    2014-01-01

    We have developed a hidden Markov model and optimization procedure for photon-based single-molecule FRET data, which takes into account the trace-dependent background intensities. This analysis technique reveals an unprecedented amount of detail in the folding kinetics of the Diels–Alderase ribozyme. We find a multitude of extended (low-FRET) and compact (high-FRET) states. Five states were consistently and independently identified in two FRET constructs and at three Mg2+ concentrations. Structures generally tend to become more compact upon addition of Mg2+. Some compact structures are observed to significantly depend on Mg2+ concentration, suggesting a tertiary fold stabilized by Mg2+ ions. One compact structure was observed to be Mg2+-independent, consistent with stabilization by tertiary Watson–Crick base pairing found in the folded Diels–Alderase structure. A hierarchy of time scales was discovered, including dynamics of 10 ms or faster, likely due to tertiary structure fluctuations, and slow dynamics on the seconds time scale, presumably associated with significant changes in secondary structure. The folding pathways proceed through a series of intermediate secondary structures. There exist both compact pathways and more complex ones, which display tertiary unfolding, then secondary refolding, and, subsequently, again tertiary refolding. PMID:24568646

  9. Development of a Fast Microfluidic Mixer for Studies of Protein Folding KineticsFinal Report Cover Page

    SciTech Connect

    Bakajin, O

    2005-02-10

    We designed and fabricated mixing devices that will help us elucidate the mechanisms of protein folding through measurements of folding reaction rates. These devices can be used in studying of other biological systems and are compatible with various spectroscopic observation methods. The project involved development of fabrication processes and setup of a laboratory for assembly and characterization of microfluidic devices, as well as measurements of protein folding kinetics. We produced three variants of the mixer: (1) The ultra fast mixer for Foerster Resonance Energy Transfer measurements (described by Anal. Chem. Article UCRL-JRNL-206676) and MicroTAS Conference Proceedings article (UCRL-JC-153057 ) included in the report; (2) The ultra fast mixer for UV measurements (described by the poster presented at MicroTAS conference (UCRL-POST-207476) included in the report); and (3) The mixer for single molecule measurements (described by the Science article UCRL-JC-153057) included in the report. In these mixers, the channels are narrow, ranging from a few to hundreds of {micro}m, so that the flow is laminar and all of the mixing is achieved through diffusion. Our goal is to develop robust microfluidic mixer with at least 100 times lower consumption rate, shorter dead time and time resolution than commercially available mixers that would be compatible with most commonly used spectroscopic methods. We are also developing mixers that can be used in combination with single molecule spectroscopy. The mixers are used to study kinetics of fast protein folding reactions using bulk fluorescence and single molecule fluorescence resonance energy transfer techniques. Capabilities for microfluidic have been developed at BSNL that will be useful for studies of interactions of DNA with proteins and other projects such as the single molecule detector for detection of low concentration of toxins.

  10. Electrostatic mechanism of nucleosomal array folding revealed by computer simulation

    NASA Astrophysics Data System (ADS)

    Sun, Jian; Zhang, Qing; Schlick, Tamar

    2005-06-01

    Although numerous experiments indicate that the chromatin fiber displays salt-dependent conformations, the associated molecular mechanism remains unclear. Here, we apply an irregular Discrete Surface Charge Optimization (DiSCO) model of the nucleosome with all histone tails incorporated to describe by Monte Carlo simulations salt-dependent rearrangements of a nucleosomal array with 12 nucleosomes. The ensemble of nucleosomal array conformations display salt-dependent condensation in good agreement with hydrodynamic measurements and suggest that the array adopts highly irregular 3D zig-zag conformations at high (physiological) salt concentrations and transitions into the extended "beads-on-a-string" conformation at low salt. Energy analyses indicate that the repulsion among linker DNA leads to this extended form, whereas internucleosome attraction drives the folding at high salt. The balance between these two contributions determines the salt-dependent condensation. Importantly, the internucleosome and linker DNA-nucleosome attractions require histone tails; we find that the H3 tails, in particular, are crucial for stabilizing the moderately folded fiber at physiological monovalent salt. chromatin modeling | irregular 3D zig-zag | Discrete Surface Charge Optimization model

  11. Electrostatic mechanism of nucleosomal array folding revealed by computer simulation

    PubMed Central

    Sun, Jian; Zhang, Qing; Schlick, Tamar

    2005-01-01

    Although numerous experiments indicate that the chromatin fiber displays salt-dependent conformations, the associated molecular mechanism remains unclear. Here, we apply an irregular Discrete Surface Charge Optimization (DiSCO) model of the nucleosome with all histone tails incorporated to describe by Monte Carlo simulations salt-dependent rearrangements of a nucleosomal array with 12 nucleosomes. The ensemble of nucleosomal array conformations display salt-dependent condensation in good agreement with hydrodynamic measurements and suggest that the array adopts highly irregular 3D zig-zag conformations at high (physiological) salt concentrations and transitions into the extended “beads-on-a-string” conformation at low salt. Energy analyses indicate that the repulsion among linker DNA leads to this extended form, whereas internucleosome attraction drives the folding at high salt. The balance between these two contributions determines the salt-dependent condensation. Importantly, the internucleosome and linker DNA–nucleosome attractions require histone tails; we find that the H3 tails, in particular, are crucial for stabilizing the moderately folded fiber at physiological monovalent salt. PMID:15919827

  12. Quantitative criteria for native energetic heterogeneity influences in the prediction of protein folding kinetics

    PubMed Central

    Cho, Samuel S.; Levy, Yaakov; Wolynes, Peter G.

    2009-01-01

    Energy landscape theory requires that the protein-folding mechanism is generally globally directed or funneled toward the native state. The collective nature of transition state ensembles further suggests that sufficient averaging of the native interactions can occur so that the knowledge of the native topology may suffice for predicting the mechanism. Nevertheless, while simple homogeneously weighted native topology-based models predict the folding mechanisms for many proteins, for other proteins knowledge of the native topology, by itself, seems not to suffice in determining the folding mechanism. Simulations of proteins with differing topologies reveal that the failure of homogeneously weighted topology-based models can, however, be completely understood within the framework of a funneled energy landscape and can be quantified by comparing the fluctuation of entropy cost for forming contacts to the expected fluctuations in contact energy. To be precise, we find the transition state ensembles of proteins with all-α topologies, which are more uniform in the specific entropy cost of contact formation, have transition state ensembles that are more readily perturbed by differences in energetic weights than are the transition state ensembles of proteins with significant amounts of β-structure, where the specific entropy costs of contact formation are more widely distributed. This behavior is consistent with a random-field Ising model analogy that follows from the free energy functional approach to folding. PMID:19075236

  13. Kinetic Dissection of the Pre-existing Conformational Equilibrium in the Trypsin Fold*

    PubMed Central

    Vogt, Austin D.; Chakraborty, Pradipta; Di Cera, Enrico

    2015-01-01

    Structural biology has recently documented the conformational plasticity of the trypsin fold for both the protease and zymogen in terms of a pre-existing equilibrium between closed (E*) and open (E) forms of the active site region. How such plasticity is manifested in solution and affects ligand recognition by the protease and zymogen is poorly understood in quantitative terms. Here we dissect the E*-E equilibrium with stopped-flow kinetics in the presence of excess ligand or macromolecule. Using the clotting protease thrombin and its zymogen precursor prethrombin-2 as relevant models we resolve the relative distribution of the E* and E forms and the underlying kinetic rates for their interconversion. In the case of thrombin, the E* and E forms are distributed in a 1:4 ratio and interconvert on a time scale of 45 ms. In the case of prethrombin-2, the equilibrium is shifted strongly (10:1 ratio) in favor of the closed E* form and unfolds over a faster time scale of 4.5 ms. The distribution of E* and E forms observed for thrombin and prethrombin-2 indicates that zymogen activation is linked to a significant shift in the pre-existing equilibrium between closed and open conformations that facilitates ligand binding to the active site. These findings broaden our mechanistic understanding of how conformational transitions control ligand recognition by thrombin and its zymogen precursor prethrombin-2 and have direct relevance to other members of the trypsin fold. PMID:26216877

  14. Spin glasses and the statistical mechanics of protein folding.

    PubMed Central

    Bryngelson, J D; Wolynes, P G

    1987-01-01

    The theory of spin glasses was used to study a simple model of protein folding. The phase diagram of the model was calculated, and the results of dynamics calculations are briefly reported. The relation of these results to folding experiments, the relation of these hypotheses to previous protein folding theories, and the implication of these hypotheses for protein folding prediction schemes are discussed. PMID:3478708

  15. Unfolding single RNA molecules by mechanical force: A stochastic kinetic method

    NASA Astrophysics Data System (ADS)

    Liu, Fei; Ou-Yang, Zhong-Can

    2004-10-01

    Using simple polymer elastic theory and known RNA free energies, we study the single RNA folding and unfolding on the secondary structure level under mechanical constant force by stochastic kinetic simulation. As a primary application, this method is used to simulate the experiment performed by Liphardt [Science 292, 733 (2001)]. The extension-force curves in equilibrium and kinetic reaction rate constants for folding and unfolding are calculated. Our results show that the agreement between simulation and experimental measurements is satisfactory.

  16. KINETICS AND MECHANISMS OF SOIL BIOGEOCHEMICAL PROCESSES

    EPA Science Inventory

    The application of kinetic studies to soil chemistry is useful to determine reaction mechanisms and fate of nutrients and environmental contaminants. How deeply one wishes to query the mechanism depends on the detail sought. Reactions that involve chemical species in more than on...

  17. KINETICS AND MECHANISMS OF SOIL BIOGEOCHEMICAL PROCESSES

    EPA Science Inventory

    The application of kinetic studies to soil chemistry is useful to determine reaction mechanisms and fate of nutrients and environmental contaminants. How deeply one wishes to query the mechanism depends on the detail sought. Reactions that involve chemical species in more than on...

  18. Kinetic network study of the diversity and temperature dependence of Trp-Cage folding pathways: combining transition path theory with stochastic simulations.

    PubMed

    Zheng, Weihua; Gallicchio, Emilio; Deng, Nanjie; Andrec, Michael; Levy, Ronald M

    2011-02-17

    We present a new approach to study a multitude of folding pathways and different folding mechanisms for the 20-residue mini-protein Trp-Cage using the combined power of replica exchange molecular dynamics (REMD) simulations for conformational sampling, transition path theory (TPT) for constructing folding pathways, and stochastic simulations for sampling the pathways in a high dimensional structure space. REMD simulations of Trp-Cage with 16 replicas at temperatures between 270 and 566 K are carried out with an all-atom force field (OPLSAA) and an implicit solvent model (AGBNP). The conformations sampled from all temperatures are collected. They form a discretized state space that can be used to model the folding process. The equilibrium population for each state at a target temperature can be calculated using the weighted-histogram-analysis method (WHAM). By connecting states with similar structures and creating edges satisfying detailed balance conditions, we construct a kinetic network that preserves the equilibrium population distribution of the state space. After defining the folded and unfolded macrostates, committor probabilities (P(fold)) are calculated by solving a set of linear equations for each node in the network and pathways are extracted together with their fluxes using the TPT algorithm. By clustering the pathways into folding "tubes", a more physically meaningful picture of the diversity of folding routes emerges. Stochastic simulations are carried out on the network, and a procedure is developed to project sampled trajectories onto the folding tubes. The fluxes through the folding tubes calculated from the stochastic trajectories are in good agreement with the corresponding values obtained from the TPT analysis. The temperature dependence of the ensemble of Trp-Cage folding pathways is investigated. Above the folding temperature, a large number of diverse folding pathways with comparable fluxes flood the energy landscape. At low temperature

  19. Mechanical Properties of Bovine Rhodopsin and Bacteriorhodopsin: Possible Roles in Folding and Function†

    PubMed Central

    Sapra, K. Tanuj; Park, Paul S.-H.; Palczewski, Krzysztof; Muller, Daniel J.

    2008-01-01

    Molecular interactions and mechanical properties that contribute to the stability and function of proteins are complex and of fundamental importance. In this study, we used single-molecule dynamic force spectroscopy (DFS) to explore the interactions and the unfolding energy landscape of bovine rhodopsin and bacteriorhodopsin. An analysis of the experimental data enabled the extraction of parameters that provided insights into the kinetic stability and mechanical properties of these membrane proteins. Individual structural segments of rhodopsin and bacteriorhodopsin have different properties. A core of rigid structural segments was observed in rhodopsin but not in bacteriorhodopsin. This core may reflect differences in mechanisms of protein folding between the two membrane proteins. The different structural rigidity of the two proteins may also reflect their adaptation to differing functions. PMID:18266338

  20. Concordant Exploration of the Kinetics of RNA Folding from Global and Local Perspectives

    SciTech Connect

    Kwok,L.; Scherbakova, I.; Lamb, J.; Park, H.; Andresen, K.; Smith, H.; Brenowitz, M.; Pollack, L.

    2006-01-01

    Time-resolved small-angle X-ray scattering (SAXS) with millisecond time-resolution reveals two discrete phases of global compaction upon Mg{sup 2+}-mediated folding of the Tetrahymena thermophila ribozyme. Electrostatic relaxation of the RNA occurs rapidly and dominates the first phase of compaction during which the observed radius of gyration (R{sub g}) decreases from 75 Angstroms to 55 Angstroms. A further decrease in R{sub g} to 45 Angstroms occurs in a well-defined second phase. An analysis of mutant ribozymes shows that the latter phase depends upon the formation of long-range tertiary contacts within the P4-P6 domain of the ribozyme; disruption of the three remaining long-range contacts linking the peripheral helices has no effect on the 55-45 Angstroms compaction transition. A better understanding of the role of specific tertiary contacts in compaction was obtained by concordant time-resolved hydroxyl radical ({center_dot}OH) analyses that report local changes in the solvent accessibility of the RNA backbone. Comparison of the global and local measures of folding shows that formation of a subset of native tertiary contacts (i.e. those defining the ribozyme core) can occur within a highly compact ensemble whose R{sub g} is close to that of the fully folded ribozyme. Analyses of additional ribozyme mutants and reaction conditions establish the generality of the rapid formation of a partially collapsed state with little to no detectable tertiary structure. These studies directly link global RNA compaction with formation of tertiary structure as the molecule acquires its biologically active structure, and underscore the strong dependence on salt of both local and global measures of folding kinetics.

  1. Kinetics of folding and association of differently glycosylated variants of invertase from Saccharomyces cerevisiae.

    PubMed Central

    Kern, G.; Kern, D.; Jaenicke, R.; Seckler, R.

    1993-01-01

    A core-glycosylated form of the dimeric enzyme invertase has been isolated from secretion mutants of Saccharomyces cerevisiae blocked in transport to the Golgi apparatus. This glycosylation variant corresponds to the form that folds and associates during biosynthesis of the protein in vivo. In the present work, its largely homogeneous subunit size and well-defined quaternary structure were utilized to characterize the folding and association pathway of this highly glycosylated protein in comparison with the nonglycosylated cytoplasmic and the high-mannose-glycosylated periplasmic forms of the same enzyme encoded by the suc2 gene. Renaturation of core-glycosylated invertase upon dilution from guanidinium-chloride solutions follows a unibimolecular reaction scheme with consecutive first-order subunit folding and second-order association reactions. The rate constant of the rate-limiting step of subunit folding, as detected by fluorescence increase, is k1 = 1.6 +/- 0.4 x 10(-3) s-1 at 20 degrees C; it is characterized by an activation enthalpy of delta H++ = 65 kJ/mol. The reaction is not catalyzed by peptidyl-prolyl cis-trans isomerase of the cyclophilin type. Reactivation of the enzyme depends on protein concentration and coincides with subunit association, as monitored by size-exclusion high-pressure liquid chromatography. The association rate constant, estimated by numerical simulation of reactivation kinetics, increases from 5 x 10(3) M-1 s-1 to 7 x 10(4) M-1 s-1 between 5 and 30 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8268797

  2. The structural basis for biphasic kinetics in the folding of the WW domain from a formin-binding protein: Lessons for protein design?

    PubMed Central

    Karanicolas, John; Brooks, Charles L.

    2003-01-01

    The mechanism of formation of β-sheets is of great importance because of the significant role of such structures in the initiation and propagation of amyloid diseases. In this study we examine the folding of a series of three-stranded antiparallel β-sheets known as WW domains. Whereas other WW domains have been shown to fold with single-exponential kinetics, the WW domain from murine formin-binding protein 28 has recently been shown to fold with biphasic kinetics. By using a combination of kinetics and thermodynamics to characterize a simple model for this protein, the origins of the biphasic kinetics is found to lie in the fact that most of the protein is able to fold without requiring one of the β-hairpins to be correctly registered. The correct register of this hairpin is enforced by a surface-exposed hydrophobic contact, which is not present in other WW domains. This finding suggests the use of judiciously chosen surface-exposed hydrophobic pairs as a protein design strategy for enforcing the desired strand registry. PMID:12655041

  3. Improvements in Mixing Time and Mixing Uniformity in Devices Designed for Studies of Protein Folding Kinetics

    SciTech Connect

    Yao, Shuhuai; Bakajin, Olgica

    2007-08-01

    Using a microfluidic laminar flow mixer designed for studies of protein folding kinetics, we demonstrate a mixing time of 1 +/- 1 micros with sample consumption on the order of femtomoles. We recognize two limitations of previously proposed designs: (1) size and shape of the mixing region, which limits mixing uniformity and (2) the formation of Dean vortices at high flow rates, which limits the mixing time. We address these limitations by using a narrow shape-optimized nozzle and by reducing the bend of the side channel streamlines. The final design, which combines both of these features, achieves the best performance. We quantified the mixing performance of the different designs by numerical simulation of coupled Navier-Stokes and convection-diffusion equations and experiments using fluorescence resonance energy-transfer (FRET)-labeled DNA.

  4. Mechanism of Coupled Folding and Binding in the siRNA-PAZ Complex.

    PubMed

    Chen, Hai-Feng

    2008-08-01

    The PAZ domain plays a key role in gene silencing pathway. The PAZ domain binds with siRNAs to form the multimeric RNA-induced silencing complex (RISC). RISC identifies mRNAs homologous to the siRNAs and promotes their degradation. It was found that binding with siRNA significantly enhances apo-PAZ folding. However, the mechanism by which folding is coupled to binding is poorly understood. Thus, the coupling relationship between binding and folding is very important for understanding the function of gene silencing. We have performed molecular dynamics (MD) of both bound and apo-PAZ to study the coupling mechanism between binding and folding in the siRNA-PAZ complex. Room-temperature MD simulations suggest that both PAZ and siRNA become more rigid and stable upon siRNA binding. Kinetic analysis of high-temperature MD simulations shows that both bound and apo-PAZ unfold via a two-state process. The unfolding pathways are different between bound and apo-PAZ: the order of helix III and helices I & II unfolding is switched. Furthermore, transition probability was used to determine the transition state ensemble for both bound and apo-PAZ. It was found that the transition state of bound PAZ is more compact than that of apo-PAZ. The predicted Φ-values suggest that the Φ-values of helix III and sheets of β3-β7 for bound PAZ are more native-like than those of apo-PAZ upon the binding of siRNA. The results can help us to understand the mechanism of gene silencing.

  5. Comparative analysis of the folding dynamics and kinetics of an engineered knotted protein and its variants derived from HP0242 of Helicobacter pylori

    NASA Astrophysics Data System (ADS)

    Wang, Liang-Wei; Liu, Yu-Nan; Lyu, Ping-Chiang; Jackson, Sophie E.; Hsu, Shang-Te Danny

    2015-09-01

    Understanding the mechanism by which a polypeptide chain thread itself spontaneously to attain a knotted conformation has been a major challenge in the field of protein folding. HP0242 is a homodimeric protein from Helicobacter pylori with intertwined helices to form a unique pseudo-knotted folding topology. A tandem HP0242 repeat has been constructed to become the first engineered trefoil-knotted protein. Its small size renders it a model system for computational analyses to examine its folding and knotting pathways. Here we report a multi-parametric study on the folding stability and kinetics of a library of HP0242 variants, including the trefoil-knotted tandem HP0242 repeat, using far-UV circular dichroism and fluorescence spectroscopy. Equilibrium chemical denaturation of HP0242 variants shows the presence of highly populated dimeric and structurally heterogeneous folding intermediates. Such equilibrium folding intermediates retain significant amount of helical structures except those at the N- and C-terminal regions in the native structure. Stopped-flow fluorescence measurements of HP0242 variants show that spontaneous refolding into knotted structures can be achieved within seconds, which is several orders of magnitude faster than previously observed for other knotted proteins. Nevertheless, the complex chevron plots indicate that HP0242 variants are prone to misfold into kinetic traps, leading to severely rolled-over refolding arms. The experimental observations are in general agreement with the previously reported molecular dynamics simulations. Based on our results, kinetic folding pathways are proposed to qualitatively describe the complex folding processes of HP0242 variants.

  6. The E. coli thioredoxin folding mechanism: the key role of the C-terminal helix.

    PubMed

    Vazquez, Diego S; Sánchez, Ignacio E; Garrote, Ana; Sica, Mauricio P; Santos, Javier

    2015-02-01

    In this work, the unfolding mechanism of oxidized Escherichia coli thioredoxin (EcTRX) was investigated experimentally and computationally. We characterized seven point mutants distributed along the C-terminal α-helix (CTH) and the preceding loop. The mutations destabilized the protein against global unfolding while leaving the native structure unchanged. Global analysis of the unfolding kinetics of all variants revealed a linear unfolding route with a high-energy on-pathway intermediate state flanked by two transition state ensembles TSE1 and TSE2. The experiments show that CTH is mainly unfolded in TSE1 and the intermediate and becomes structured in TSE2. Structure-based molecular dynamics are in agreement with these experiments and provide protein-wide structural information on transient states. In our model, EcTRX folding starts with structure formation in the β-sheet, while the protein helices coalesce later. As a whole, our results indicate that the CTH is a critical module in the folding process, restraining a heterogeneous intermediate ensemble into a biologically active native state and providing the native protein with thermodynamic and kinetic stability. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Alpha/Beta-hydrolase fold enzymes: structures, functions and mechanisms.

    PubMed

    Holmquist, M

    2000-09-01

    The alpha/beta-hydrolase fold family of enzymes is rapidly becoming one of the largest group of structurally related enzymes with diverse catalytic functions. Members in this family include acetylcholinesterase, dienelactone hydrolase, lipase, thioesterase, serine carboxypeptidase, proline iminopeptidase, proline oligopeptidase, haloalkane dehalogenase, haloperoxidase, epoxide hydrolase, hydroxynitrile lyase and others. The enzymes all have a Nucleophile-His-Acid catalytic triad evolved to efficiently operate on substrates with different chemical composition or physicochemical properties and in various biological contexts. For example, acetylcholine esterase catalyzes the cleavage of the neurotransmitter acetylcholine, at a rate close to the limits of diffusion of substrate to the active site of the enzyme. Dienelactone hydrolase uses substrate-assisted catalysis to degrade aromatic compounds. Lipases act adsorbed at the water/lipid interface of their neutral water-insoluble ester substrates. Most lipases have their active site buried under secondary structure elements, a flap, which must change conformation to allow substrate to access the active site. Thioesterases are involved in a multitude of biochemical processes including bioluminiscence, fatty acid- and polyketide biosynthesis and metabolism. Serine carboxypeptidases recognize the negatively charged carboxylate terminus of their peptide substrates. Haloalkane dehalogenase is a detoxifying enzyme that converts halogenated aliphatics to the corresponding alcohols, while haloperoxidase catalyzes the halogenation of organic compounds. Hydroxynitrile lyase cleaves carbon-carbon bonds in cyanohydrins with concomitant hydrogen cyanide formation as a defense mechanism in plants. This paper gives an overview of catalytic activities reported for this family of enzymes by discussing selected examples. The current state of knowledge of the molecular basis for catalysis and substrate specificity is outlined

  8. Entanglement in correlated random spin chains, RNA folding and kinetic roughening

    NASA Astrophysics Data System (ADS)

    Rodríguez-Laguna, Javier; Santalla, Silvia N.; Ramírez, Giovanni; Sierra, Germán

    2016-07-01

    Average block entanglement in the 1D XX-model with uncorrelated random couplings is known to grow as the logarithm of the block size, in similarity to conformal systems. In this work we study random spin chains whose couplings present long range correlations, generated as gaussian fields with a power-law spectral function. Ground states are always planar valence bond states, and their statistical ensembles are characterized in terms of their block entropy and their bond-length distribution, which follow power-laws. We conjecture the existence of a critical value for the spectral exponent, below which the system behavior is identical to the case of uncorrelated couplings. Above that critical value, the entanglement entropy violates the area law and grows as a power law of the block size, with an exponent which increases from zero to one. Interestingly, we show that XXZ models with positive anisotropy present the opposite behavior, and strong correlations in the couplings lead to lower entropies. Similar planar bond structures are also found in statistical models of RNA folding and kinetic roughening, and we trace an analogy between them and quantum valence bond states. Using an inverse renormalization procedure we determine the optimal spin-chain couplings which give rise to a given planar bond structure, and study the statistical properties of the couplings whose bond structures mimic those found in RNA folding.

  9. Reduced methanol kinetic mechanisms for combustion applications

    SciTech Connect

    Yalamanchili, S.; Sirignano, W.A.; Seiser, R.; Seshadri, K.

    2005-08-01

    Reduced chemical kinetic mechanisms for methanol combustion were investigated by evaluating ignition delay magnitudes and combustion in a continuously stirred reactor. Unsteady computations were made to study the characteristics of the kinetic mechanisms proposed in the literature and to compare the dependence of various parameters on methanol combustion. All computations were done under isobaric conditions, and, to capture the influence of all the reactions involved in the mechanism, a very small time step was used. Finite-difference methods were used to solve the coupled differential equations. The five-step mechanism developed by C.M. Mueller and N. Peters [in: N. Peters, B. Rogg (Eds.), Reduced Kinetic Mechanisms for Applications in Combustion Systems, Springer-Verlag, New York, 1993, pp. 143-155] for premixed flames and both the five-step mechanism and the four-step mechanisms developed by C.M. Mueller, K. Seshadri, J.Y. Chen [ibid, pp. 284-307] for non-premixed flames were considered. It was found that the Mueller et al. five-step mechanism, with some modifications, best supported the spontaneous ignition and continuous stirred reactor combustion. The results were validated by comparing calculated ignition delays with available experimental data of C.T. Bowman [Combust. Flame 25 (1975) 343-354], and calculated final steady-state concentrations with chemical equilibrium calculations [J.-Y. Chen, Combust. Sci. Technol. 78 (1991) 127]. Initial temperature and concentration and the operating pressure of the system have a major effect on the delay of methanol ignition. The residence time of the continuous stirred reactor affects ignition delay and also changes the transient characteristic of chemical composition of the fuel-vapor mixture. The computations are intended to guide and explain many combustion studies that require a methanol kinetic mechanism.

  10. GroEL mediates protein folding with a two successive timer mechanism.

    PubMed

    Ueno, Taro; Taguchi, Hideki; Tadakuma, Hisashi; Yoshida, Masasuke; Funatsu, Takashi

    2004-05-21

    GroEL encapsulates nonnative substrate proteins in a central cavity capped by GroES, providing a safe folding cage. Conventional models assume that a single timer lasting approximately 8 s governs the ATP hydrolysis-driven GroEL chaperonin cycle. We examine single molecule imaging of GFP folding within the cavity, binding release dynamics of GroEL-GroES, ensemble measurements of GroEL/substrate FRET, and the initial kinetics of GroEL ATPase activity. We conclude that the cycle consists of two successive timers of approximately 3 s and approximately 5 s duration. During the first timer, GroEL is bound to ATP, substrate protein, and GroES. When the first timer ends, the substrate protein is released into the central cavity and folding begins. ATP hydrolysis and phosphate release immediately follow this transition. ADP, GroES, and substrate depart GroEL after the second timer is complete. This mechanism explains how GroES binding to a GroEL-substrate complex encapsulates the substrate rather than allowing it to escape into solution.

  11. Kinetic modelling indicates that fast-translating codons can coordinate cotranslational protein folding by avoiding misfolded intermediates

    NASA Astrophysics Data System (ADS)

    O'Brien, Edward P.; Vendruscolo, Michele; Dobson, Christopher M.

    2014-01-01

    It has been observed for several proteins that slowing down the rate at which individual codons are translated can increase their probability of cotranslational protein folding, while speeding up codon translation can decrease it. Here we investigate whether or not this inverse relationship between translation speed and the cotranslational folding probability is a general phenomenon or if other scenarios are possible. We first derive chemical kinetic equations that relate individual codon translation rates to the probability that a domain will fold, populate an intermediate or misfold, and examine the cotranslational folding scenarios that are possible within these models. We find that speeding up codon translation through misfolding-prone segments can, in some cases, increase the folding probability of a domain immediately before the nascent protein is released from the ribosome and decrease its chances of misfolding. Thus, for some proteins fast-translating codons could be as important as slow-translating codons in coordinating cotranslational protein folding.

  12. Absence of kinetic thermal stabilization in a hyperthermophile rubredoxin indicated by 40 microsecond folding in the presence of irreversible denaturation.

    PubMed

    LeMaster, David M; Tang, Jianzhong; Hernández, Griselda

    2004-10-01

    The striking kinetic stability of many proteins derived from hyperthermophilic organisms has led to the proposal that such stability may result from a heightened activation barrier for unfolding independent of a corresponding increase in the thermodynamic stability. This in turn implies a corresponding retardation of the folding reaction. A commonly cited model for kinetic thermal stabilization is the rubredoxin from Pyrococcus furiosus (Pf), which exhibits an irreversible denaturation lifetime at 100 degrees C of nearly a week. Utilizing protein resonances shifted well outside of the random coil chemical shift envelope, nuclear magnetic resonance (NMR) chemical exchange measurements on Pf rubredoxin as well as on the mesophile Clostridium pasteurianum (Cp) rubredoxin demonstrate reversible thermal transition temperatures of 144 degrees C (137 degrees C for the N-terminal modified A2K variant) and 104 degrees C, respectively, with similar (un)folding rates of approximately 25,000 s(-1), only modestly slower than the diffusion controlled rate. The absence of a substantial activation barrier to rubredoxin folding as well as the similar folding kinetics of the mesophile protein indicate that kinetic stabilization has not been utilized by the hyperthermophile rubredoxin in achieving its extreme thermal stability. The two-state folding kinetics observed for Pf rubredoxin contradict a previous assertion of multiphasic folding based on hydrogen exchange data extrapolated to an estimated midpoint of transition temperature (T(m)) of nearly 200 degrees C. This discrepancy is resolved by the observation that the base-catalyzed hydrogen exchange of the model dipeptide (N-acetyl-L-cysteine-N-methylamide)4-Cd2+ is 23-fold slower than that of the free cysteine model dipeptide used to normalize the Pf rubredoxin hydrogen exchange data.

  13. Kinetic mechanism of pulmonary carbonyl reductase.

    PubMed

    Matsuura, K; Nakayama, T; Nakagawa, M; Hara, A; Sawada, H

    1988-05-15

    The kinetic mechanism of guinea-pig lung carbonyl reductase was studied at pH 7 in the forward reaction with five carbonyl substrates and NAD(P)H and in the reverse reaction with propan-2-ol and NAD(P)+. In each case the enzyme mechanism was sequential, and product-inhibition studies were consistent with a di-iso ordered bi bi mechanism, in which NAD(P)H binds to the enzyme first and NAD(P)+ leaves last and the binding of cofactor induces isomerization. The kinetic and binding studies of the cofactors and several inhibitors such as pyrazole, benzoic acid, Cibacron Blue and benzamide indicate that the cofactor and Cibacron Blue bind to the free enzyme whereas the other inhibitors bind to the binary and/or ternary complexes.

  14. Kinetic mechanism of pulmonary carbonyl reductase.

    PubMed Central

    Matsuura, K; Nakayama, T; Nakagawa, M; Hara, A; Sawada, H

    1988-01-01

    The kinetic mechanism of guinea-pig lung carbonyl reductase was studied at pH 7 in the forward reaction with five carbonyl substrates and NAD(P)H and in the reverse reaction with propan-2-ol and NAD(P)+. In each case the enzyme mechanism was sequential, and product-inhibition studies were consistent with a di-iso ordered bi bi mechanism, in which NAD(P)H binds to the enzyme first and NAD(P)+ leaves last and the binding of cofactor induces isomerization. The kinetic and binding studies of the cofactors and several inhibitors such as pyrazole, benzoic acid, Cibacron Blue and benzamide indicate that the cofactor and Cibacron Blue bind to the free enzyme whereas the other inhibitors bind to the binary and/or ternary complexes. PMID:3048244

  15. Integration of Inhibition Kinetics and Molecular Dynamics Simulations: A Urea-Mediated Folding Study on Acetaldehyde Dehydrogenase 1.

    PubMed

    Xu, Yingying; Lee, Jinhyuk; Lü, Zhi-Rong; Mu, Hang; Zhang, Qian; Park, Yong-Doo

    2016-07-01

    Understanding the mechanism of acetaldehyde dehydrogenase 1 (ALDH1) folding is important because this enzyme is directly involved in several types of cancers and other diseases. We investigated the urea-mediated unfolding of ALDH1 by integrating kinetic inhibition studies with computational molecular dynamics (MD) simulations. Conformational changes in the enzyme structure were also analyzed using intrinsic and 1-anilinonaphthalene-8-sulfonate (ANS)-binding fluorescence measurements. Kinetic studies revealed that the direct binding of urea to ALDH1 induces inactivation of ALDH1 in a manner of mixed-type inhibition. Tertiary structural changes associated with regional hydrophobic exposure of the active site were observed. The urea binding regions on ALDH1 were predicted by docking simulations and were partly shared with active site residues of ALDH1 and with interface residues of the oligomerization domain for tetramer formation. The docking results suggest that urea prevents formation of the ALDH1 normal shape for the tetramer state as well as entrance of the substrate into the active site. Our study provides insight into the structural changes that accompany urea-mediated unfolding of ALDH1 and the catalytic role associated with conformational changes.

  16. Statistical mechanics of RNA folding: Importance of alphabet size

    NASA Astrophysics Data System (ADS)

    Mukhopadhyay, Ranjan; Emberly, Eldon; Tang, Chao; Wingreen, Ned S.

    2003-10-01

    We construct a base-stacking model of RNA secondary-structure formation and use it to study the mapping from sequence to structure. There are strong, qualitative differences between two-letter and four- or six-letter alphabets. With only two kinds of bases, most sequences have many alternative folding configurations and are consequently thermally unstable. Stable ground states are found only for a small set of structures of high designability, i.e., total number of associated sequences. In contrast, sequences made from four bases, as found in nature, or six bases have far fewer competing folding configurations, resulting in a much greater average stability of the ground state.

  17. Mechanism of development of inversion folding in the subsalt

    NASA Astrophysics Data System (ADS)

    Lunev, B. V.; Lapkovsky, V. V.

    2014-01-01

    The numerical modeling of the Archimedean upwelling of a salt bed predicts that, with mature diapirs (from the finger stage and later), an about 2-km-thick zone of inversion (mirror) folding, where the suprasalt diapirs correspond to the subsalt synclines while the suprasalt inter-diapir sags correspond to the anticlines in the subsalt, should be expected immediately below the bottom of the salt deposits. These deformations decay with depth. The development of the inversion folding is exceptionally due to the flow caused by the rising of the unstable layer.

  18. The Fast-Folding Mechanism of Villin Headpiece Subdomain Studied by Multiscale Distributed Computing.

    PubMed

    Harada, Ryuhei; Kitao, Akio

    2012-01-10

    The fast-folding mechanism of a 35-residue mini-protein, villin headpiece subdomain (HP35), was investigated using folding free energy landscape analysis with the multiscale free energy landscape calculation method (MSFEL). A major and a minor folding pathway were deduced from the folding free energy landscape. In the major folding pathway, the formation of helices II and III was the rate-limiting step in the transition to an intermediate state, triggered by the folding of the PLWK motif. HP35 then folds into the native structure through the formation of the hydrophobic core located at the center of the three-helix bundle. Mutations in the motif and hydrophobic core that suppressed folding into the native state drastically changed the folding free energy landscape compared to the wild type protein. In the minor folding pathway, nucleation of the hydrophobic core preceded formation of the motif.

  19. Mechanical Modeling and Computer Simulation of Protein Folding

    ERIC Educational Resources Information Center

    Prigozhin, Maxim B.; Scott, Gregory E.; Denos, Sharlene

    2014-01-01

    In this activity, science education and modern technology are bridged to teach students at the high school and undergraduate levels about protein folding and to strengthen their model building skills. Students are guided from a textbook picture of a protein as a rigid crystal structure to a more realistic view: proteins are highly dynamic…

  20. Mechanical Modeling and Computer Simulation of Protein Folding

    ERIC Educational Resources Information Center

    Prigozhin, Maxim B.; Scott, Gregory E.; Denos, Sharlene

    2014-01-01

    In this activity, science education and modern technology are bridged to teach students at the high school and undergraduate levels about protein folding and to strengthen their model building skills. Students are guided from a textbook picture of a protein as a rigid crystal structure to a more realistic view: proteins are highly dynamic…

  1. Multiple-probe analysis of folding and unfolding pathways of human serum albumin. Evidence for a framework mechanism of folding.

    PubMed

    Santra, Manas Kumar; Banerjee, Abhijit; Krishnakumar, Shyam Sundar; Rahaman, Obaidur; Panda, Dulal

    2004-05-01

    The changes in the far-UV CD signal, intrinsic tryptophan fluorescence and bilirubin absorbance showed that the guanidine hydrochloride (GdnHCl)-induced unfolding of a multidomain protein, human serum albumin (HSA), followed a two-state process. However, using environment sensitive Nile red fluorescence, the unfolding and folding pathways of HSA were found to follow a three-state process and an intermediate was detected in the range 0.25-1.5 m GdnHCl. The intermediate state displayed 45% higher fluorescence intensity than that of the native state. The increase in the Nile red fluorescence was found to be due to an increase in the quantum yield of the HSA-bound Nile red. Low concentrations of GdnHCl neither altered the binding affinity of Nile red to HSA nor induced the aggregation of HSA. In addition, the secondary structure of HSA was not perturbed during the first unfolding transition (<1.5 m GdnHCl); however, the secondary structure was completely lost during the second transition. The data together showed that the half maximal loss of the tertiary structure occurred at a lower GdnHCl concentration than the loss of the secondary structure. Further kinetic studies of the refolding process of HSA using multiple spectroscopic techniques showed that the folding occurred in two phases, a burst phase followed by a slow phase. An intermediate with native-like secondary structure but only a partial tertiary structure was found to form in the burst phase of refolding. Then, the intermediate slowly folded into the native state. An analysis of the refolding data suggested that the folding of HSA could be best explained by the framework model.

  2. Kinetic barriers and the role of topology in protein and RNA folding

    PubMed Central

    Sosnick, Tobin R.

    2008-01-01

    This review compares the folding behavior of proteins and RNAs. Topics covered include the role of topology in the determination of folding rates, major folding events including collapse, properties of denatured states, pathway heterogeneity, and the influence of the mode of initiation on the folding pathway. PMID:18502978

  3. Folding kinetics and thermodynamics of Pseudomonas syringae effector protein AvrPto provide insight into translocation via the type III secretion system.

    PubMed

    Dawson, Jennifer E; Nicholson, Linda K

    2008-07-01

    In order to infect their hosts, many Gram-negative bacteria translocate agents of infection, called effector proteins, through the type III secretion system (TTSS) into the host cytoplasm. This process is thought to require at least partial unfolding of these agents, raising the question of how an effector protein might unfold to enable its translocation and then refold once it reaches the host cytoplasm. AvrPto is a well-studied effector protein of Pseudomonas syringae pv tomato. The presence of a readily observed unfolded population of AvrPto in aqueous solution and the lack of a known secretion chaperone make it ideal for studying the kinetic and thermodynamic characteristics that facilitate translocation. Application of Nzz exchange spectroscopy revealed a global, two-state folding equilibrium with 16% unfolded population, a folding rate of 1.8 s(-1), and an unfolding rate of 0.33 s(-1) at pH 6.1. TrAvrPto stability increases with increasing pH, with only 2% unfolded population observed at pH 7.0. The R(1) relaxation of TrAvrPto, which is sensitive to both the global anisotropy of folded TrAvrPto and slow exchange between folded and unfolded conformations, provided independent verification of the global kinetic rate constants. Given the acidic apoplast in which the pathogen resides and the more basic host cytoplasm, these results offer an intriguing mechanism by which the pH dependence of stability and slow folding kinetics of AvrPto would allow efficient translocation of the unfolded form through the TTSS and refolding into its functional folded form once inside the host.

  4. Evolution of ribonuclease in relation to polypeptide folding mechanisms.

    NASA Technical Reports Server (NTRS)

    Barnard, E. A.; Cohen, M. S.; Gold, M. H.; Kim, J.-K.

    1972-01-01

    Comparisons of the N-terminal region of pancreatic RNAase in seven species are presented, taking into account cow, bison, deer, rat, pig, kangaroo, and turtle. The available limited evidence on hypervariable regions indicates that there is still an evolutionary constraint on them. It is proposed that there is a selection pressure acting on all regions of a protein sequence in evolution. Mutations that tend to obstruct the folding process can lead to various intensities of selection pressure.

  5. Evolution of ribonuclease in relation to polypeptide folding mechanisms.

    NASA Technical Reports Server (NTRS)

    Barnard, E. A.; Cohen, M. S.; Gold, M. H.; Kim, J.-K.

    1972-01-01

    Comparisons of the N-terminal region of pancreatic RNAase in seven species are presented, taking into account cow, bison, deer, rat, pig, kangaroo, and turtle. The available limited evidence on hypervariable regions indicates that there is still an evolutionary constraint on them. It is proposed that there is a selection pressure acting on all regions of a protein sequence in evolution. Mutations that tend to obstruct the folding process can lead to various intensities of selection pressure.

  6. Kinetics and mechanism of autohydrolysis of hardwoods.

    PubMed

    Chen, Xiaowen; Lawoko, Martin; Heiningen, Adriaan van

    2010-10-01

    Autohydrolysis using water is a promising method to extract hemicelluloses from wood prior to pulping in order to make co-products such as ethanol and acetic acid besides pulp. Many studies have been carried out on the kinetics and mechanism of autohydrolysis using batch reactors. The present study was performed in a continuous mixed flow reactor where the wood chips are retained in a basket inside the reactor. This reactor is well suited to determine intrinsic kinetics of hemicellulose dissolution because the dissolved products are rapidly removed from the reactor, thus minimizing further hydrolysis and degradation of the hemicelluloses in solution. The xylan removal rate follows an S-shaped behavior. GPC analysis of the continuously removed extract shows that the dissolved xylan oligomers have a DP smaller than about 25. Lignin-free xylan oligomers and cellulose oligomers are the major components dissolved in the initial stage of autohydrolysis, while xylan covalently bound to lignin (i.e. an LCC) is the major component removed during the later stage of autohydrolysis. The molecular weight of the dissolved components decreases with time in the second stage. The kinetics of xylan removal are explained in terms of a mechanism based on recent knowledge of the ultrastructure of the cell fibre wall.

  7. Kinetic study and mechanism of Niclosamide degradation.

    PubMed

    Zaazaa, Hala E; Abdelrahman, Maha M; Ali, Nouruddin W; Magdy, Maimana A; Abdelkawy, M

    2014-11-11

    A spectrophotometric kinetic study of Niclosamide alkaline degradation as a function of drug concentration, alkaline concentration and temperature has been established utilizing double divisor-ratio spectra spectrophotometric method. The developed method allowed determination of Niclosamide in presence of its alkaline degradation products; namely; 2-chloro-4-nitro aniline (DEG I) and 5-chloro salicylic acid (DEG II) with characterization of its degradation mechanism. It was found that degradation kinetic of Niclosamide followed pseudo-first order under the established experimental conditions with a degradation rate constant (k) of 0.0829 mol/h and half life (t1/2) of 8.35 h. The overall degradation rate constant as a function of the temperature under the given conditions obeyed Arrhenius equation where the activation energy was calculated to be 3.41 kcal/mol.

  8. Kinetic study and mechanism of Niclosamide degradation

    NASA Astrophysics Data System (ADS)

    Zaazaa, Hala E.; Abdelrahman, Maha M.; Ali, Nouruddin W.; Magdy, Maimana A.; Abdelkawy, M.

    2014-11-01

    A spectrophotometric kinetic study of Niclosamide alkaline degradation as a function of drug concentration, alkaline concentration and temperature has been established utilizing double divisor-ratio spectra spectrophotometric method. The developed method allowed determination of Niclosamide in presence of its alkaline degradation products; namely; 2-chloro-4-nitro aniline (DEG I) and 5-chloro salicylic acid (DEG II) with characterization of its degradation mechanism. It was found that degradation kinetic of Niclosamide followed pseudo-first order under the established experimental conditions with a degradation rate constant (k) of 0.0829 mol/h and half life (t1/2) of 8.35 h. The overall degradation rate constant as a function of the temperature under the given conditions obeyed Arrhenius equation where the activation energy was calculated to be 3.41 kcal/mol.

  9. The energy landscape of modular repeat proteins: topology determines folding mechanism in the ankyrin family.

    PubMed

    Ferreiro, Diego U; Cho, Samuel S; Komives, Elizabeth A; Wolynes, Peter G

    2005-12-02

    Proteins consisting of repeating amino acid motifs are abundant in all kingdoms of life, especially in higher eukaryotes. Repeat-containing proteins self-organize into elongated non-globular structures. Do the same general underlying principles that dictate the folding of globular domains apply also to these extended topologies? Using a simplified structure-based model capturing a perfectly funneled energy landscape, we surveyed the predicted mechanism of folding for ankyrin repeat containing proteins. The ankyrin family is one of the most extensively studied classes of non-globular folds. The model based only on native contacts reproduces most of the experimental observations on the folding of these proteins, including a folding mechanism that is reminiscent of a nucleation propagation growth. The confluence of simulation and experimental results suggests that the folding of non-globular proteins is accurately described by a funneled energy landscape, in which topology plays a determinant role in the folding mechanism.

  10. Single-molecule fluorescence resonance energy transfer studies of the human telomerase RNA pseudoknot: temperature-/urea-dependent folding kinetics and thermodynamics.

    PubMed

    Holmstrom, Erik D; Nesbitt, David J

    2014-04-10

    The ribonucleoprotein telomerase is an RNA-dependent DNA polymerase that catalyzes the repetitive addition of a short, species-specific, DNA sequence to the ends of linear eukaryotic chromosomes. The single RNA component of telomerase contains both the template sequence for DNA synthesis and a functionally critical pseudoknot motif, which can also exist as a less stable hairpin. Here we use a minimal version of the human telomerase RNA pseudoknot to study this hairpin-pseudoknot structural equilibrium using temperature-controlled single-molecule fluorescence resonance energy transfer (smFRET) experiments. The urea dependence of these experiments aids in determination of the folding kinetics and thermodynamics. The wild-type pseudoknot behavior is compared and contrasted to a mutant pseudoknot sequence implicated in a genetic disorder-dyskeratosis congenita. These findings clearly identify that this 2nt noncomplementary mutation destabilizes the folding of the wild-type pseudoknot by substantially reducing the folding rate constant (≈ 400-fold) while only nominally increasing the unfolding rate constant (≈ 5-fold). Furthermore, the urea dependence of the equilibrium and rate constants is used to develop a free energy landscape for this unimolecular equilibrium and propose details about the structure of the transition state. Finally, the urea-dependent folding experiments provide valuable physical insights into the mechanism for destabilization of RNA pseudoknots by such chemical denaturants.

  11. Single-Molecule Fluorescence Resonance Energy Transfer Studies of the Human Telomerase RNA Pseudoknot: Temperature-/Urea-Dependent Folding Kinetics and Thermodynamics

    PubMed Central

    2015-01-01

    The ribonucleoprotein telomerase is an RNA-dependent DNA polymerase that catalyzes the repetitive addition of a short, species-specific, DNA sequence to the ends of linear eukaryotic chromosomes. The single RNA component of telomerase contains both the template sequence for DNA synthesis and a functionally critical pseudoknot motif, which can also exist as a less stable hairpin. Here we use a minimal version of the human telomerase RNA pseudoknot to study this hairpin–pseudoknot structural equilibrium using temperature-controlled single-molecule fluorescence resonance energy transfer (smFRET) experiments. The urea dependence of these experiments aids in determination of the folding kinetics and thermodynamics. The wild-type pseudoknot behavior is compared and contrasted to a mutant pseudoknot sequence implicated in a genetic disorder–dyskeratosis congenita. These findings clearly identify that this 2nt noncomplementary mutation destabilizes the folding of the wild-type pseudoknot by substantially reducing the folding rate constant (≈ 400-fold) while only nominally increasing the unfolding rate constant (≈ 5-fold). Furthermore, the urea dependence of the equilibrium and rate constants is used to develop a free energy landscape for this unimolecular equilibrium and propose details about the structure of the transition state. Finally, the urea-dependent folding experiments provide valuable physical insights into the mechanism for destabilization of RNA pseudoknots by such chemical denaturants. PMID:24617561

  12. Studies of combustion kinetics and mechanisms

    SciTech Connect

    Gutman, D.

    1993-12-01

    The objective of the current research is to gain new quantitative knowledge of the kinetics and mechanisms of polyatomic free radicals which are important in hydrocarbon combustion processes. The special facility designed and built for these (which includes a heatable tubular reactor coupled to a photoionization mass spectrometer) is continually being improved. Where possible, these experimental studies are coupled with theoretical ones, sometimes conducted in collaboration with others, to obtain an improved understanding of the factors determining reactivity. The decomposition of acetyl radicals, isopropyl radicals, and n-propyl radicals have been studied as well as the oxidation of methylpropargyl radicals.

  13. Effect of surfaces in modulating protein folding mechanisms

    NASA Astrophysics Data System (ADS)

    Shea, Joan

    2014-03-01

    Protein-surface interactions are ubiquitous in the crowded cytosol, where proteins encounter a variety of surfaces, ranging from membranes surfaces, to the surfaces presented by chaperone molecules. Protein-surface interactions are also at the heart of a number of emerging technologies, including protein micro-arrays, biosensors and biomaterials. The effect of surfaces on protein structure and stability can vary substantially depending on the chemical composition of the surface. In this talk, I will present detailed atomistic simulations of the folding of a small beta-sheet protein in the presence of graphite and titanium oxide surfaces. The role of water-mediated and direct protein-surface interactions in governing protein conformations will be discussed.

  14. Mechanism of folding chamber closure in a group II chaperonin.

    PubMed

    Zhang, Junjie; Baker, Matthew L; Schröder, Gunnar F; Douglas, Nicholai R; Reissmann, Stefanie; Jakana, Joanita; Dougherty, Matthew; Fu, Caroline J; Levitt, Michael; Ludtke, Steven J; Frydman, Judith; Chiu, Wah

    2010-01-21

    Group II chaperonins are essential mediators of cellular protein folding in eukaryotes and archaea. These oligomeric protein machines, approximately 1 megadalton, consist of two back-to-back rings encompassing a central cavity that accommodates polypeptide substrates. Chaperonin-mediated protein folding is critically dependent on the closure of a built-in lid, which is triggered by ATP hydrolysis. The structural rearrangements and molecular events leading to lid closure are still unknown. Here we report four single particle cryo-electron microscopy (cryo-EM) structures of Mm-cpn, an archaeal group II chaperonin, in the nucleotide-free (open) and nucleotide-induced (closed) states. The 4.3 A resolution of the closed conformation allowed building of the first ever atomic model directly from the single particle cryo-EM density map, in which we were able to visualize the nucleotide and more than 70% of the side chains. The model of the open conformation was obtained by using the deformable elastic network modelling with the 8 A resolution open-state cryo-EM density restraints. Together, the open and closed structures show how local conformational changes triggered by ATP hydrolysis lead to an alteration of intersubunit contacts within and across the rings, ultimately causing a rocking motion that closes the ring. Our analyses show that there is an intricate and unforeseen set of interactions controlling allosteric communication and inter-ring signalling, driving the conformational cycle of group II chaperonins. Beyond this, we anticipate that our methodology of combining single particle cryo-EM and computational modelling will become a powerful tool in the determination of atomic details involved in the dynamic processes of macromolecular machines in solution.

  15. Using D-amino acids to delineate the mechanism of protein folding: Application to Trp-cage

    NASA Astrophysics Data System (ADS)

    Culik, Robert M.; Annavarapu, Srinivas; Nanda, Vikas; Gai, Feng

    2013-08-01

    Using the miniprotein Trp-cage as a model, we show that D-amino acids can be used to facilitate the delineation of protein folding mechanism. Specifically, we study the folding-unfolding kinetics of three Trp-cage mutants where the native glycine residue near the C-terminus of the α-helix is replaced by a D-amino acid. A previous study showed that these mutations increase the Trp-cage stability, due to a terminal capping effect. Our results show that the stabilizing effect of D-asparagine and D-glutamine originates almost exclusively from a decrease in the unfolding rate, while the D-alanine mutation results in a similar decrease in the unfolding rate, but it also increases the folding rate. Together, these results support a folding mechanism wherein the α-helix formation in the transition state is nucleated at the N-terminus, whereas those long-range native interactions stabilizing this helix are developed at the downhill side of the folding free energy barrier.

  16. Numerical impact simulation of gradually increased kinetic energy transfer has the potential to break up folded protein structures resulting in cytotoxic brain tissue edema.

    PubMed

    von Holst, Hans; Li, Xiaogai

    2013-07-01

    Although the consequences of traumatic brain injury (TBI) and its treatment have been improved, there is still a substantial lack of understanding the mechanisms. Numerical simulation of the impact can throw further lights on site and mechanism of action. A finite element model of the human head and brain tissue was used to simulate TBI. The consequences of gradually increased kinetic energy transfer was analyzed by evaluating the impact intracranial pressure (ICP), strain level, and their potential influences on binding forces in folded protein structures. The gradually increased kinetic energy was found to have the potential to break apart bonds of Van der Waals in all impacts and hydrogen bonds at simulated impacts from 6 m/s and higher, thereby superseding the energy in folded protein structures. Further, impacts below 6 m/s showed none or very slight increase in impact ICP and strain levels, whereas impacts of 6 m/s or higher showed a gradual increase of the impact ICP and strain levels reaching over 1000 KPa and over 30%, respectively. The present simulation study shows that the free kinetic energy transfer, impact ICP, and strain levels all have the potential to initiate cytotoxic brain tissue edema by unfolding protein structures. The definition of mild, moderate, and severe TBI should thus be looked upon as the same condition and separated only by a gradual severity of impact.

  17. Analysis of the kinetics of folding of proteins and peptides using circular dichroism

    PubMed Central

    Greenfield, Norma J.

    2009-01-01

    Circular dichroism (CD) is a useful spectroscopic technique for studying the secondary structure, folding and binding properties of proteins. This protocol covers how to use the intrinsic circular dichroic properties of proteins to follow their folding and unfolding as a function of time. Included will be methods of obtaining data and how to analyze the folding and unfolding data to determine the rate constants and the order of the folding/unfolding reactions. The protocol focuses on the use of CD to follow folding when it is relatively slow, on the order of minutes to days. The methods for analyzing the data, however, can also be applied to data collected with a CD machine equipped with stopped-flow accessories in the millisecond to second range and folding analyzed by other spectroscopic methods including changes in absorption or fluorescence spectra as a function of time. PMID:17406548

  18. How Kinetics within the Unfolded State Affects Protein Folding: an Analysis Based on Markov State Models and an Ultra-Long MD Trajectory

    PubMed Central

    Deng, Nan-jie; Dai, Wei

    2013-01-01

    Understanding how kinetics in the unfolded state affects protein folding is a fundamentally important yet less well-understood issue. Here we employ three different models to analyze the unfolded landscape and folding kinetics of the miniprotein Trp-cage. The first is a 208 μs explicit solvent molecular dynamics (MD) simulation from D. E. Shaw Research containing tens of folding events. The second is a Markov state model (MSM-MD) constructed from the same ultra-long MD simulation; MSM-MD can be used to generate thousands of folding events. The third is a Markov state model built from temperature replica exchange MD simulations in implicit solvent (MSM-REMD). All the models exhibit multiple folding pathways, and there is a good correspondence between the folding pathways from direct MD and those computed from the MSMs. The unfolded populations interconvert rapidly between extended and collapsed conformations on time scales ≤ 40 ns, compared with the folding time of ≈ 5 μs. The folding rates are independent of where the folding is initiated from within the unfolded ensemble. About 90 % of the unfolded states are sampled within the first 40 μs of the ultra-long MD trajectory, which on average explores ~27 % of the unfolded state ensemble between consecutive folding events. We clustered the folding pathways according to structural similarity into “tubes”, and kinetically partitioned the unfolded state into populations that fold along different tubes. From our analysis of the simulations and a simple kinetic model, we find that when the mixing within the unfolded state is comparable to or faster than folding, the folding waiting times for all the folding tubes are similar and the folding kinetics is essentially single exponential despite the presence of heterogeneous folding paths with non-uniform barriers. When the mixing is much slower than folding, different unfolded populations fold independently leading to non-exponential kinetics. A kinetic partition of

  19. Direct observation of an ensemble of stable collapsed states in the mechanical folding of ubiquitin

    PubMed Central

    Garcia-Manyes, Sergi; Dougan, Lorna; Badilla, Carmen L.; Brujić, Jasna; Fernández, Julio M.

    2009-01-01

    Statistical theories of protein folding have long predicted plausible mechanisms for reducing the vast conformational space through distinct ensembles of structures. However, these predictions have remained untested by bulk techniques, because the conformational diversity of folding molecules has been experimentally unapproachable. Owing to recent advances in single molecule force-clamp spectroscopy, we are now able to probe the structure and dynamics of the small protein ubiquitin by measuring its length and mechanical stability during each stage of folding. Here, we discover that upon hydrophobic collapse, the protein rapidly selects a subset of minimum energy structures that are mechanically weak and essential precursors of the native fold. From this much reduced ensemble, the native state is acquired through a barrier-limited transition. Our results support the validity of statistical mechanics models in describing the folding of a small protein on biological timescales. PMID:19541635

  20. Understanding the kinetic mechanism of RNA single base pair formation

    PubMed Central

    Xu, Xiaojun; Yu, Tao; Chen, Shi-Jie

    2016-01-01

    RNA functions are intrinsically tied to folding kinetics. The most elementary step in RNA folding is the closing and opening of a base pair. Understanding this elementary rate process is the basis for RNA folding kinetics studies. Previous studies mostly focused on the unfolding of base pairs. Here, based on a hybrid approach, we investigate the folding process at level of single base pairing/stacking. The study, which integrates molecular dynamics simulation, kinetic Monte Carlo simulation, and master equation methods, uncovers two alternative dominant pathways: Starting from the unfolded state, the nucleotide backbone first folds to the native conformation, followed by subsequent adjustment of the base conformation. During the base conformational rearrangement, the backbone either retains the native conformation or switches to nonnative conformations in order to lower the kinetic barrier for base rearrangement. The method enables quantification of kinetic partitioning among the different pathways. Moreover, the simulation reveals several intriguing ion binding/dissociation signatures for the conformational changes. Our approach may be useful for developing a base pair opening/closing rate model. PMID:26699466

  1. Thermodynamics and kinetics of protein folding on the ribosome: Alteration in energy landscapes, denatured state, and transition state ensembles

    NASA Astrophysics Data System (ADS)

    O'Brien, Edward; Vendruscolo, Michele; Dobson, Christopher

    2010-03-01

    In vitro experiments examining cotranslational folding utilize ribosome-nascent chain complexes (RNCs) in which the nascent chain is stalled at different points of its biosynthesis on the ribosome. We investigate the thermodynamics, kinetics, and structural properties of RNCs containing five different globular and repeat proteins stalled at ten different nascent chain lengths using coarse grained replica exchange simulations. We find that when the proteins are stalled near the ribosome exit tunnel opening they exhibit altered folding coopserativity, quantified by the van't Hoff enthalpy criterion; a significantly altered denatured state ensemble, in terms of Rg and shape parameters (Rg tensor); and the appearance of partially folded intermediates during cotranslation, evidenced by the appearance of a third basin in the free energy profile. These trends are due in part to excluded volume (crowding) interactions between the ribosome and nascent chain. We perform in silico temperature-jump experiments on the RNCs and examine nascent chain folding kinetics and structural changes in the transition state ensemble at various stall lengths.

  2. Trp zipper folding kinetics by molecular dynamics and temperature-jump spectroscopy

    NASA Astrophysics Data System (ADS)

    Snow, Christopher D.; Qiu, Linlin; Du, Deguo; Gai, Feng; Hagen, Stephen J.; Pande, Vijay S.

    2004-03-01

    We studied the microsecond folding dynamics of three hairpins (Trp zippers 1-3, TZ1-TZ3) by using temperature-jump fluorescence and atomistic molecular dynamics in implicit solvent. In addition, we studied TZ2 by using time-resolved IR spectroscopy. By using distributed computing, we obtained an aggregate simulation time of 22 ms. The simulations included 150, 212, and 48 folding events at room temperature for TZ1, TZ2, and TZ3, respectively. The all-atom optimized potentials for liquid simulations (OPLSaa) potential set predicted TZ1 and TZ2 properties well; the estimated folding rates agreed with the experimentally determined folding rates and native conformations were the global potential-energy minimum. The simulations also predicted reasonable unfolding activation enthalpies. This work, directly comparing large simulated folding ensembles with multiple spectroscopic probes, revealed both the surprising predictive ability of current models as well as their shortcomings. Specifically, for TZ1-TZ3, OPLS for united atom models had a nonnative free-energy minimum, and the folding rate for OPLSaa TZ3 was sensitive to the initial conformation. Finally, we characterized the transition state; all TZs fold by means of similar, native-like transition-state conformations.

  3. Bend, buckle, and fold: mechanical engineering with nanomembranes.

    PubMed

    Kim, Dae-Hyeong; Rogers, John A

    2009-03-24

    Research on nanomembranes and graphene sheets represents the "third wave" of work on nanomaterials, following earlier studies of nanoparticles/fullerenes and, somewhat later, nanowires/nanotubes. Inorganic semiconductor nanomembranes are particularly appealing due to their materials diversity, the ease with which they can be grown with high quality over large areas, and the ability to exploit them in unique, high-performance electronic and optoelectronic systems. The mechanics of such nanomembranes and the coupling of strain to their electronic properties are topics of considerable current interest. A new paper by the Lagally group in this issue combines single-crystalline silicon nanomembranes with chemical vapor deposition techniques to form "mechano-electronic" superlattices whose properties could lead to unusual classes of electronic devices.

  4. Deciphering the mechanisms of binding induced folding at nearly atomic resolution: The Φ value analysis applied to IDPs

    PubMed Central

    Gianni, Stefano; Dogan, Jakob; Jemth, Per

    2014-01-01

    The Φ value analysis is a method to analyze the structure of metastable states in reaction pathways. Such a methodology is based on the quantitative analysis of the effect of point mutations on the kinetics and thermodynamics of the probed reaction. The Φ value analysis is routinely used in protein folding studies and is potentially an extremely powerful tool to analyze the mechanism of binding induced folding of intrinsically disordered proteins. In this review we recapitulate the key equations and experimental advices to perform the Φ value analysis in the perspective of the possible caveats arising in intrinsically disordered systems. Finally, we briefly discuss some few examples already available in the literature. PMID:28232881

  5. Effect of hydrogen bond networks on the nucleation mechanism of protein folding

    NASA Astrophysics Data System (ADS)

    Djikaev, Y. S.; Ruckenstein, Eli

    2009-12-01

    We have recently developed a kinetic model for the nucleation mechanism of protein folding (NMPF) in terms of ternary nucleation by using the first passage time analysis. A protein was considered as a random heteropolymer consisting of hydrophobic, hydrophilic (some of which are negatively or positively ionizable), and neutral beads. The main idea of the NMPF model consisted of averaging the dihedral potential in which a selected residue is involved over all possible configurations of all neighboring residues along the protein chain. The combination of the average dihedral, effective pairwise (due to Lennard-Jones-type and electrostatic interactions), and confining (due to the polymer connectivity constraint) potentials gives rise to an overall potential around the cluster that, as a function of the distance from the cluster center, has a double-well shape. This allows one to evaluate the protein folding time. In the original NMPF model hydrogen bonding was not taken into account explicitly. To improve the NMPF model and make it more realistic, in this paper we modify our (previously developed) probabilistic hydrogen bond model and combine it with the former. Thus, a contribution due to the disruption of hydrogen bond networks around the interacting particles (cluster of native residues and residue in the protein unfolded part) appears in the overall potential field around a cluster. The modified model is applied to the folding of the same model proteins that were examined in the original model: a short protein consisting of 124 residues (roughly mimicking bovine pancreatic ribonuclease) and a long one consisting of 2500 residues (as a representative of large proteins with superlong polypeptide chains), at pH=8.3 , 7.3, and 6.3. The hydrogen bond contribution now plays a dominant role in the total potential field around the cluster (except for very short distances thereto where the repulsive energy tends to infinity). It is by an order of magnitude stronger for

  6. Characterization of the kinetic and thermodynamic landscape of RNA folding using a novel application of isothermal titration calorimetry.

    PubMed

    Vander Meulen, Kirk A; Butcher, Samuel E

    2012-03-01

    A novel isothermal titration calorimetry (ITC) method was applied to investigate RNA helical packing driven by the GAAA tetraloop-receptor interaction in magnesium and potassium solutions. Both the kinetics and thermodynamics were obtained in individual ITC experiments, and analysis of the kinetic data over a range of temperatures provided Arrhenius activation energies (ΔH(‡)) and Eyring transition state entropies (ΔS(‡)). The resulting rich dataset reveals strongly contrasting kinetic and thermodynamic profiles for this RNA folding system when stabilized by potassium versus magnesium. In potassium, association is highly exothermic (ΔH(25°C) = -41.6 ± 1.2 kcal/mol in 150 mM KCl) and the transition state is enthalpically barrierless (ΔH(‡) = -0.6 ± 0.5). These parameters are significantly positively shifted in magnesium (ΔH(25°C) = -20.5 ± 2.1 kcal/mol, ΔH(‡) = 7.3 ± 2.2 kcal/mol in 0.5 mM MgCl(2)). Mixed salt solutions approximating physiological conditions exhibit an intermediate thermodynamic character. The cation-dependent thermodynamic landscape may reflect either a salt-dependent unbound receptor conformation, or alternatively and more generally, it may reflect a small per-cation enthalpic penalty associated with folding-coupled magnesium uptake.

  7. Swelling and folding as mechanisms of 3D shape formation in thin elastic sheets

    NASA Astrophysics Data System (ADS)

    Dias, Marcelo A.

    We work with two different mechanisms to generate geometric frustration on thin elastic sheets; isotropic differential growth and folding. We describe how controlled growth and prescribing folding patterns are useful tools for designing three-dimensional objects from information printed in two dimensions. The first mechanism is inspired by the possibility to control shapes by swelling polymer films, where we propose a solution for the problem of shape formation by asking the question, “what 2D metric should be prescribed to achieve a given 3D shape?”', namely the reverse problem. We choose two different types of initial configurations of sheets, disk-like with one boundary and annular with two boundaries. We demonstrate our technique by choosing four examples of 3D axisymmetric shapes and finding the respective swelling factors to achieve the desired shape. Second, we present a mechanical model for a single curved fold that explains both the buckled shape of a closed fold and its mechanical stiffness. The buckling arises from the geometrical frustration between the prescribed crease angle and the bending energy of the sheet away from the crease. This frustration increases as the sheet's area increases. Stiff folds result in creases with constant space curvature while softer folds inherit the broken symmetry of the buckled shape. We extend the application of our numerical model to show the potential to study multiple fold structures.

  8. Reaction Mechanism Generator: Automatic construction of chemical kinetic mechanisms

    NASA Astrophysics Data System (ADS)

    Gao, Connie W.; Allen, Joshua W.; Green, William H.; West, Richard H.

    2016-06-01

    Reaction Mechanism Generator (RMG) constructs kinetic models composed of elementary chemical reaction steps using a general understanding of how molecules react. Species thermochemistry is estimated through Benson group additivity and reaction rate coefficients are estimated using a database of known rate rules and reaction templates. At its core, RMG relies on two fundamental data structures: graphs and trees. Graphs are used to represent chemical structures, and trees are used to represent thermodynamic and kinetic data. Models are generated using a rate-based algorithm which excludes species from the model based on reaction fluxes. RMG can generate reaction mechanisms for species involving carbon, hydrogen, oxygen, sulfur, and nitrogen. It also has capabilities for estimating transport and solvation properties, and it automatically computes pressure-dependent rate coefficients and identifies chemically-activated reaction paths. RMG is an object-oriented program written in Python, which provides a stable, robust programming architecture for developing an extensible and modular code base with a large suite of unit tests. Computationally intensive functions are cythonized for speed improvements.

  9. Architecture and Folding Mechanism of the Azoarcus Group I Pre-tRNA

    SciTech Connect

    Rangan,P.; Masquida, B.; Westhof, E.; Woodson, S.

    2004-01-01

    Self-splicing RNAs must evolve to function in their specific exon context. The conformation of a group I pre-tRNA{sup ile} from the bacterium Azoarcus was probed by ribonuclease T1 and hydroxyl radical cleavage, and by native gel electrophoresis. Biochemical data and three-dimensional models of the pre-tRNA showed that the tRNA is folded, and that the tRNA and intron sequences form separate tertiary domains. Models of the active site before steps 1 and 2 of the splicing reaction predict that exchange of the external G-cofactor and the 3{prime}-terminal G is accomplished by a slight conformational change in P9.0 of the Azoarcus group I intron. Kinetic assays showed that the pre-tRNA folds in minutes, much more slowly than the intron alone. The dependence of the folding kinetics on Mg{sup 2+} and the concentration of urea, and RNase T1 experiments showed that formation of native pre-tRNA is delayed by misfolding of P3-P9, including mispairing between residues in P9 and the tRNA. Thus, although the intron and tRNA sequences form separate domains in the native pre-tRNA, their folding is coupled via metastable non-native base-pairs. This could help prevent premature processing of the 5{prime} and 3{prime} ends of unspliced pre-tRNA.

  10. Multiple mechanisms mediate aluminum kinetics and neurotoxicity

    SciTech Connect

    Provan, S.D.

    1988-01-01

    There is considerable evidence suggesting that calcium (Ca) and membrane Ca-regulating systems may influence the kinetics and neurotoxicity of aluminum (Al). These interactions likely occur at the membrane level under physiological conditions. To more fully examine these mechanisms, the hypothesis that Al would interact with membranes under physiological conditions was tested. The results suggest that Al, under these conditions, could interact with and traverse biological membranes. A biological membrane model was used to test the hypothesis that Al interacts with an intestinal Ca transporting system. To evaluate the effects of a Ca deficient diet on Al absorption, rats were maintained on a Ca-deficient or Ca-replete diet for up to four weeks. A modified in situ preparations was prepared. Rats maintained on the Ca-deficient diet exhibited a more rapid disappearance rate from the gut and greater extent of absorption into the portal blood. To evaluate Al distribution, rats were injected with Al and maintained on the above diets for four weeks. There was generally more Al in the tissues of rats maintained on a Ca-deficient diet with the exception of heart and muscle. More Al accumulated within the hippocampus than the cerebral cortex in those rats fed a Ca-deficient diet and injected with 400 {mu}mol Al/kg. The release of radiolabeled glutamate from transverse, rat hippocampal slices was used as a model of Al neurotoxicity.

  11. Kinetic mechanism of chicken liver xanthine dehydrogenase.

    PubMed Central

    Bruguera, P; Lopez-Cabrera, A; Canela, E I

    1988-01-01

    The kinetic behaviour of chicken-liver xanthine dehydrogenase (xanthine/NAD+ oxidoreductase; EC 1.2.1.37) has been studied. Steady-state results, obtained from a wide range of concentrations of substrates and products, were fitted by rational functions of degree 1:1, 1:2, 2:2 and 3:3 with respect to substrates, and 0:1, 1:1, 0:2 and 1:2 with regard to products, using a non-linear regression program which guarantees the fit. The goodness of fit was improved using a computer program that combines model discrimination, parameter refinement and sequential experimental design. The AIC and F tests were also used for model discrimination. For comparative purposes, the xanthine/oxygen oxidoreductase reaction was also studied. From the functions which give the maximum improvement, the complete rate equation was deduced. The significance of the terms was stated by the above methods. It was concluded that xanthine dehydrogenase requires a minimum mechanism of degree 1:1 for xanthine, 2:2 for NAD+, 1:1 for uric acid and 1:2 for NADH in the xanthine/NAD+ oxidoreductase reaction. These are the minimum degrees required but a rate equation of higher degree is not excluded. PMID:3422556

  12. Kinetics and Mechanisms of Nanosilver Oxysulfidation

    PubMed Central

    Liu, Jingyu; Pennell, Kelly G.; Hurt, Robert H.

    2011-01-01

    Among the many new engineered nanomaterials, nanosilver is one of the highest priority cases for environmental risk assessment. Recent analysis of field samples from water treatment facilities suggests that silver is converted to silver sulfide, whose very low solubility may limit the bioavailability and adverse impact of silver in the environment. The present study demonstrates that silver nanoparticles react with dissolved sulfide species (HS−, S2−) under relevant but controlled laboratory conditions to produce silver sulfide nanostructures similar to those observed in the field. The reaction is tracked by time-resolved sulfide depletion measurements to yield quantitative reaction rates and stoichiometries. The reaction requires dissolved oxygen, and it is sensitive to pH and natural organic matter. Focusedion-beam analysis of surface films reveals an irregular coarse-grained sulfide phase that allows deep (> 1 μm) conversion of silver surfaces without passivation. At high sulfide concentrations, nanosilver oxysulfidation occurs by a direct particle-fluid reaction. At low sulfide concentration, quantitative kinetic analysis suggests a mechanistic switch to an oxidative dissolution/precipitation mechanism, in which the biologically active Ag+ ion is generated as an intermediate. The environmental transformation pathways for nanosilver will vary depending on the media-specific competing rates of oxidative dissolution and direct oxysulfidation. PMID:21770469

  13. Mesophile versus thermophile: insights into the structural mechanisms of kinetic stability.

    PubMed

    Kelch, Brian A; Agard, David A

    2007-07-20

    Obtaining detailed knowledge of folding intermediate and transition state (TS) structures is critical for understanding protein folding mechanisms. Comparisons between proteins adapted to survive extreme temperatures with their mesophilic homologs are likely to provide valuable information on the interactions relevant to the unfolding transition. For kinetically stable proteins such as alpha-lytic protease (alphaLP) and its family members, their large free energy barrier to unfolding is central to their biological function. To gain new insights into the mechanisms that underlie kinetic stability, we have determined the structure and high temperature unfolding kinetics of a thermophilic homolog, Thermobifida fusca protease A (TFPA). These studies led to the identification of a specific structural element bridging the N and C-terminal domains of the protease (the "domain bridge") proposed to be associated with the enhanced high temperature kinetic stability in TFPA. Mutagenesis experiments exchanging the TFPA domain bridge into alphaLP validate this hypothesis and illustrate key structural details that contribute to TFPA's increased kinetic thermostability. These results lead to an updated model for the unfolding transition state structure for this important class of proteases in which domain bridge undocking and unfolding occurs at or before the TS. The domain bridge appears to be a structural element that can modulate the degree of kinetic stability of the different members of this class of proteases.

  14. KINETICS AND MECHANISMS OF NOx - CHAR REDUCTION

    SciTech Connect

    Suuberg, E.M.

    1998-06-19

    This study was undertaken in order to improve understanding of several aspects of the NO-carbon reaction. This reaction is of practical importance in combustion systems, but its close examination also provides some fundamental insight into oxidizing gas-carbon reactions. As part of this study, a comprehensive literature review of earlier work on this reaction has been published (Aarna and Suuberg, Fuel, 1997, 76, 475-491). It has been thought for some time that the kinetics of the NO-carbon reaction are unusual, in that they often show a two-regime Arrhenius behavior. It has, however, turned out during this work that NO is not alone in this regard. In this laboratory, we also uncovered evidence of two kinetic regime behavior in CO{sub 2} gasification. In another laboratory, a former colleague has identified the same behavior in N{sub 2}O. The low temperature reaction regime always shows an activation energy which is lower than that in the high temperature regime, leaving little doubt that a shift in mechanism, as opposed to transport limitations, dictates the behavior. The activation energy of the low temperature regime of these reactions is typically less than 100 kJ/mol, and the activation energy of the high temperature regime is generally considerably in excess of this value. In this study, we have resolved some apparent inconsistencies in the explanation of the low temperature regime, whose rate has generally been ascribed to desorption-controlled processes. Part of the problem in characterization of the different temperature regimes is that they overlap to a high degree. It is difficult to probe the low temperature regime experimentally, because of slow relaxation of the surface oxides in that regime. Using careful experimental techniques, we were able to demonstrate that the low temperature regime is indeed characterized by zero order in NO, as it must be. A separate study is being carried out to model the behavior in this regime in NO and in other gases, and

  15. Kinematics, structural mechanics, and design of origami structures with smooth folds

    NASA Astrophysics Data System (ADS)

    Peraza Hernandez, Edwin Alexander

    model for the structural mechanics of origami continuum bodies with smooth folds is presented. Such a model entails the integration of the presented kinematic model and existing plate theories in order to obtain a structural representation for folds having non-zero thickness and comprised of arbitrary materials. The model is validated against finite element analysis. The last contribution addresses the design and analysis of active material-based self-folding structures that morph via simultaneous folding towards a given three-dimensional goal shape starting from a planar configuration. Implementation examples including shape memory alloy (SMA)-based self-folding structures are provided.

  16. Mechanical versus kinematical shortening reconstructions of the Zagros High Folded Zone (Kurdistan region of Iraq)

    NASA Astrophysics Data System (ADS)

    Frehner, Marcel; Reif, Daniel; Grasemann, Bernhard

    2012-06-01

    This paper compares kinematical and mechanical techniques for the palinspastic reconstruction of folded cross sections in collision orogens. The studied area and the reconstructed NE-SW trending, 55.5 km long cross section is located in the High Folded Zone of the Zagros fold-and-thrust belt in the Kurdistan region of Iraq. The present-day geometry of the cross section has been constructed from field as well as remote sensing data. In a first step, the structures and the stratigraphy are simplified and summarized in eight units trying to identify the main geometric and mechanical parameters. In a second step, the shortening is kinematically estimated using the dip domain method to 11%-15%. Then the same cross section is used in a numerical finite element model to perform dynamical unfolding simulations taking various rheological parameters into account. The main factor allowing for an efficient dynamic unfolding is the presence of interfacial slip conditions between the mechanically strong units. Other factors, such as Newtonian versus power law viscous rheology or the presence of a basement, affect the numerical simulations much less strongly. If interfacial slip is accounted for, fold amplitudes are reduced efficiently during the dynamical unfolding simulations, while welded layer interfaces lead to unrealistic shortening estimates. It is suggested that interfacial slip and decoupling of the deformation along detachment horizons is an important mechanical parameter that controlled the folding processes in the Zagros High Folded Zone.

  17. Mechanical versus kinematical shortening reconstructions of the Zagros High Folded Zone (Kurdistan Region of Iraq)

    NASA Astrophysics Data System (ADS)

    Frehner, M.; Reif, D.; Grasemann, B.

    2012-04-01

    Our study compares kinematical and mechanical techniques for the palinspastic reconstruction of folded cross-sections in collision orogens. The studied area and the reconstructed NE-SW-trending, 55.5 km long cross-section is located in the High Folded Zone of the Zagros fold-and-thrust belt in the Kurdistan Region of Iraq. The present-day geometry of the cross-section has been constructed from field, as well as remote sensing data. In a first step, the structures and the stratigraphy are simplified and summarized in eight units trying to identify the main geometric and mechanical parameters. In a second step, the shortening is kinematically estimated using the dip-domain method to 11%-15%. Then the same cross-section is used in a numerical finite-element model to perform dynamical unfolding simulations taking various rheological parameters into account. The main factor allowing for an efficient dynamic unfolding is the presence of interfacial slip conditions between the mechanically strong units. Other factors, such as Newtonian vs. power-law viscous rheology or the presence of a basement affect the numerical simulations much less strongly. If interfacial slip is accounted for, fold amplitudes are reduced efficiently during the dynamical unfolding simulations, while welded layer interfaces lead to unrealistic shortening estimates. It is suggested that interfacial slip and decoupling of the deformation along detachment horizons is an important mechanical parameter that controlled the folding processes in the Zagros High Folded Zone.

  18. Design of a mechanical larynx with agarose as a soft tissue substitute for vocal fold applications.

    PubMed

    Choo, J Q; Lau, D P C; Chui, C K; Yang, T; Chng, C B; Teoh, S H

    2010-06-01

    Mechanical and computational models consisting of flow channels with convergent and oscillating constrictions have been applied to study the dynamics of human vocal fold vibration. To the best of our knowledge, no mechanical model has been studied using a material substitute with similar physical properties to the human vocal fold for surgical experimentation. In this study, we design and develop a mechanical larynx with agarose as a vocal fold substitute, and assess its suitability for surgical experimentation. Agarose is selected as a substitute for the vocal fold as it exhibits similar nonlinear hyperelastic characteristics to biological soft tissue. Through uniaxial compression and extension tests, we determined that agarose of 0.375% concentration most closely resembles the vocal fold mucosa and ligament of a 20-year old male for small tensile strain with an R(2) value of 0.9634 and root mean square error of 344.05±39.84 Pa. Incisions of 10 mm lengthwise and 3 mm in depth were created parallel to the medial edge on the superior surface of agar phantom. These were subjected to vibrations of 80, 130, and 180 Hz, at constant amplitude of 0.9 mm over a period of 10 min each in the mechanical larynx model. Lateral expansion of the incision was observed to be most significant for the lower frequency of 80 Hz. This model serves as a basis for future assessments of wound closure techniques during microsurgery to the vocal fold.

  19. Kinetic regulation mechanism of pbuE riboswitch

    NASA Astrophysics Data System (ADS)

    Gong, Sha; Wang, Yujie; Zhang, Wenbing

    2015-01-01

    Riboswitches are RNA residue segments located in untranslated regions of messenger RNAs. These folded segments directly bind ligands through shape complementarity and specific interactions in cells and alter the expression of genes at the transcriptional or translational level through conformation change. Using the recently developed systematic helix-based computational method to predict the cotranscription folding kinetics, we theoretically studied the cotranscription folding behavior of the Bacillus subtilis pbuE riboswitch in the absence and presence of the ligand. The ligand concentration, the transcription speed, and the transcription pausing are incorporated into the method. The results are in good agreement with the experimental results. We find that there are no obvious misfolded structures formed during the transcription and the formation of the ligand bound state is rate-limited by the association of the ligand and the RNA. For this kinetically driven riboswitch, the ligand concentration, the transcription speed, and the transcription pausing are coupled to perform regulatory activity.

  20. Effect of hydrophobic interactions on the folding mechanism of β-hairpins.

    PubMed

    Popp, Alexander; Wu, Ling; Keiderling, Timothy A; Hauser, Karin

    2014-12-11

    Hydrophobic interactions are essential in stabilizing protein structures. How they affect the folding pathway and kinetics, however, is less clear. We used time-resolved infrared spectroscopy to study the dynamics of hydrophobic interactions of β-hairpin variants of the sequence Trpzip2 (SWTWENGKWTWK-NH2) that is stabilized by two cross-strand Trp-Trp pairs. The hydrophobicity strength was varied by substituting the tryptophans pairwise by either tyrosines or valines. Relaxation dynamics were induced by a laser-excited temperature jump, which separately probed for the loss of the cross-strand β-hairpin interaction and the rise of the disordered structure. All substitutions tested result in reduced thermal stability, lower transition temperatures, and faster dynamics compared to Trpzip2. However, the changes in folding dynamics depend on the amino acid substituted for Trp. The aromatic substitution of Tyr for Trp results in the same kinetics for the unfolding of sheet and growth of disorder, with similar activation energies, independent of the substitution position. Substitution of Trp with a solely hydrophobic Val results in even faster kinetics than substitution with Tyr but is additionally site-dependent. If the hairpin has a Val pair close to its termini, the rate constants for loss of sheet and gain of disorder are the same, but if the pair is close to the turn, the sheet and disorder components show different relaxation kinetics. The Trp → Val substitutions reveal that hydrophobic interactions alone weakly stabilize the hairpin structure, but adding edge-to-face aromatic interaction strengthens it, and both modify the complex folding process.

  1. Understanding the Mechanism of Prosegment-catalyzed Folding by Solution NMR Spectroscopy*

    PubMed Central

    Wang, Shenlin; Horimoto, Yasumi; Dee, Derek R.; Yada, Rickey Y.

    2014-01-01

    Multidomain protein folding is often more complex than a two-state process, which leads to the spontaneous folding of the native state. Pepsin, a zymogen-derived enzyme, without its prosegment (PS), is irreversibly denatured and folds to a thermodynamically stable, non-native conformation, termed refolded pepsin, which is separated from native pepsin by a large activation barrier. While it is known that PS binds refolded pepsin and catalyzes its conversion to the native form, little structural details are known regarding this conversion. In this study, solution NMR was used to elucidate the PS-catalyzed folding mechanism by examining the key equilibrium states, e.g. native and refolded pepsin, both in the free and PS-bound states, and pepsinogen, the zymogen form of pepsin. Refolded pepsin was found to be partially structured and lacked the correct domain-domain structure and active-site cleft formed in the native state. Analysis of chemical shift data revealed that upon PS binding refolded pepsin folds into a state more similar to that of pepsinogen than to native pepsin. Comparison of pepsin folding by wild-type and mutant PSs, including a double mutant PS, indicated that hydrophobic interactions between residues of prosegment and refolded pepsin lower the folding activation barrier. A mechanism is proposed for the binding of PS to refolded pepsin and how the formation of the native structure is mediated. PMID:24265313

  2. Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics.

    PubMed

    Aliye, Naser; Fabbretti, Attilio; Lupidi, Giulio; Tsekoa, Tsepo; Spurio, Roberto

    2015-02-01

    A number of studies have been conducted to improve chromophore maturation, folding kinetics, thermostability, and other traits of green fluorescent protein (GFP). However, no specific work aimed at improving the thermostability of the yellow fluorescent protein (YFP) and of the pH-sensitive, yet thermostable color variants of GFP has so far been done. The protein variants reported in this study were improved through rational multiple site-directed mutagenesis of GFP (ASV) by introducing up to ten point mutations including the mutations near and at the chromophore region. Therefore, we report the development and characterization of fast folder and thermo-tolerant green variant (FF-GFP), and a fast folder thermostable yellow fluorescent protein (FFTS-YFP) endowed with remarkably improved thermostability and folding kinetics. We demonstrate that the fluorescence intensity of this yellow variant is not affected by heating at 75 °C. Moreover, we have developed a pH-unresponsive cyan variant AcS-CFP, which has potential use as part of in vivo imaging irrespective of intracellular pH. The combined improved properties make these fluorescent variants ideal tools to study protein expression and function under different pH environments, in mesophiles and thermophiles. Furthermore, coupling of the FFTS-YFP and AcS-CFP could potentially serve as an ideal tool to perform functional analysis of live cells by multicolor labeling.

  3. Modeling of the Kinetics of Metal Film Growth on 5-Fold Surfaces of Icosohedral Quasicrystals

    NASA Astrophysics Data System (ADS)

    Evans, J. W.; Unal, B.; Fournee, V.; Ghosh, C.; Liu, D.-J.; Jenks, C. J.; Thiel, P. A.

    2007-03-01

    During submonolayer deposition of metals on 5-f icosohedral Al- Pd-Mn and Al-Cu-Fe surfaces, experimental evidence for several system points to heterogeneous nucleation of islands at specific ``dark star'' trap sites. We model this phenomenon using a mean-field rate equation formulation for Ag on Al-Pd-Mn, where data is available for both the flux and temperature dependence of the island density. We also utilize a more sophisticated kinetic Monte Carlo simulation approach to analyze an atomistic lattice-gas model (for an appropriate ``disordered-bond-network'' of nearest-neighbor adsorption sites) describing nucleation of starfish islands observed by STM for Al on Al-Cu-Fe. Finally, we briefly describe multilayer growth morphologies (which can display kinetic roughening or quantum size effects), but which also generally reflect the submonolayer island distribution. B. Unal et al. PRB 75 (2007); C. Ghosh et al. Phil. Mag. 86 (2006) 831; Surf. Sci. 600 (2006) 1110; V. Fournee et al. PRL 95 (2005) 155504.

  4. Infrared study of the stability and folding kinetics of a series of β-hairpin peptides with a common NPDG turn.

    PubMed

    Xu, Yao; Du, Deguo; Oyola, Rolando

    2011-12-29

    The thermal stability and folding kinetics of a series of 15-residue β-hairpins with a common Type I [3:5] NPDG turn were studied using Fourier transform infrared spectroscopy (FTIR) and laser-induced temperature jump (T-jump) with infrared detection, respectively. Mutations at positions 3, 5, or 13 in the peptide sequence SEXYXNPDGTWTXTE, where X represents the position of mutation, were performed to study the roles of hydrophobic interactions in determining the thermodynamic and kinetic properties of β-hairpin folding. The thermal stability studies show a broad thermal folding/unfolding transition for all the peptides. T-jump studies indicate that these β-hairpin peptides fold in less than 2 μs. In addition, both folding and unfolding rate constants decrease with increasing strength of hydrophobic interactions. Kinetically, the hydrophobic interactions have more significant influence on the unfolding rate than the folding rate. Φ-value analysis indicates that the hydrophobic interactions between the side chains are mainly formed at the latter part of the transition-state region during the folding process. In summary, the results suggest that the formation of the native structure of these β-hairpins depends on the correct topology of the hydrophobic cluster. Besides the formation of the turn region as a key process for folding as suggested by previous studies, a hydrophobic collapse process may also play a crucial role during β-hairpin folding.

  5. Ring Separation Highlights the Protein-Folding Mechanism Used by the Phage EL-Encoded Chaperonin.

    PubMed

    Molugu, Sudheer K; Hildenbrand, Zacariah L; Morgan, David Gene; Sherman, Michael B; He, Lilin; Georgopoulos, Costa; Sernova, Natalia V; Kurochkina, Lidia P; Mesyanzhinov, Vadim V; Miroshnikov, Konstantin A; Bernal, Ricardo A

    2016-04-05

    Chaperonins are ubiquitous, ATP-dependent protein-folding molecular machines that are essential for all forms of life. Bacteriophage φEL encodes its own chaperonin to presumably fold exceedingly large viral proteins via profoundly different nucleotide-binding conformations. Our structural investigations indicate that ATP likely binds to both rings simultaneously and that a misfolded substrate acts as the trigger for ATP hydrolysis. More importantly, the φEL complex dissociates into two single rings resulting from an evolutionarily altered residue in the highly conserved ATP-binding pocket. Conformational changes also more than double the volume of the single-ring internal chamber such that larger viral proteins are accommodated. This is illustrated by the fact that φEL is capable of folding β-galactosidase, a 116-kDa protein. Collectively, the architecture and protein-folding mechanism of the φEL chaperonin are significantly different from those observed in group I and II chaperonins.

  6. Critical State Finite Element Models of Contractional Fault-Related Folding: Structural and Mechanical Analyses

    NASA Astrophysics Data System (ADS)

    Albertz, M.; Lingrey, S.; Sanz, P. F.

    2011-12-01

    Geometric/kinematic models of the common fault-related fold types (fault-bend, fault-propagation, detachment folding) typically assume a simplified flexural-slip based bed-parallel simple shear mechanism. The magnitude of local strain is a function of layer dip change irrespective of material properties. Line-lengths parallel to and layer thicknesses orthogonal to the flexural-slip surface remain constant. This study reports on a range of more complicated kinematic and mechanical responses observed in fourteen idealized forward numerical models of contractional fault-related folding. The models test the effects of material properties, initial fault dip, and the presence of weak inter-layer detachment horizons. We employ a Lagrangian finite element method with adaptive remeshing and a constitutive model that is based on critical state mechanics. This approach allows for large, volumetric deformation and realistic evolution of the failure envelope during progressive deformation. We demonstrate that material properties affect the way faults propagate and thus exert a significant control on resultant fold layer geometry. In most cases, these geometries differ from the flexural-slip based kinematic idealizations. For instance, models of uniform sandstone properties exhibit efficient strain localization and clear patterns of fault tip propagation. Uniform shale properties tends to inhibit fault propagation due to distributed plastic deformation. Models with mixed inter-layered sandstone and shale deform in a disharmonic manner, resembling lobate-cuspate arrangements that are common to many outcrop-scale folds. Inter-layer detachments accommodate shortening by bed-parallel slip, resulting in fault-bend fold kinematics, imbrication of sand layers, and a general absence of fault propagation across layers. Constant area based plane strain restoration of the deformed models recovers the first-order contractional deformation (80-90% of true contractional strain). Constant line

  7. The natural history of recoverable vocal fold paralysis: Implications for kinetics of reinnervation.

    PubMed

    Mau, Ted; Pan, Hao-Min; Childs, Lesley F

    2017-06-13

    Patients with unilateral vocal fold paralysis (UVFP) are commonly told to wait 12 months for spontaneous recovery. This study aims to 1) determine the time to vocal recovery in UVFP, 2) use that data to develop a neurophysiologically plausible model for recovery, and 3) use the model to generate meaningful predictions for patient counseling. Case series with de novo mathematical modeling. Patients with UVFP who could pinpoint a discrete onset of vocal improvement were identified. The time-to-recovery data were modeled by assuming an "early" recovery group with neuropraxia and a "late" recovery group with more severe nerve injury. For the late group, a two-stage model was developed to explain the time to recovery: regenerating axons must cross the site of injury in stage 1 (probabilistic), followed by unimpeded regrowth to the larynx in stage 2 (deterministic). Of 727 cases of UVFP over a 7-year period, 44 reported spontaneous recovery with a discrete onset of vocal improvement. A hybrid distribution incorporating the two stages (exponentially modified Gaussian) accurately modeled the time-to-recovery data (R(2) = 0.918). The model predicts 86% of patients with recoverable UVFP will recover within 6 months, with 96% recovering within 9 months. Earlier vocal recovery is associated with recovery of vocal fold motion and younger age. Waiting 12 months for spontaneous recovery is probably too conservative. Repair across the site of injury, and not regrowth to larynx, is likely the rate-determining step in reinnervation, consistent with other works on peripheral nerve regeneration. 4. Laryngoscope, 2017. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.

  8. On the Signaling of Electrochemical Aptamer-Based Sensors: Collision- and Folding-Based Mechanisms

    PubMed Central

    Xiao, Yi; Uzawa, Takanori; White, Ryan J.; DeMartini, Daniel; Plaxco, Kevin W.

    2010-01-01

    Recent years have seen the emergence of a new class of electrochemical sensors predicated on target binding-induced folding of electrode-bound redox-modified aptamers and directed against targets ranging from small molecules to proteins. Previous studies of the relationship between gain and probe-density for these electrochemical, aptamer-based (E-AB) sensors suggest that signal transduction is linked to binding-induced changes in the efficiency with which the attached redox tag strikes the electrode. This, in turn, suggests that even well folded aptamers may support E-AB signaling if target binding sufficiently alters their flexibility. Here we investigate this using a thrombin-binding aptamer that undergoes binding-induced folding at low ionic strength but can be forced to adopt a folded conformation at higher ionic strength even in the absence of its protein target. We find that, under conditions in which the thrombin aptamer is fully folded prior to target binding, we still obtain a ca. 30% change in E-AB signal upon saturated target levels. In contrast, however, under conditions in which the aptamer is unfolded in the absence of target and thus undergoes binding-induced folding the observed signal change is twice as great. The ability of folded aptamers to support E-AB signaling, however, is not universal: a fully folded anti-IgE aptamer, for example, produces only an extremely small, ca. 2.5% signal change in the presence of target despite the larger steric bulk of this protein. Thus, while it appears that binding-induced changes in the dynamics in fully folded aptamers can support E-AB signaling, this signaling mechanism may not be general, and in order to ensure the design of high-gain sensors binding must be linked to a large-scale conformational change. PMID:20436787

  9. Temperature dependence of the folding and unfolding kinetics of the GCN4 leucine zipper via 13C(alpha)-NMR.

    PubMed Central

    Holtzer, M E; Bretthorst, G L; d'Avignon, D A; Angeletti, R H; Mints, L; Holtzer, A

    2001-01-01

    Studies by one-dimensional NMR are reported on the interconversion of folded and unfolded forms of the GCN4 leucine zipper in neutral saline buffer. The peptide bears 99% 13C(alpha) labels at three sites: V9, L12, and G31. Time-domain 13C(alpha)-NMR spectra are interpreted by global Bayesian lineshape analysis to extract the rate constants for both unfolding and folding as functions of temperature in the range 47-71 degrees C. The data are well fit by the assumption that the same rate constants apply at each labeled site, confirming that only two conformational states need be considered. Results show that 1) both processes require a free energy of activation; 2) unfolding is kinetically enthalpy-opposed and entropy-driven, while folding is the opposite; and 3) the transition state dimer ensemble averages approximately 40% helical. The activation parameters for unfolding, derived from NMR data at the elevated temperatures where both conformations are populated, lead to estimates of the rate constant at low temperatures (5-15 degrees C) that agree with extant values determined by stopped-flow CD via dilution from denaturing media. However, the corresponding estimated values for the folding rate constant are larger by two to three orders of magnitude than those obtained by stopped flow. We propose that this apparent disagreement is caused by the necessity, in the stopped-flow experiment, for initiation of new helices as the highly denaturant-unfolded molecule adjusts to the newly created benign solvent conditions. This must reduce the success rate of collisions in producing the folded molecule. In the NMR determinations, however, the unfolded chains always have a small, but essential, helix content that makes such initiation unnecessary. Support for this hypothesis is adduced from recent extant experiments on the helix-coil transition in single-chain helical peptides and from demonstration that the folding rate constants for coiled coils, as obtained by stopped flow

  10. Molecular Dynamics Simulation for the Dynamics and Kinetics of Folding Peptides in the Gas Phase.

    PubMed

    Litinas, Iraklis; Koutselos, Andreas D

    2015-12-31

    The conformations of flexible molecular species, such as oligomers and oligopeptides, and their interconversion in the gas phase have been probed by ion mobility spectrometry measurements. The ion motion is interpreted through the calculation of effective cross sections in the case of stable conformations of the macromolecules. However, when the molecular structures transform to each other as the ions collide with gas atoms during their flight through the drift tube, the introduction of an average cross section is required. To provide a direct way for the reproduction of the ion motion, we employ a nonequilibrium molecular dynamics simulation method and consider a molecular model that consists of two connected stiff cylindrical bodies interacting through an intramolecular model potential. With this procedure we have calculated the ion mobility as a function of temperature for a prototype peptide that converts between a helical and an extended globular form. The results are in good agreement with ion mobility spectrometry data confirming that an angular vibration coordinate can be used for the interpretation of the shifting of the drift-time distributions at high temperatures. The approach produces mean kinetic energies as well as various combined distributions of the ion degrees of freedom. It is easily applied to flexible macromolecular ions and can be extended to include additional degrees of freedom.

  11. Probing Protein Folding Kinetics with High-resolution, Stabilized Optical Tweezers

    NASA Astrophysics Data System (ADS)

    Wong, Wesley; Halvorsen, Ken

    2009-03-01

    Single-molecule techniques provide a powerful means of exploring molecular transitions such as the unfolding and refolding of a protein. However, the quantification of bi-directional transitions and near-equilibrium phenomena poses unique challenges, and is often limited by the detection resolution and long-term stability of the instrument. We have developed unique optical tweezers methods that address these problems, including an interference-based method for high-resolution 3D bead tracking (˜1 nm laterally, ˜0.3 nm vertically, at > 100 Hz), and a continuous autofocus system that stabilizes the trap height to within 1-2 nm longterm [1,2]. We have used our instruments to quantify the force-dependent unfolding and refolding kinetics of single protein domains (e.g. spectrin in collaboration with E. Evans). These single-molecule studies are presented, together with the accompanying probabilistic analysis that we have developed. References: 1. W.P. Wong, V. Heinrich, E. Evans, Mat. Res. Soc. Symp. Proc., 790, P5.1-P5.10 (2004). 2. V. Heinrich, W.P. Wong, K. Halvorsen, E. Evans, Langmuir, 24, 1194-1203 (2008).

  12. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  13. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  14. Impact of Superparamagnetic Iron Oxide Nanoparticles on Vocal Fold Fibroblasts: Cell Behavior and Cellular Iron Kinetics.

    PubMed

    Pöttler, Marina; Fliedner, Anna; Schreiber, Eveline; Janko, Christina; Friedrich, Ralf Philipp; Bohr, Christopher; Döllinger, Michael; Alexiou, Christoph; Dürr, Stephan

    2017-12-01

    The voice is the most important instrument of communication. Tissue defects in the vocal fold (VF) area lead to serious reduction in quality of life, but thus far, no satisfactory VF implant exists. Therefore, we aim to establish a functional VF implant in a rabbit model by magnetic tissue engineering (MTE) using superparamagnetic iron oxide nanoparticles (SPION). Hence, iron quantification over time as well as cell behavior studies upon SPION treatment are of great importance. Rabbit VF fibroblasts (VFF) were treated with different concentrations of SPIONs (20, 40, and 80 μg/cm(2)), and iron content was examined for up to 40 days using microwave plasma-atom emission spectroscopy. The effects of SPION treatment on VFF (adhesion, spreading, and migration), which are important for the formation of 3D structures, were tested. Cellular SPION quantification revealed that there was no residual iron remaining in VFFs after 40 days. SPIONs had a dose-dependent effect on cell adhesion, with good tolerability observed up to 20 μg/cm(2). Migration and spreading were not significantly influenced by SPION treatment up to 80 μg/cm(2). To develop 3D structures, cell behavior should not be affected by SPION uptake. After 40 days, cells were free of iron as a result of metabolism or rarefication during cell division. Cell functions including adhesion, spreading, and migration were proven to be intact in a dose-dependent manner after SPION treatment, suggesting a safe usage of MTE for voice rehabilitation. Our results thus constitute a solid basis for a successful transfer of this technique into 3D constructs, in order to provide an individual and personalized human VF implant in the future.

  15. Detecting Selection on Protein Stability through Statistical Mechanical Models of Folding and Evolution

    PubMed Central

    Bastolla, Ugo

    2014-01-01

    The properties of biomolecules depend both on physics and on the evolutionary process that formed them. These two points of view produce a powerful synergism. Physics sets the stage and the constraints that molecular evolution has to obey, and evolutionary theory helps in rationalizing the physical properties of biomolecules, including protein folding thermodynamics. To complete the parallelism, protein thermodynamics is founded on the statistical mechanics in the space of protein structures, and molecular evolution can be viewed as statistical mechanics in the space of protein sequences. In this review, we will integrate both points of view, applying them to detecting selection on the stability of the folded state of proteins. We will start discussing positive design, which strengthens the stability of the folded against the unfolded state of proteins. Positive design justifies why statistical potentials for protein folding can be obtained from the frequencies of structural motifs. Stability against unfolding is easier to achieve for longer proteins. On the contrary, negative design, which consists in destabilizing frequently formed misfolded conformations, is more difficult to achieve for longer proteins. The folding rate can be enhanced by strengthening short-range native interactions, but this requirement contrasts with negative design, and evolution has to trade-off between them. Finally, selection can accelerate functional movements by favoring low frequency normal modes of the dynamics of the native state that strongly correlate with the functional conformation change. PMID:24970217

  16. Surface folding in metals: a mechanism for delamination wear in sliding

    PubMed Central

    Mahato, Anirban; Guo, Yang; Sundaram, Narayan K.; Chandrasekar, Srinivasan

    2014-01-01

    Using high-resolution, in situ imaging of a hard, wedge-shaped model asperity sliding against a metal surface, we demonstrate a new mechanism for particle formation and delamination wear. Damage to the residual surface is caused by the occurrence of folds on the free surface of the prow-shaped region ahead of the wedge. This damage manifests itself as shallow crack-like features and surface tears, which are inclined at very acute angles to the surface. The transformation of folds into cracks, tears and particles is directly captured. Notably, a single sliding pass is sufficient to damage the surface, and subsequent passes result in the generation of platelet-like wear particles. Tracking the folding process at every stage from surface bumps to folds to cracks/tears/particles ensures that there is no ambiguity in capturing the mechanism of wear. Because fold formation and consequent delamination are quite general, our findings have broad applicability beyond wear itself, including implications for design of surface generation and conditioning processes. PMID:25197251

  17. Kinetics and mechanism of olefin catalytic hydroalumination by organoaluminum compounds

    NASA Astrophysics Data System (ADS)

    Koledina, K. F.; Gubaidullin, I. M.

    2016-05-01

    The complex reaction mechanism of α-olefin catalytic hydroalumination by alkylalanes is investigated via mathematical modeling that involves plotting the kinetic models for the individual reactions that make up a complex system and a separate study of their principles. Kinetic parameters of olefin catalytic hydroalumination are estimated. Activation energies of the possible steps of the schemes of complex reaction mechanisms are compared and possible reaction pathways are determined.

  18. Kinetic mechanism of DNA polymerase I (Klenow)

    SciTech Connect

    Kuchta, R.D.; Mizrahi, V.; Benkovic, P.A.; Johnson, K.A.; Benkovic, S.J.

    1987-12-15

    The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence, labeled with (/sup 32/P)-nucleotides. A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF-DNA/sub n/-dNTP and KF-DNA/sub n+1/-PP/sub i/ complexes. The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments (the microscopic reverse of polymerization). Data analysis then provides an estimate of the internal equilibrium constant. The dissociations of DNA, dNTP, and PP/sub i/ from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments. The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis. The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences.

  19. Mechanical restoration of large-scale folded multilayers using the finite element method: Application to the Zagros Simply Folded Belt, N-Iraq

    NASA Astrophysics Data System (ADS)

    Frehner, Marcel; Reif, Daniel; Grasemann, Bernhard

    2010-05-01

    There are a large number of numerical finite element studies concerned with modeling the evolution of folded geological layers through time. This body of research includes many aspects of folding and many different approaches, such as two- and three-dimensional studies, single-layer folding, detachment folding, development of chevron folds, Newtonian, power-law viscous and more complex rheologies, influence of anisotropy, pure-shear, simple-shear and other boundary conditions and so forth. In recent years, studies of multilayer folding emerged, thanks to more advanced mesh generator software and increased computational power. Common to all of these studies is the fact that they consider a forward directed time evolution, as in nature. Very few studies use the finite element method for reverse-time simulations. In such studies, folded geological layers are taken as initial conditions for the numerical simulation. The folding process is reversed by changing the signs of the boundary conditions that supposedly drove the folding process. In such studies, the geometry of the geological layers before the folding process is searched and the amount of shortening necessary for the final folded geometry can be calculated. In contrast to a kinematic or geometric fold restoration procedure, the described approach takes the mechanical behavior of the geological layers into account, such as rheology and the relative strength of the individual layers. This approach is therefore called mechanical restoration of folds. In this study, the concept of mechanical restoration is applied to a two-dimensional 50km long NE-SW-cross-section through the Zagros Simply Folded Belt in Iraqi Kurdistan, NE from the city of Erbil. The Simply Folded Belt is dominated by gentle to open folding and faults are either absent or record only minor offset. Therefore, this region is ideal for testing the concept of mechanical restoration. The profile used is constructed from structural field measurements

  20. Redesigning the type II' β-turn in green fluorescent protein to type I': implications for folding kinetics and stability.

    PubMed

    Madan, Bharat; Sokalingam, Sriram; Raghunathan, Govindan; Lee, Sun-Gu

    2014-10-01

    Both Type I' and Type II' β-turns have the same sense of the β-turn twist that is compatible with the β-sheet twist. They occur predominantly in two residue β-hairpins, but the occurrence of Type I' β-turns is two times higher than Type II' β-turns. This suggests that Type I' β-turns may be more stable than Type II' β-turns, and Type I' β-turn sequence and structure can be more favorable for protein folding than Type II' β-turns. Here, we redesigned the native Type II' β-turn in GFP to Type I' β-turn, and investigated its effect on protein folding and stability. The Type I' β-turns were designed based on the statistical analysis of residues in natural Type I' β-turns. The substitution of the native "GD" sequence of i+1 and i+2 residues with Type I' preferred "(N/D)G" sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' β-turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' β-turns in natural β-hairpins can be further optimized by converting the sequence to Type I'.

  1. Early Events in RNA Folding

    NASA Astrophysics Data System (ADS)

    Thirumalai, D.; Lee, Namkyung; Woodson, Sarah A.; Klimov, Dk

    2001-10-01

    We describe a conceptual framework for understanding the way large RNA molecules fold based on the notion that their free-energy landscape is rugged. A key prediction of our theory is that RNA folding can be described by the kinetic partitioning mechanism (KPM). According to KPM a small fraction of molecules folds rapidly to the native state whereas the remaining fraction is kinetically trapped in a low free-energy non-native state. This model provides a unified description of the way RNA and proteins fold. Single-molecule experiments on Tetrahymena ribozyme, which directly validate our theory, are analyzed using KPM. We also describe the earliest events that occur on microsecond time scales in RNA folding. These must involve collapse of RNA molecules that are mediated by counterion-condensation. Estimates of time scales for the initial events in RNA folding are provided for the Tetrahymena ribozyme.

  2. Single-Molecule Chemo-Mechanical Spectroscopy Provides Structural Identity of Folding Intermediates

    PubMed Central

    Motlagh, Hesam N.; Toptygin, Dmitri; Kaiser, Christian M.; Hilser, Vincent J.

    2016-01-01

    Single-molecule force spectroscopy has emerged as a powerful tool for studying the folding of biological macromolecules. Mechanical manipulation has revealed a wealth of mechanistic information on transient and intermediate states. To date, the majority of state assignment of intermediates has relied on empirical demarcation. However, performing such experiments in the presence of different osmolytes provides an alternative approach that reports on the structural properties of intermediates. Here, we analyze the folding and unfolding of T4 lysozyme with optical tweezers under a chemo-mechanical perturbation by adding osmolytes. We find that two unrelated protective osmolytes, sorbitol and trimethylamine-n-oxide, function by marginally decelerating unfolding rates and specifically modulating early events in the folding process, stabilizing formation of an on-pathway intermediate. The chemo-mechanical perturbation provides access to two independent metrics of the relevant states during folding trajectories, the contour length, and the solvent-accessible surface area. We demonstrate that the dependence of the population of the intermediate in different osmolytes, in conjunction with its measured contour length, provides the ability to discriminate between potential structural models of intermediate states. Our study represents a general strategy that may be employed in the structural modeling of equilibrium intermediate states observed in single-molecule experiments. PMID:27028638

  3. Mechanisms of kinetic stabilization by the drugs paclitaxel and vinblastine

    PubMed Central

    Castle, Brian T.; McCubbin, Seth; Prahl, Louis S.; Bernens, Jordan N.; Sept, David; Odde, David J.

    2017-01-01

    Microtubule-targeting agents (MTAs), widely used as biological probes and chemotherapeutic drugs, bind directly to tubulin subunits and “kinetically stabilize” microtubules, suppressing the characteristic self-assembly process of dynamic instability. However, the molecular-level mechanisms of kinetic stabilization are unclear, and the fundamental thermodynamic and kinetic requirements for dynamic instability and its elimination by MTAs have yet to be defined. Here we integrate a computational model for microtubule assembly with nanometer-scale fluorescence microscopy measurements to identify the kinetic and thermodynamic basis of kinetic stabilization by the MTAs paclitaxel, an assembly promoter, and vinblastine, a disassembly promoter. We identify two distinct modes of kinetic stabilization in live cells, one that truly suppresses on-off kinetics, characteristic of vinblastine, and the other a “pseudo” kinetic stabilization, characteristic of paclitaxel, that nearly eliminates the energy difference between the GTP- and GDP-tubulin thermodynamic states. By either mechanism, the main effect of both MTAs is to effectively stabilize the microtubule against disassembly in the absence of a robust GTP cap. PMID:28298489

  4. Kinetic mechanism of DNA polymerase I

    SciTech Connect

    Kuchta, R.D.; Mizrahi, V.; Benkovic, S.J.; Benkovic, P.; Johnson, K.A.

    1987-05-01

    The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from E. coli has been determined with short DNA oligomers of defined sequence. A key feature of this scheme is a minimal two step sequence for the interconversion of the ternary KF x DNA/sub n/ x dNTP and KF x DNA/sub n+1/ x PP/sub i/ complexes. The rate is not limited by the actual polymerization process but a separate step possibly important in insuring fidelity. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments. Data analysis provides an estimate of the internal equilibrium constant. The dissociation constants of DNA (5 nM), dNTP (5 ..mu..M) and PP/sub i/ (100 ..mu..M) from the various complexes were measured by isotope trapping experiments. The rate constant for DNA dissociation from KF is sequence dependent and rate limiting when the next required nucleotide is missing. Finally, this scheme can be used to describe the incorporation of incorrect nucleotides; there is no change in the rate determining step during misincorporation, and the 3' ..-->.. 5' exonuclease does not rapidly remove misincorporated nucleotides.

  5. Kinetic and thermodynamic consequences of the removal of the Cys-77-Cys-123 disulphide bond for the folding of TEM-1 beta-lactamase.

    PubMed Central

    Vanhove, M; Guillaume, G; Ledent, P; Richards, J H; Pain, R H; Frère, J M

    1997-01-01

    Class A beta-lactamases of the TEM family contain a single disulphide bond which connects cysteine residues 77 and 123. To clarify the possible role of the disulphide bond in the stability and folding kinetics of the TEM-1 beta-lactamase, this bond was removed by introducing a Cys-77-->Ser mutation, and the enzymically active mutant protein was studied by reversible guanidine hydrochloride-induced denaturation. The unfolding and refolding rates were monitored using tryptophan fluorescence. At low guanidine hydrochloride concentrations, the refolding of the wild-type and mutant enzymes followed biphasic time courses. The characteristics of the two phases were not significantly affected by the mutation. Double-jump experiments, in which the protein was unfolded in a high concentration of guanidine hydrochloride for a short time period and then refolded by diluting out the denaturant, indicated that, for both the wild-type and mutant enzymes, the two refolding phases could be ascribed to proline isomerization reactions. Equilibrium unfolding experiments monitored by fluorescence spectroscopy and far-UV CD indicated a three-state mechanism (N<-->H<--U). Both the folded mutant protein (N) and, to a lesser extent the thermodynamically stable intermediate, H. were destabilized relative to the fully unfolded state, U. Removal of the disulphide bond resulted in a decrease of 14.2 kJ/mol (3.4 kcal/mol) in the global free energy of stabilization. Similarly, the mutation also induced a drastic increase in the rate of thermal inactivation. PMID:9020874

  6. Kinetic cooperativity of tyrosinase. A general mechanism.

    PubMed

    Muñoz-Muñoz, Jose Luis; Garcia-Molina, Francisco; Varon, Ramón; Tudela, Jose; Garcia-Cánovas, Francisco; Rodríguez-López, Jose N

    2011-01-01

    Tyrosinase shows kinetic cooperativity in its action on o-diphenols, but not when it acts on monophenols, confirming that the slow step is the hydroxylation of monophenols to o-diphenols. This model can be generalised to a wide range of substrates; for example, type S(A) substrates, which give rise to a stable product as the o-quinone evolves by means of a first or pseudo first order reaction (α-methyl dopa, dopa methyl ester, dopamine, 3,4-dihydroxyphenylpropionic acid, 3,4-dihydroxyphenylacetic acid, α-methyl-tyrosine, tyrosine methyl ester, tyramine, 4-hydroxyphenylpropionic acid and 4-hydroxyphenylacetic acid), type S(B) substrates, which include those whose o-quinone evolves with no clear stoichiometry (catechol, 4-methylcatechol, phenol and p-cresol) and, lastly, type S(C) substrates, which give rise to stable o-quinones (4-tert-butylcatechol/4-tert-butylphenol).

  7. Kinetic mechanism of octopus hepatopancreatic glutathione transferase in reverse micelles.

    PubMed Central

    Tang, S S; Chang, G G

    1996-01-01

    Octopus glutathione transferase (GST) was enzymically active in aerosol-OT [sodium bis-(2-ethylhexyl)sulphosuccinate]/iso-octane reverse micelles albeit with lowered catalytic constant (kcat). The enzyme reaction rate was found to be dependent on the [H2O]/[surfactant] ratio (omega(o)) of the system with maximum rate observed at omega(o) 13.88, which corresponded to vesicles with a core volume of 64 nm3. According to the physical examinations, a vesicle of this size is barely large enough to accommodate a monomeric enzyme subunit. Dissociation of the enzyme in reverse micelles was confirmed by cross-linking of the associated subunits with glutaraldehyde and separation of the monomers and dimers with electrophoresis in the presence of SDS. The kinetic properties of the enzyme were investigated by steady-state kinetic analysis. Both GSH and 1-chloro-2,4-dinitrobenzene (CDNB) showed substrate inhibition and the Michaelis constant for CDNB was increased by 36-fold to 11.05 mM in reverse micelles. Results on the initial-velocity and product-inhibition studies indicate that the octopus GST conforms to a steady-state sequential random Bi Bi mechanism. The results from a log kcat versus pH plot suggest that amino acid residues with pKa values of 6.56 0.07 and 8.81 0.17 should be deprotonated to give optimum catalytic function. In contrast, the amino acid residue with a pKa value of 9.69 0.16 in aqueous solution had to be protonated for the reaction to proceed. We propose that the pKa1 (6.56) is that for the enzyme-bound GSH, which has a pKa value lowered by 1.40-1.54 pH units compared with that of free GSH in reverse micelles. The most probable candidate for the observed pKa2 (8.81) is Tyr7 of GST. The pKa of Tyr7 is 0.88 pH unit lower than that in aqueous solution and is about 2 pH units below the normal tyrosine. This tyrosyl residue may act as a base catalyst facilitating the dissociation of enzyme-bound GSH. The possible interaction of GST with plasma membrane in vivo

  8. Kinetic mechanism of octopus hepatopancreatic glutathione transferase in reverse micelles.

    PubMed

    Tang, S S; Chang, G G

    1996-04-15

    Octopus glutathione transferase (GST) was enzymically active in aerosol-OT [sodium bis-(2-ethylhexyl)sulphosuccinate]/iso-octane reverse micelles albeit with lowered catalytic constant (kcat). The enzyme reaction rate was found to be dependent on the [H2O]/[surfactant] ratio (omega(o)) of the system with maximum rate observed at omega(o) 13.88, which corresponded to vesicles with a core volume of 64 nm3. According to the physical examinations, a vesicle of this size is barely large enough to accommodate a monomeric enzyme subunit. Dissociation of the enzyme in reverse micelles was confirmed by cross-linking of the associated subunits with glutaraldehyde and separation of the monomers and dimers with electrophoresis in the presence of SDS. The kinetic properties of the enzyme were investigated by steady-state kinetic analysis. Both GSH and 1-chloro-2,4-dinitrobenzene (CDNB) showed substrate inhibition and the Michaelis constant for CDNB was increased by 36-fold to 11.05 mM in reverse micelles. Results on the initial-velocity and product-inhibition studies indicate that the octopus GST conforms to a steady-state sequential random Bi Bi mechanism. The results from a log kcat versus pH plot suggest that amino acid residues with pKa values of 6.56 0.07 and 8.81 0.17 should be deprotonated to give optimum catalytic function. In contrast, the amino acid residue with a pKa value of 9.69 0.16 in aqueous solution had to be protonated for the reaction to proceed. We propose that the pKa1 (6.56) is that for the enzyme-bound GSH, which has a pKa value lowered by 1.40-1.54 pH units compared with that of free GSH in reverse micelles. The most probable candidate for the observed pKa2 (8.81) is Tyr7 of GST. The pKa of Tyr7 is 0.88 pH unit lower than that in aqueous solution and is about 2 pH units below the normal tyrosine. This tyrosyl residue may act as a base catalyst facilitating the dissociation of enzyme-bound GSH. The possible interaction of GST with plasma membrane in vivo

  9. Local Kinetic Measures of Macromolecular Structure Reveal Partitioning Among Multiple Parallel Pathways from the Earliest Steps in the Folding of a Large RNA Molecule

    SciTech Connect

    Laederach,A.; Shcherbakova, I.; Liang, M.; Brenowitz, M.; Altman, R.

    2006-01-01

    At the heart of the RNA folding problem is the number, structures, and relationships among the intermediates that populate the folding pathways of most large RNA molecules. Unique insight into the structural dynamics of these intermediates can be gleaned from the time-dependent changes in local probes of macromolecular conformation (e.g. reports on individual nucleotide solvent accessibility offered by hydroxyl radical ({center_dot}OH) footprinting). Local measures distributed around a macromolecule individually illuminate the ensemble of separate changes that constitute a folding reaction. Folding pathway reconstruction from a multitude of these individual measures is daunting due to the combinatorial explosion of possible kinetic models as the number of independent local measures increases. Fortunately, clustering of time progress curves sufficiently reduces the dimensionality of the data so as to make reconstruction computationally tractable. The most likely folding topology and intermediates can then be identified by exhaustively enumerating all possible kinetic models on a super-computer grid. The folding pathways and measures of the relative flux through them were determined for Mg{sup 2+} and Na{sup +}-mediated folding of the Tetrahymena thermophila group I intron using this combined experimental and computational approach. The flux during Mg{sup 2+}-mediated folding is divided among numerous parallel pathways. In contrast, the flux during the Na{sup +}-mediated reaction is predominantly restricted through three pathways, one of which is without detectable passage through intermediates. Under both conditions, the folding reaction is highly parallel with no single pathway accounting for more than 50% of the molecular flux. This suggests that RNA folding is non-sequential under a variety of different experimental conditions even at the earliest stages of folding. This study provides a template for the systematic analysis of the time-evolution of RNA structure

  10. Kinetic mechanism of indole-3-glycerol phosphate synthase.

    PubMed

    Schlee, Sandra; Dietrich, Susanne; Kurćon, Tomasz; Delaney, Pamela; Goodey, Nina M; Sterner, Reinhard

    2013-01-08

    The (βα)(8)-barrel enzyme indole-3-glycerol phosphate synthase (IGPS) catalyzes the multistep transformation of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) into indole-3-glycerol phosphate (IGP) in tryptophan biosynthesis. Mutagenesis data and crystal structure analysis of IGPS from Sulfolobus solfataricus (sIGPS) allowed for the formulation of a plausible chemical mechanism of the reaction, and molecular dynamics simulations suggested that flexibility of active site loops might be important for catalysis. Here we developed a method that uses extrinsic fluorophores attached to active site loops to connect the kinetic mechanism of sIGPS to structure and conformational motions. Specifically, we elucidated the kinetic mechanism of sIGPS and correlated individual steps in the mechanism to conformational motions of flexible loops. Pre-steady-state kinetic measurements of CdRP to IGP conversion monitoring changes in intrinsic tryptophan and IGP fluorescence provided a minimal three-step kinetic model in which fast substrate binding and chemical transformation are followed by slow product release. The role of sIGPS loop conformational motion during substrate binding and catalysis was examined via variants that were covalently labeled with fluorescent dyes at the N-terminal extension of the enzyme and mobile active site loop β1α1. Analysis of kinetic data monitoring dye fluorescence revealed a conformational change that follows substrate binding, suggesting an induced-fit-type binding mechanism for the substrate CdRP. Global fitting of all kinetic results obtained with wild-type sIGPS and the labeled variants was best accommodated by a four-step kinetic model. In this model, both the binding of CdRP and its on-enzyme conversion to IGP are accompanied by conformational transitions. The liberation of the product from the active site is the rate-limiting step of the overall reaction. Our results confirm the importance of flexible active loops for substrate

  11. Optical Measurements of Vocal Fold Tensile Properties: Implications for Phonatory Mechanics

    PubMed Central

    Kelleher, Jordan E.; Siegmund, Thomas; Chan, Roger W.; Henslee, Erin A.

    2011-01-01

    In voice research, in vitro tensile stretch experiments of vocal fold tissues are commonly employed to determine the tissue biomechanical properties. In the standard stretch-release protocol, tissue deformation is computed from displacements applied to sutures inserted through the thyroid and arytenoid cartilages, with the cartilages assumed to be rigid. Here, a non-contact optical method was employed to determine the actual tissue deformation of vocal fold lamina propria specimens from three excised human larynges in uniaxial tensile tests. Specimen deformation was found to consist not only of deformation of the tissue itself, but also deformation of the cartilages, as well as suture alignment and tightening. Stress-stretch curves of a representative load cycle were characterized by an incompressible Ogden model. The initial longitudinal elastic modulus was found to be considerably higher if determined based on optical displacement measurements than typical values reported in the literature. The present findings could change the understanding of the mechanics underlying vocal fold vibration. Given the high longitudinal elastic modulus the lamina propria appeared to demonstrate a substantial level of anisotropy. Consequently, transverse shear could play a significant role in vocal fold vibration, and fundamental frequencies of phonation should be predicted by beam theories accounting for such effects. PMID:21497355

  12. Fractal features of a crumpling network in randomly folded thin matter and mechanics of sheet crushing.

    PubMed

    Balankin, Alexander S; Horta Rangel, Antonio; García Pérez, Gregorio; Gayosso Martinez, Felipe; Sanchez Chavez, Hugo; Martínez-González, Claudia L

    2013-05-01

    We study the static and dynamic properties of networks of crumpled creases formed in hand crushed sheets of paper. The fractal dimensionalities of crumpling networks in the unfolded (flat) and folded configurations are determined. Some other noteworthy features of crumpling networks are established. The physical implications of these findings are discussed. Specifically, we state that self-avoiding interactions introduce a characteristic length scale of sheet crumpling. A framework to model the crumpling phenomena is suggested. Mechanics of sheet crushing under external confinement is developed. The effect of compaction geometry on the crushing mechanics is revealed.

  13. Fractal features of a crumpling network in randomly folded thin matter and mechanics of sheet crushing

    NASA Astrophysics Data System (ADS)

    Balankin, Alexander S.; Horta Rangel, Antonio; García Pérez, Gregorio; Gayosso Martinez, Felipe; Sanchez Chavez, Hugo; Martínez-González, Claudia L.

    2013-05-01

    We study the static and dynamic properties of networks of crumpled creases formed in hand crushed sheets of paper. The fractal dimensionalities of crumpling networks in the unfolded (flat) and folded configurations are determined. Some other noteworthy features of crumpling networks are established. The physical implications of these findings are discussed. Specifically, we state that self-avoiding interactions introduce a characteristic length scale of sheet crumpling. A framework to model the crumpling phenomena is suggested. Mechanics of sheet crushing under external confinement is developed. The effect of compaction geometry on the crushing mechanics is revealed.

  14. Mechanisms and Kinetics of Diphthalocyanine Electrode Processes.

    DTIC Science & Technology

    1983-07-01

    Science and Engineering, to be published by Pergamon Press in 1983. In condensed style, this paper describes electrochromic display devices and their...operating mechanisms and compares the electrochromic materials tungsten oxide, iridium oxide, n-heptyl viologen bromide, rare-earth diphthalocyanines...Scheme .. .. ... .... 14 E. Electrochromics .. .. ... ... ... ... . .. ..... 21 *F. Multicolor Electrochromic Display Technology .. .. ...... 22 S.IV

  15. Chemical kinetic reaction mechanism for the combustion of propane

    NASA Technical Reports Server (NTRS)

    Jachimowski, C. J.

    1984-01-01

    A detailed chemical kinetic reaction mechanism for the combustion of propane is presented and discussed. The mechanism consists of 27 chemical species and 83 elementary chemical reactions. Ignition and combustion data as determined in shock tube studies were used to evaluate the mechanism. Numerical simulation of the shock tube experiments showed that the kinetic behavior predicted by the mechanism for stoichiometric mixtures is in good agrement with the experimental results over the entire temperature range examined (1150-2600K). Sensitivity and theoretical studies carried out using the mechanism revealed that hydrocarbon reactions which are involved in the formation of the HO2 radical and the H2O2 molecule are very important in the mechanism and that the observed nonlinear behavior of ignition delay time with decreasing temperature can be interpreted in terms of the increased importance of the HO2 and H2O2 reactions at the lower temperatures.

  16. Determination of enzyme mechanisms by radiationless energy transfer kinetics.

    PubMed

    Lobb, R R; Auld, D S

    1979-06-01

    Rigorous definition of the elementary steps of an enzymatic reaction requires visualization of transient enzyme-substrate (ES) complexes. Measurement of radiationless energy transfer (RET) between enzyme tryptophan residues and a fluorescent dansyl (5-dimethylaminonaphthalene-1-sulfonyl) substrate provides a sensitive means to observe ES complexes directly. Analysis of the rate of formation and breakdown of ES complexes by RET can serve as the basis of a rapid kinetic approach to enzyme mechanisms. Both pre-steady-state and steady-state kinetics can be performed in the same RET experiment. Analysis at steady state precisely determines k(cat) and K(m) values by multiple means. Analysis at pre-steady state determines the number of intermediates, the type of reaction mechanism, and all the individual binding and rate constants. Chymotrypsin was chosen as a standard of reference for RET kinetics because extensive investigations have established both the existence of transient intermediates in the course of its catalytic process and the range of values to be expected for pertinent kinetic constants. As predicted, RET kinetics readily detects the two known intermediates in the alpha-chymotrypsincatalyzed hydrolysis of specific ester substrates. The results are both qualitatively and quantitatively in accord with data derived for this enzyme from classical kinetics. Hence, this experimental study both validates and demonstrates the theoretical advantages and potential of RET kinetics. The generality of the approach has been investigated by synthesizing a family of dansyl-labeled substrates designed to meet the specificity requirements of a number of metallo- and nonmetallo- exo- and endopeptidases. In all cases, the ES complex is observed readily at micromolar or lower concentrations of enzyme under stopped-flow conditions. The success of the RET kinetic approach on proteolytic enzymes shows its broad utility.

  17. Applied origami. Using origami design principles to fold reprogrammable mechanical metamaterials.

    PubMed

    Silverberg, Jesse L; Evans, Arthur A; McLeod, Lauren; Hayward, Ryan C; Hull, Thomas; Santangelo, Christian D; Cohen, Itai

    2014-08-08

    Although broadly admired for its aesthetic qualities, the art of origami is now being recognized also as a framework for mechanical metamaterial design. Working with the Miura-ori tessellation, we find that each unit cell of this crease pattern is mechanically bistable, and by switching between states, the compressive modulus of the overall structure can be rationally and reversibly tuned. By virtue of their interactions, these mechanically stable lattice defects also lead to emergent crystallographic structures such as vacancies, dislocations, and grain boundaries. Each of these structures comes from an arrangement of reversible folds, highlighting a connection between mechanical metamaterials and programmable matter. Given origami's scale-free geometric character, this framework for metamaterial design can be directly transferred to milli-, micro-, and nanometer-size systems.

  18. Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments.

    PubMed

    Mizukami, Takuya; Abe, Yukiko; Maki, Kosuke

    2015-01-01

    In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0-2.2 M) than the formation of the native state (0-1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7-2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.

  19. Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

    PubMed Central

    Mizukami, Takuya; Abe, Yukiko; Maki, Kosuke

    2015-01-01

    In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0–2.2 M) than the formation of the native state (0–1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7–2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin. PMID:26244984

  20. Nonlinear kinetics and new approaches to complex reaction mechanisms.

    PubMed

    Ross, J; Vlad, M O

    1999-01-01

    This paper reviews recent developments in the field of nonlinear chemical kinetics. Five topics are dealt with: (a) new approaches to complex reaction mechanisms, stoichiometric network analysis, classification of chemical oscillators and formulation of their mechanisms by deduction from experiments, and correlation metric construction of reaction pathways from measurements; (b) thermodynamic and stochastic theory of nonequilibrium processes, the eikonal approximation, the evaluation of stochastic potentials, experimental tests of the thermodynamic and stochastic theory of relative stability, and fluctuation-dissipation relations in nonequilibrium chemical systems; (c) chemical kinetics and cellular automata and lattice gas automata; (d) theoretical approaches and experimental studies of stochastic resonance in chemical kinetics; and (e) rate processes in disordered systems, stochastic Liouville equations, stretched exponential relaxation in disordered systems, and universality classes for rate processes in systems with static or dynamic disorder.

  1. Two-state folding of horse ferrocytochrome c: analyses of linear free energy relationship, chevron curvature, and stopped-flow burst relaxation kinetics.

    PubMed

    Kumar, Rajesh; Bhuyan, Abani K

    2005-03-01

    Ferrocytchrome c is a classic two-state fast folder. This assurance comes from extensive equilibrium and kinetic folding studies carried out under strictly anaerobic conditions at 22 degrees C. Conventional guanidine hydrochloride (GdnHCl)-induced unfolding transitions monitored by the use of a sizable set of optical probes do not reveal the accumulation of any intermediate to a detectable level. The GdnHCl dependence of unfolding free energy (DeltaG(D)) is linear over the full range of the denaturant concentration. The GdnHCl folding chevron is characterized by curvatures in both folding and unfolding limbs. However, refolding rates as a function of urea in the presence of different concentrations of GdnHCl yield m(++)f values (the kinetic m-value) that are quantitatively identical. This result, analyzed in terms of the denaturant dependence of the difference in the extent of solvent exposure between a relatively fixed transition state and the preceding state involved in the transition, suggests that the chevron curvature is not related to differential accumulation of a folding intermediate with varying concentration of GdnHCl in the refolding medium. Denaturant dependence of stopped-flow burst signals recorded in normal refolding experiments (pH 7, 22 degrees C) is essentially identical with that recorded in simulating experiments in which the protein stays steadily unfolded even in the denaturant-diluted medium (pH 1.5-2, 22 or 43 degrees C depending on the use of urea or GdnHCl), and they match the denaturant dependence of equilibrium signals for the unfolded protein. The results demonstrate that the burst phase does not entail an early folding intermediate. Rather, the folding kinetics are essentially two-state. These results are central to the phenomenological description of protein folding.

  2. Detailed chemical kinetic oxidation mechanism for a biodiesel surrogate

    SciTech Connect

    Herbinet, O; Pitz, W J; Westbrook, C K

    2007-09-20

    A detailed chemical kinetic mechanism has been developed and used to study the oxidation of methyl decanoate, a surrogate for biodiesel fuels. This model has been built by following the rules established by Curran et al. for the oxidation of n-heptane and it includes all the reactions known to be pertinent to both low and high temperatures. Computed results have been compared with methyl decanoate experiments in an engine and oxidation of rapeseed oil methyl esters in a jet stirred reactor. An important feature of this mechanism is its ability to reproduce the early formation of carbon dioxide that is unique to biofuels and due to the presence of the ester group in the reactant. The model also predicts ignition delay times and OH profiles very close to observed values in shock tube experiments fueled by n-decane. These model capabilities indicate that large n-alkanes can be good surrogates for large methyl esters and biodiesel fuels to predict overall reactivity, but some kinetic details, including early CO{sub 2} production from biodiesel fuels, can be predicted only by a detailed kinetic mechanism for a true methyl ester fuel. The present methyl decanoate mechanism provides a realistic kinetic tool for simulation of biodiesel fuels.

  3. Detailed chemical kinetic oxidation mechanism for a biodiesel surrogate

    SciTech Connect

    Herbinet, O; Pitz, W J; Westbrook, C K

    2007-09-17

    A detailed chemical kinetic mechanism has been developed and used to study the oxidation of methyl decanoate, a surrogate for biodiesel fuels. This model has been built by following the rules established by Curran et al. for the oxidation of n-heptane and it includes all the reactions known to be pertinent to both low and high temperatures. Computed results have been compared with methyl decanoate experiments in an engine and oxidation of rapeseed oil methyl esters in a jet stirred reactor. An important feature of this mechanism is its ability to reproduce the early formation of carbon dioxide that is unique to biofuels and due to the presence of the ester group in the reactant. The model also predicts ignition delay times and OH profiles very close to observed values in shock tube experiments fueled by n-decane. These model capabilities indicate that large n-alkanes can be good surrogates for large methyl esters and biodiesel fuels to predict overall reactivity, but some kinetic details, including early CO2 production from biodiesel fuels, can be predicted only by a detailed kinetic mechanism for a true methyl ester fuel. The present methyl decanoate mechanism provides a realistic kinetic tool for simulation of biodiesel fuels.

  4. Detailed chemical kinetic oxidation mechanism for a biodiesel surrogate

    SciTech Connect

    Herbinet, Olivier; Pitz, William J.; Westbrook, Charles K.

    2008-08-15

    A detailed chemical kinetic mechanism has been developed and used to study the oxidation of methyl decanoate, a surrogate for biodiesel fuels. This model has been built by following the rules established by Curran and co-workers for the oxidation of n-heptane and it includes all the reactions known to be pertinent to both low and high temperatures. Computed results have been compared with methyl decanoate experiments in an engine and oxidation of rapeseed oil methyl esters in a jet-stirred reactor. An important feature of this mechanism is its ability to reproduce the early formation of carbon dioxide that is unique to biofuels and due to the presence of the ester group in the reactant. The model also predicts ignition delay times and OH profiles very close to observed values in shock tube experiments fueled by n-decane. These model capabilities indicate that large n-alkanes can be good surrogates for large methyl esters and biodiesel fuels to predict overall reactivity, but some kinetic details, including early CO{sub 2} production from biodiesel fuels, can be predicted only by a detailed kinetic mechanism for a true methyl ester fuel. The present methyl decanoate mechanism provides a realistic kinetic tool for simulation of biodiesel fuels. (author)

  5. A mechanical model of vocal-fold collision with high spatial and temporal resolution

    NASA Astrophysics Data System (ADS)

    Gunter, Heather E.

    2003-02-01

    The tissue mechanics governing vocal-fold closure and collision during phonation are modeled in order to evaluate the role of elastic forces in glottal closure and in the development of stresses that may be a risk factor for pathology development. The model is a nonlinear dynamic contact problem that incorporates a three-dimensional, linear elastic, finite-element representation of a single vocal fold, a rigid midline surface, and quasistatic air pressure boundary conditions. Qualitative behavior of the model agrees with observations of glottal closure during normal voice production. The predicted relationship between subglottal pressure and peak collision force agrees with published experimental measurements. Accurate predictions of tissue dynamics during collision suggest that elastic forces play an important role during glottal closure and are an important determinant of aerodynamic variables that are associated with voice quality. Model predictions of contact force between the vocal folds are directly proportional to compressive stress (r2=0.79), vertical shear stress (r2=0.69), and Von Mises stress (r2=0.83) in the tissue. These results guide the interpretation of experimental measurements by relating them to a quantity that is important in tissue damage.

  6. A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism.

    PubMed

    Jin, Yi; Petricevic, Marija; John, Alan; Raich, Lluís; Jenkins, Huw; Portela De Souza, Leticia; Cuskin, Fiona; Gilbert, Harry J; Rovira, Carme; Goddard-Borger, Ethan D; Williams, Spencer J; Davies, Gideon J

    2016-12-28

    The enzymatic cleavage of β-1,4-mannans is achieved by endo-β-1,4-mannanases, enzymes involved in germination of seeds and microbial hemicellulose degradation, and which have increasing industrial and consumer product applications. β-Mannanases occur in a range of families of the CAZy sequence-based glycoside hydrolase (GH) classification scheme including families 5, 26, and 113. In this work we reveal that β-mannanases of the newly described GH family 134 differ from other mannanase families in both their mechanism and tertiary structure. A representative GH family 134 endo-β-1,4-mannanase from a Streptomyces sp. displays a fold closely related to that of hen egg white lysozyme but acts with inversion of stereochemistry. A Michaelis complex with mannopentaose, and a product complex with mannotriose, reveal ligands with pyranose rings distorted in an unusual inverted chair conformation. Ab initio quantum mechanics/molecular mechanics metadynamics quantified the energetically accessible ring conformations and provided evidence in support of a (1)C4 → (3)H4(‡) → (3)S1 conformational itinerary along the reaction coordinate. This work, in concert with that on GH family 124 cellulases, reveals how the lysozyme fold can be co-opted to catalyze the hydrolysis of different polysaccharides in a mechanistically distinct manner.

  7. A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism

    PubMed Central

    2016-01-01

    The enzymatic cleavage of β-1,4-mannans is achieved by endo-β-1,4-mannanases, enzymes involved in germination of seeds and microbial hemicellulose degradation, and which have increasing industrial and consumer product applications. β-Mannanases occur in a range of families of the CAZy sequence-based glycoside hydrolase (GH) classification scheme including families 5, 26, and 113. In this work we reveal that β-mannanases of the newly described GH family 134 differ from other mannanase families in both their mechanism and tertiary structure. A representative GH family 134 endo-β-1,4-mannanase from a Streptomyces sp. displays a fold closely related to that of hen egg white lysozyme but acts with inversion of stereochemistry. A Michaelis complex with mannopentaose, and a product complex with mannotriose, reveal ligands with pyranose rings distorted in an unusual inverted chair conformation. Ab initio quantum mechanics/molecular mechanics metadynamics quantified the energetically accessible ring conformations and provided evidence in support of a 1C4 → 3H4‡ → 3S1 conformational itinerary along the reaction coordinate. This work, in concert with that on GH family 124 cellulases, reveals how the lysozyme fold can be co-opted to catalyze the hydrolysis of different polysaccharides in a mechanistically distinct manner. PMID:28058278

  8. Three-fold Symmetric Doping Mechanism in GaAs Nanowires.

    PubMed

    Dastjerdi, M H T; Fiordaliso, E M; Leshchenko, E D; Akhtari-Zavareh, A; Kasama, T; Aagesen, M; Dubrovskii, V G; LaPierre, R R

    2017-10-11

    A new dopant incorporation mechanism in Ga-assisted GaAs nanowires grown by molecular beam epitaxy is reported. Off-axis electron holography revealed that p-type Be dopants introduced in situ during molecular beam epitaxy growth of the nanowires were distributed inhomogeneously in the nanowire cross-section, perpendicular to the growth direction. The active dopants showed a remarkable azimuthal distribution along the (111)B flat top of the nanowires, which is attributed to preferred incorporation along 3-fold symmetric truncated facets under the Ga droplet. A diffusion model is presented to explain the unique radial and azimuthal variation of the active dopants in the GaAs nanowires.

  9. Kinetics and Mechanisms of Calcite Reactions with Saline Waters

    SciTech Connect

    Chapman, Piers; *Morse, John W.

    2010-11-15

    1. Objective The general objective of this research was to determine the kinetics and mechanisms of calcite reactions with saline waters over a wide range of saline water composition, carbon dioxide partial pressure (pCO2), and modest ranges of T and P. This would be done by studying both reaction rates and solubility from changes in solution chemistry. Also, nanoscale observations of calcite surface morphology and composition would be made to provide an understanding of rate controlling mechanisms.

  10. A three-dimensional statistical mechanical model of folding double-stranded chain molecules

    NASA Astrophysics Data System (ADS)

    Zhang, Wenbing; Chen, Shi-Jie

    2001-05-01

    Based on a graphical representation of intrachain contacts, we have developed a new three-dimensional model for the statistical mechanics of double-stranded chain molecules. The theory has been tested and validated for the cubic lattice chain conformations. The statistical mechanical model can be applied to the equilibrium folding thermodynamics of a large class of chain molecules, including protein β-hairpin conformations and RNA secondary structures. The application of a previously developed two-dimensional model to RNA secondary structure folding thermodynamics generally overestimates the breadth of the melting curves [S-J. Chen and K. A. Dill, Proc. Natl. Acad. Sci. U.S.A. 97, 646 (2000)], suggesting an underestimation for the sharpness of the conformational transitions. In this work, we show that the new three-dimensional model gives much sharper melting curves than the two-dimensional model. We believe that the new three-dimensional model may give much improved predictions for the thermodynamic properties of RNA conformational changes than the previous two-dimensional model.

  11. Structural Characterization of the Early Events in the Nucleation-Condensation Mechanism in a Protein Folding Process.

    PubMed

    Kukic, Predrag; Pustovalova, Yulia; Camilloni, Carlo; Gianni, Stefano; Korzhnev, Dmitry M; Vendruscolo, Michele

    2017-05-24

    The nucleation-condensation mechanism represents a major paradigm to understand the folding process of many small globular proteins. Although substantial evidence has been acquired for this mechanism, it has remained very challenging to characterize the initial events leading to the formation of a folding nucleus. To achieve this goal, we used a combination of relaxation dispersion NMR spectroscopy and molecular dynamics simulations to determine ensembles of conformations corresponding to the denatured, transition, and native states in the folding of the activation domain of human procarboxypeptidase A2 (ADA2h). We found that the residues making up the folding nucleus tend to interact in the denatured state in a transient manner and not simultaneously, thereby forming incomplete and distorted versions of the folding nucleus. Only when all the contacts between these key residues are eventually formed can the protein reach the transition state and continue folding. Overall, our results elucidate the mechanism of formation of the folding nucleus of a protein and provide insights into how its folding rate can be modified during evolution by mutations that modulate the strength of the interactions between the residues forming the folding nucleus.

  12. Origami mechanical metamaterials based on the Miura-derivative fold patterns.

    PubMed

    Zhou, Xiang; Zang, Shixi; You, Zhong

    2016-07-01

    This paper presents two new types of origami-inspired mechanical metamaterials based on the Miura-derivative fold patterns that consist of non-identical parallelogram facets. The analytical models to predict dimension changes and deformation kinematics of the proposed metamaterials are developed. Furthermore, by modelling the creases as revolute hinges with certain rotational spring constants, we derived analytical models for stretching and bulk moduli. The analytical models are validated through finite-element simulation results. Numerical examples reveal that the proposed metamaterials possess some intriguing properties, including negative Poisson's ratios and bulk modulus. The work presented in this paper can provide a highly flexible framework for the design of versatile tunable mechanical metamaterials.

  13. Origami mechanical metamaterials based on the Miura-derivative fold patterns

    PubMed Central

    Zhou, Xiang; Zang, Shixi

    2016-01-01

    This paper presents two new types of origami-inspired mechanical metamaterials based on the Miura-derivative fold patterns that consist of non-identical parallelogram facets. The analytical models to predict dimension changes and deformation kinematics of the proposed metamaterials are developed. Furthermore, by modelling the creases as revolute hinges with certain rotational spring constants, we derived analytical models for stretching and bulk moduli. The analytical models are validated through finite-element simulation results. Numerical examples reveal that the proposed metamaterials possess some intriguing properties, including negative Poisson’s ratios and bulk modulus. The work presented in this paper can provide a highly flexible framework for the design of versatile tunable mechanical metamaterials. PMID:27493581

  14. Origami mechanical metamaterials based on the Miura-derivative fold patterns

    NASA Astrophysics Data System (ADS)

    Zhou, Xiang; Zang, Shixi; You, Zhong

    2016-07-01

    This paper presents two new types of origami-inspired mechanical metamaterials based on the Miura-derivative fold patterns that consist of non-identical parallelogram facets. The analytical models to predict dimension changes and deformation kinematics of the proposed metamaterials are developed. Furthermore, by modelling the creases as revolute hinges with certain rotational spring constants, we derived analytical models for stretching and bulk moduli. The analytical models are validated through finite-element simulation results. Numerical examples reveal that the proposed metamaterials possess some intriguing properties, including negative Poisson's ratios and bulk modulus. The work presented in this paper can provide a highly flexible framework for the design of versatile tunable mechanical metamaterials.

  15. Folding to Curved Surfaces: A Generalized Design Method and Mechanics of Origami-based Cylindrical Structures.

    PubMed

    Wang, Fei; Gong, Haoran; Chen, Xi; Chen, C Q

    2016-09-14

    Origami structures enrich the field of mechanical metamaterials with the ability to convert morphologically and systematically between two-dimensional (2D) thin sheets and three-dimensional (3D) spatial structures. In this study, an in-plane design method is proposed to approximate curved surfaces of interest with generalized Miura-ori units. Using this method, two combination types of crease lines are unified in one reprogrammable procedure, generating multiple types of cylindrical structures. Structural completeness conditions of the finite-thickness counterparts to the two types are also proposed. As an example of the design method, the kinematics and elastic properties of an origami-based circular cylindrical shell are analysed. The concept of Poisson's ratio is extended to the cylindrical structures, demonstrating their auxetic property. An analytical model of rigid plates linked by elastic hinges, consistent with numerical simulations, is employed to describe the mechanical response of the structures. Under particular load patterns, the circular shells display novel mechanical behaviour such as snap-through and limiting folding positions. By analysing the geometry and mechanics of the origami structures, we extend the design space of mechanical metamaterials and provide a basis for their practical applications in science and engineering.

  16. Folding to Curved Surfaces: A Generalized Design Method and Mechanics of Origami-based Cylindrical Structures

    NASA Astrophysics Data System (ADS)

    Wang, Fei; Gong, Haoran; Chen, Xi; Chen, C. Q.

    2016-09-01

    Origami structures enrich the field of mechanical metamaterials with the ability to convert morphologically and systematically between two-dimensional (2D) thin sheets and three-dimensional (3D) spatial structures. In this study, an in-plane design method is proposed to approximate curved surfaces of interest with generalized Miura-ori units. Using this method, two combination types of crease lines are unified in one reprogrammable procedure, generating multiple types of cylindrical structures. Structural completeness conditions of the finite-thickness counterparts to the two types are also proposed. As an example of the design method, the kinematics and elastic properties of an origami-based circular cylindrical shell are analysed. The concept of Poisson’s ratio is extended to the cylindrical structures, demonstrating their auxetic property. An analytical model of rigid plates linked by elastic hinges, consistent with numerical simulations, is employed to describe the mechanical response of the structures. Under particular load patterns, the circular shells display novel mechanical behaviour such as snap-through and limiting folding positions. By analysing the geometry and mechanics of the origami structures, we extend the design space of mechanical metamaterials and provide a basis for their practical applications in science and engineering.

  17. Folding to Curved Surfaces: A Generalized Design Method and Mechanics of Origami-based Cylindrical Structures

    PubMed Central

    Wang, Fei; Gong, Haoran; Chen, Xi; Chen, C. Q.

    2016-01-01

    Origami structures enrich the field of mechanical metamaterials with the ability to convert morphologically and systematically between two-dimensional (2D) thin sheets and three-dimensional (3D) spatial structures. In this study, an in-plane design method is proposed to approximate curved surfaces of interest with generalized Miura-ori units. Using this method, two combination types of crease lines are unified in one reprogrammable procedure, generating multiple types of cylindrical structures. Structural completeness conditions of the finite-thickness counterparts to the two types are also proposed. As an example of the design method, the kinematics and elastic properties of an origami-based circular cylindrical shell are analysed. The concept of Poisson’s ratio is extended to the cylindrical structures, demonstrating their auxetic property. An analytical model of rigid plates linked by elastic hinges, consistent with numerical simulations, is employed to describe the mechanical response of the structures. Under particular load patterns, the circular shells display novel mechanical behaviour such as snap-through and limiting folding positions. By analysing the geometry and mechanics of the origami structures, we extend the design space of mechanical metamaterials and provide a basis for their practical applications in science and engineering. PMID:27624892

  18. Mechanical analysis of fault activation in southern Longmen Shan fold-and- thrust belt

    NASA Astrophysics Data System (ADS)

    Zhang, Zhen; Zhang, Huai; Wang, Liangshu; Shi, Yaolin; Leroy, Yves M.

    2017-04-01

    A mixed fault activation mode with obvious hinterland rupture in the southern Longmen Shan, the eastern margin of Tibetan Plateau, is revealed by recent 2008 Mw7.9 Wenchuan and 2013 Mw6.6 Lushan earthquakes together with GPS measurements. How to systematically understand the coexistence and competition mechanisms of fault activation, especially the principal-subordinate relationship on deformation absorption, in essence, involves mechanical onset analysis of this fold- and-thrust belt. However, due to the two-décollement- level thrust system with active 'flat-ramp- flat' geometry décollement, the predication of fault activation in the LMS has beyond the scope of Critical Coulomb wedge theory, not to mention the synchronous listric-type splay fault rupturing in the Beichuan fault (BCF) and Pengguan fault (PGF). For that purpose, we adopted maximum strength theorem, the kinematic approach of limit analysis, to deal with mechanical analysis of fault activation. Four end-member failure modes, or collapse mechanisms (CMs) in classical limit analysis, are proposed corresponding to the rupture of BCF, PGF, Range Frontal Blind Fault (RFBF) and the rupture of the flat-ramp- flat décollement into Sichuan Basin via RFBF. By selecting the available CMs via finite element limit analysis, the listric geometry of BCF and PGF is demonstrated to the dominant factor in trapping deformation in the hinterland. To activate the high-angle Beichuan splay fault, low cohesion and low friction angle on the BCF are combined effects on the rupturing of BCF. The change in cohesion and friction on BCF eventually forms the transition state between high angle BCF and low-angle PGF. Besides, due to the existence of low frictional upper décollement layer in Sichuan Basin (the Triassic evaporate layer), small amount of deformation is attracted into the Sichuan Basin forming small-scale thrusting folding. Moreover, favorable deformation migration toward Sichuan Basin is jointly influenced by

  19. Web-based toolkits for topology prediction of transmembrane helical proteins, fold recognition, structure and binding scoring, folding-kinetics analysis and comparative analysis of domain combinations.

    PubMed

    Zhou, Hongyi; Zhang, Chi; Liu, Song; Zhou, Yaoqi

    2005-07-01

    We have developed the following web servers for protein structural modeling and analysis at http://theory.med.buffalo.edu: THUMBUP, UMDHMM(TMHP) and TUPS, predictors of transmembrane helical protein topology based on a mean-burial-propensity scale of amino acid residues (THUMBUP), hidden Markov model (UMDHMM(TMHP)) and their combinations (TUPS); SPARKS 2.0 and SP3, two profile-profile alignment methods, that match input query sequence(s) to structural templates by integrating sequence profile with knowledge-based structural score (SPARKS 2.0) and structure-derived profile (SP3); DFIRE, a knowledge-based potential for scoring free energy of monomers (DMONOMER), loop conformations (DLOOP), mutant stability (DMUTANT) and binding affinity of protein-protein/peptide/DNA complexes (DCOMPLEX & DDNA); TCD, a program for protein-folding rate and transition-state analysis of small globular proteins; and DOGMA, a web-server that allows comparative analysis of domain combinations between plant and other 55 organisms. These servers provide tools for prediction and/or analysis of proteins on the secondary structure, tertiary structure and interaction levels, respectively.

  20. Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

    PubMed Central

    Bisaria, Namita; Greenfeld, Max; Limouse, Charles; Pavlichin, Dmitri S.; Mabuchi, Hideo; Herschlag, Daniel

    2016-01-01

    The past decade has seen a wealth of 3D structural information about complex structured RNAs and identification of functional intermediates. Nevertheless, developing a complete and predictive understanding of the folding and function of these RNAs in biology will require connection of individual rate and equilibrium constants to structural changes that occur in individual folding steps and further relating these steps to the properties and behavior of isolated, simplified systems. To accomplish these goals we used the considerable structural knowledge of the folded, unfolded, and intermediate states of P4-P6 RNA. We enumerated structural states and possible folding transitions and determined rate and equilibrium constants for the transitions between these states using single-molecule FRET with a series of mutant P4-P6 variants. Comparisons with simplified constructs containing an isolated tertiary contact suggest that a given tertiary interaction has a stereotyped rate for breaking that may help identify structural transitions within complex RNAs and simplify the prediction of folding kinetics and thermodynamics for structured RNAs from their parts. The preferred folding pathway involves initial formation of the proximal tertiary contact. However, this preference was only ∼10 fold and could be reversed by a single point mutation, indicating that a model akin to a protein-folding contact order model will not suffice to describe RNA folding. Instead, our results suggest a strong analogy with a modified RNA diffusion-collision model in which tertiary elements within preformed secondary structures collide, with the success of these collisions dependent on whether the tertiary elements are in their rare binding-competent conformations. PMID:27493222

  1. Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism

    PubMed Central

    Arias-Cartin, Rodrigo; Ceccaldi, Pierre; Schoepp-Cothenet, Barbara; Frick, Klaudia; Blanc, Jean-Michel; Guigliarelli, Bruno; Walburger, Anne; Grimaldi, Stéphane; Friedrich, Thorsten; Receveur-Brechot, Véronique; Magalon, Axel

    2016-01-01

    A major gap of knowledge in metalloproteins is the identity of the prefolded state of the protein before cofactor insertion. This holds for molybdoenzymes serving multiple purposes for life, especially in energy harvesting. This large group of prokaryotic enzymes allows for coordination of molybdenum or tungsten cofactors (Mo/W-bisPGD) and Fe/S clusters. Here we report the structural data on a cofactor-less enzyme, the nitrate reductase respiratory complex and characterize the conformational changes accompanying Mo/W-bisPGD and Fe/S cofactors insertion. Identified conformational changes are shown to be essential for recognition of the dedicated chaperone involved in cofactors insertion. A solvent-exposed salt bridge is shown to play a key role in enzyme folding after cofactors insertion. Furthermore, this salt bridge is shown to be strictly conserved within this prokaryotic molybdoenzyme family as deduced from a phylogenetic analysis issued from 3D structure-guided multiple sequence alignment. A biochemical analysis with a distantly-related member of the family, respiratory complex I, confirmed the critical importance of the salt bridge for folding. Overall, our results point to a conserved cofactors insertion mechanism within the Mo/W-bisPGD family. PMID:27886223

  2. Direct Observation of Folding Energy Landscape of RNA Hairpin at Mechanical Loading Rates.

    PubMed

    Xu, Huizhong; Plaut, Benjamin; Zhu, Xiran; Chen, Maverick; Mavinkurve, Udit; Maiti, Anindita; Song, Guangtao; Murari, Krishna; Mandal, Maumita

    2017-03-16

    By applying a controlled mechanical load using optical tweezers, we measured the diffusive barrier crossing in a 49 nt long P5ab RNA hairpin. We find that in the free-energy landscape the barrier height (G(‡)) and transition distance (x(‡)) are dependent on the loading rate (r) along the pulling direction, x, as predicted by Bell. The barrier shifted toward the initial state, whereas ΔG(‡) reduced significantly from 50 to 5 kT, as r increased from 0 to 32 pN/s. However, the equilibrium work (ΔG) during strand separation, as estimated by Crook's fluctuation theorem, remained unchanged at different rates. Previously, helix formation and denaturation have been described as two-state (F ↔ U) transitions for P5ab. Herein, we report three intermediate states I1, I, and I2 located at 4, 11, and 16 nm respectively, from the folded conformation. The intermediates were observed only when the hairpin was subjected to an optimal r, 7.6 pN/s. The results indicate that the complementary strands in P5ab can zip and unzip through complex routes, whereby mismatches act as checkpoints and often impose barriers. The study highlights the significance of loading rates in force-spectroscopy experiments that are increasingly being used to measure the folding properties of biomolecules.

  3. Role of five-fold twin boundary on the enhanced mechanical properties of fcc Fe nanowires.

    PubMed

    Wu, J Y; Nagao, S; He, J Y; Zhang, Z L

    2011-12-14

    The role of 5-fold twin boundary on the structural and mechanical properties of fcc Fe nanowire under tension is explored by classical molecular dynamics. Twin-stabilized fcc nanowire with various diameters (6-24 nm) are examined by tension tests at several temperatures ranging from 0.01 to 1100 K. Significant increase in the Young's modulus of the smaller nanowires is revealed to originate from the central area of quinquefoliolate-like stress-distribution over the 5-fold twin, rather than from the surface tension that is often considered as the main source of such size-effects found in nanostructures. Because of the excess compressive stress caused by crossing twin-boundaries, the atoms in the center behave stiffer than those in bulk and even expand laterally under axial tension, providing locally negative Poisson's ratio. The yield strength of nanowire is also enhanced by the twin boundary that suppresses dislocation nucleation within a fcc twin-domain; therefore, the plasticity of nanowire is initiated by strain-induced fcc→bcc phase transformation that destroys the twin structure. After the yield, the nucleated bcc phase immediately spreads to the entire area, and forms a multigrain structure to realize ductile deformation followed by necking. As temperature elevated close to the critical temperature between bcc and fcc phases, the increased stability of fcc phase competes with the phase transformation under tension, and hence dislocation nucleations in fcc phase are observed exclusively at the highest temperature in our study.

  4. Protein roles in group I intron RNA folding: The tyrosyl-tRNA synthetase CYT-18 stabilizes the native state relative to a long-lived misfolded structure without compromising folding kinetics

    PubMed Central

    Chadee, Amanda B.; Bhaskaran, Hari; Russell, Rick

    2009-01-01

    The Neurospora crassa CYT-18 protein is a mitochondrial tyrosyl-tRNA synthetase that also promotes self-splicing of group I intron RNAs by stabilizing functional structure in the conserved core. CYT-18 binds the core along the same surface as a common peripheral element, P5abc, suggesting that CYT-18 can replace P5abc functionally. In addition to stabilizing structure generally, P5abc stabilizes the native conformation of the Tetrahymena group I intron relative to a globally-similar misfolded conformation, which has only local differences within the core and is populated significantly at equilibrium by a ribozyme variant lacking P5abc (EΔP5abc). Here we show that CYT-18 specifically promotes formation of the native group I intron core from this misfolded conformation. Catalytic activity assays demonstrate that CYT-18 shifts the equilibrium of EΔP5abc toward the native state by at least 35-fold, and binding assays suggest an even larger effect. Thus, like P5abc, CYT-18 preferentially recognizes the native core, despite the global similarity of the misfolded core and despite forming crudely similar complexes, as revealed by DMS footprinting. Interestingly, the effects of CYT-18 and P5abc on folding kinetics differ. Whereas P5abc inhibits refolding of the misfolded conformation by forming peripheral contacts that must break during refolding, CYT-18 does not display analogous inhibition, most likely because it relies to a greater extent on direct interactions with the core. Although CYT-18 does not encounter this RNA in vivo, our results suggest that it stabilizes its cognate group I introns relative to analogous misfolded intermediates. By specifically recognizing native structure, CYT-18 may also interact with earlier folding intermediates to avoid RNA misfolding or to trap native structure as it forms. More generally, our results highlight the ability of a protein cofactor to stabilize a functional RNA structure specifically without incurring associated costs in

  5. Purification and kinetic mechanism of the major glutathione S-transferase from bovine brain.

    PubMed Central

    Young, P R; Briedis, A V

    1989-01-01

    The major glutathione S-transferase isoenzyme from bovine brain was isolated and purified approx. 500-fold. The enzyme has a pI of 7.39 +/- 0.02 and consists of two non-identical subunits having apparent Mr values of 22,000 and 24,000. The enzyme is uniformly distributed in brain, and kinetic data at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate suggest a random rapid-equilibrium mechanism. The kinetics of inhibition by product, by GSH analogues and by NADH are consistent with the suggested mechanism and require inhibitor binding to several different enzyme forms. Long-chain fatty acids are excellent inhibitors of the enzyme, and values of 1nKi for hexanoic acid, octanoic acid, decanoic acid and lauric acid form a linear series when plotted as a function of alkyl chain length. A free-energy change of -1900 J/mol (-455 cal/mol) per CH2 unit is calculated for the contribution of hydrophobic binding energy to the inhibition constants. The turnover number of the purified enzyme dimer is approx. 3400/min. When compared with the second-order rate constant for the reaction between CDNB and GSH, the enzyme is providing a rate acceleration of about 1000-fold. The role of entropic contributions to this small rate acceleration is discussed. PMID:2930465

  6. Kinetics and mechanisms of some atomic oxygen reactions

    NASA Technical Reports Server (NTRS)

    Cvetanovic, R. J.

    1987-01-01

    Mechanisms and kinetics of some reactions of the ground state of oxygen atoms, O(3P), are briefly summarized. Attention is given to reactions of oxygen atoms with several different types of organic and inorganic compounds such as alkanes, alkenes, alkynes, aromatics, and some oxygen, nitrogen, halogen and sulfur derivatives of these compounds. References to some recent compilations and critical evaluations of reaction rate constants are given.

  7. Rapid three-dimensional microfluidic mixer for high viscosity solutions to unravel earlier folding kinetics of G-quadruplex under molecular crowding conditions.

    PubMed

    Liu, Chao; Li, Ying; Li, Yiwei; Chen, Peng; Feng, Xiaojun; Du, Wei; Liu, Bi-Feng

    2016-01-01

    Rapid mixing of highly viscous solutions is a great challenge, which helps to analyze the reaction kinetics in viscous liquid phase, particularly to discover the folding kinetics of macromolecules under molecular crowding conditions mimicking the conditions inside cells. Here, we demonstrated a novel microfluidic mixer based on Dean flows with three-dimensional (3D) microchannel configuration for fast mixing of high-viscosity fluids. The main structure contained three consecutive subunits, each consisting of a "U"-type channel followed by a chamber with different width and height. Thus, the two solutions injected from the two inlets would undergo a mixing in the first "U"-type channel due to the Dean flow effect, and simultaneous vortices expansions in both horizontal and vertical directions in the following chamber. Numerical simulations and experimental characterizations confirmed that the micromixer could achieve a mixing time of 122.4μs for solutions with viscosities about 33.6 times that of pure water. It was the fastest micromixer for high viscosity solutions compared with previous reports. With this highly efficient 3D microfluidic mixer, we further characterized the early folding kinetics of human telomere G-quadruplex under molecular crowding conditions, and unravelled a new folding process within 550μs.

  8. Biomineralization mechanisms: a kinetics and interfacial energy approach

    NASA Astrophysics Data System (ADS)

    Nancollas, George H.; Wu, Wenju

    2000-04-01

    The calcium phosphates and oxalates are among the most frequently encountered biomineral phases and numerous kinetics studies have been made of their crystallization and dissolution in supersaturated and undersaturated solutions, respectively. These have focused mainly on parameters such as solution composition, ionic strength, pH, temperature, and solid surface characteristics. There is considerable interest in extending such studies to solutions more closely simulating the biological milieu. The constant composition method is especially useful for investigating the mechanisms of these reactions, and in the present work, the interfacial tensions between water and each of these surfaces have been calculated from measured contact angles using surface tension component theory. Values for the calcium phosphate phases such as dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP), hydroxyapatite (HAP), and fluorapatite (FAP) may be compared with data calculated from dissolution kinetics experiments invoking different reaction mechanisms. Agreement between the directly measured interfacial energies and those calculated from the kinetics experiments provides valuable corroborative information about individual growth and dissolution mechanisms. For the calcium phosphates, the much smaller interfacial tensions of OCP and DCPD in contact with water as compared with those of HAP and FAP support the suggestion that the former phases are precursors in HAP and FAP biomineralization. The ability of a surface to nucleate mineral phases is closely related to the magnitude of the interfacial energies. Constant composition studies have also shown that HAP is an effective nucleator of calcium oxalate monohydrate, both of which are frequently observed in renal stones.

  9. Mechanics of fold-and-thrust belts and accretionary wedges Cohesive Coulomb theory

    NASA Technical Reports Server (NTRS)

    Dahlen, F. A.; Suppe, J.; Davis, D.

    1984-01-01

    A self-consistent theory for the mechanics of thin-skinned accretionary Coulomb wedges is developed and applied to the active fold-and-thrust belt of western Taiwan. The state of stress everywhere within a critical wedge is determined by solving the static equilibrium equations subject to the appropriate boundary conditions. The influence of wedge cohesion, which gives rise to a concave curvature of the critical topographic surface and affects the orientation of the principal stresses and Coulomb fracture within the wedge, is considered. The shape of the topographic surface and the angles at which thrust faults step up from the basal decollement in the Taiwanese belt is analyzed taking into account the extensive structural and fluid-pressure data available there. It is concluded that the gross geometry and structure of the Taiwan wedge are consistent with normal laboratory frictional and fracture strengths of sedimentary rocks.

  10. Alternating access mechanisms of LeuT-fold transporters: trailblazing towards the promised energy landscapes.

    PubMed

    Kazmier, Kelli; Claxton, Derek P; Mchaourab, Hassane S

    2016-12-29

    Secondary active transporters couple the uphill translocation of substrates to electrochemical ion gradients. Transporter conformational motion, generically referred to as alternating access, enables a central ligand binding site to change its orientation relative to the membrane. Here we review themes of alternating access and the transduction of ion gradient energy to power this process in the LeuT-fold class of transporters where crystallographic, computational and spectroscopic approaches have converged to yield detailed models of transport cycles. Specifically, we compare findings for the Na(+)-coupled amino acid transporter LeuT and the Na(+)-coupled hydantoin transporter Mhp1. Although these studies have illuminated multiple aspects of transporter structures and dynamics, a number of questions remain unresolved that so far hinder understanding transport mechanisms in an energy landscape perspective.

  11. Kinetic mechanism of protein N-terminal methyltransferase 1.

    PubMed

    Richardson, Stacie L; Mao, Yunfei; Zhang, Gang; Hanjra, Pahul; Peterson, Darrell L; Huang, Rong

    2015-05-01

    The protein N-terminal methyltransferase 1 (NTMT1) catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine to the protein α-amine, resulting in formation of S-adenosyl-l-homocysteine and α-N-methylated proteins. NTMT1 is an interesting potential anticancer target because it is overexpressed in gastrointestinal cancers and plays an important role in cell mitosis. To gain insight into the biochemical mechanism of NTMT1, we have characterized the kinetic mechanism of recombinant NTMT1 using a fluorescence assay and mass spectrometry. The results of initial velocity, product, and dead-end inhibition studies indicate that methylation by NTMT1 proceeds via a random sequential Bi Bi mechanism. In addition, our processivity studies demonstrate that NTMT1 proceeds via a distributive mechanism for multiple methylations. Together, our studies provide new knowledge about the kinetic mechanism of NTMT1 and lay the foundation for the development of mechanism-based inhibitors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Kinetic Mechanism of Protein N-terminal Methyltransferase 1*

    PubMed Central

    Richardson, Stacie L.; Mao, Yunfei; Zhang, Gang; Hanjra, Pahul; Peterson, Darrell L.; Huang, Rong

    2015-01-01

    The protein N-terminal methyltransferase 1 (NTMT1) catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine to the protein α-amine, resulting in formation of S-adenosyl-l-homocysteine and α-N-methylated proteins. NTMT1 is an interesting potential anticancer target because it is overexpressed in gastrointestinal cancers and plays an important role in cell mitosis. To gain insight into the biochemical mechanism of NTMT1, we have characterized the kinetic mechanism of recombinant NTMT1 using a fluorescence assay and mass spectrometry. The results of initial velocity, product, and dead-end inhibition studies indicate that methylation by NTMT1 proceeds via a random sequential Bi Bi mechanism. In addition, our processivity studies demonstrate that NTMT1 proceeds via a distributive mechanism for multiple methylations. Together, our studies provide new knowledge about the kinetic mechanism of NTMT1 and lay the foundation for the development of mechanism-based inhibitors. PMID:25771539

  13. The carbon-bond mechanism: a condensed kinetic mechanism for photochemical smog

    SciTech Connect

    Whitten, G.Z.; Hog, H.; Killus, J.P.

    1980-06-01

    Efforts to develop a model that can simulate photochemical smog with kinetic mechanisms are discussed. The carbon-bond mechanism is a set of generalized reactions that can be used to model photochemical oxidant formation. The theoretical framework of carbon-bond mechanism is outlined. Chemical variables that are incorporated into the carbon-bond mechanism model are described. Further work that is needed on the carbon-bond mechanism model is considered. (1 diagram, 13 graphs, 30 references, 2 tables)

  14. Effects of heme on the structure of the denatured state and folding kinetics of cytochrome b562.

    PubMed

    Garcia, Pascal; Bruix, Marta; Rico, Manuel; Ciofi-Baffoni, Simone; Banci, Lucia; Ramachandra Shastry, M C; Roder, Heinrich; de Lumley Woodyear, Thierry; Johnson, Christopher M; Fersht, Alan R; Barker, Paul D

    2005-02-11

    Heme-linked proteins, such as cytochromes, are popular subjects for protein folding studies. There is the underlying question of whether the heme affects the structure of the denatured state by cross-linking it and forming other interactions, which would perturb the folding pathway. We have studied wild-type and mutant cytochrome b562 from Escherichia coli, a 106 residue four-alpha-helical bundle. The holo protein apparently refolds with a half-life of 4 micros in its ferrous state. We have analysed the folding of the apo protein using continuous-flow fluorescence as well as stopped-flow fluorescence and CD. The apo protein folded much more slowly with a half-life of 270 micros that was unaffected by the presence of exogenous heme. We examined the nature of the denatured states of both holo and apo proteins by NMR methods over a range of concentrations of guanidine hydrochloride. The starting point for folding of the holo protein in concentrations of denaturant around the denaturation transition was a highly ordered native-like species with heme bound. Fully denatured holo protein at higher concentrations of denaturant consisted of denatured apo protein and free heme. Our results suggest that the very fast folding species of denatured holo protein is in a compact state, whereas the normal folding pathway from fully denatured holo protein consists of the slower folding of the apo protein followed by the binding of heme. These data should be considered in the analysis of folding of heme proteins.

  15. Constraints on bed scale fracture chronology with a FEM mechanical model of folding: The case of Split Mountain (Utah, USA)

    NASA Astrophysics Data System (ADS)

    Sassi, W.; Guiton, M. L. E.; Leroy, Y. M.; Daniel, J.-M.; Callot, J.-P.

    2012-11-01

    A technique is presented for improving the structural analysis of natural fractures development in large scale fold structures. A 3D restoration of a fold provides the external displacement loading conditions to solve, by the finite element method, the forward mechanical problem of an idealized rock material with a stress-strain relationship based on the activation of pervasive fracture sets. In this elasto-plasticity constitutive law, any activated fracture set contributes to the total plastic strain by either an opening or a sliding mode of rock failure. Inherited versus syn-folding fracture sets development can be studied using this mechanical model. The workflow of this methodology was applied to the Weber sandstone formation deformed by forced folding at Split Mountain Anticline, Utah for which the different fracture sets were created and developed successively during the Sevier and the syn-folding Laramide orogenic phases. The field observations at the top stratigraphic surface of the Weber sandstone lead to classify the fracture sets into a pre-fold WNW-ESE fracture set, and a NE-SW fracture set post-dating the former. The development and relative chronology of the fracture sets are discussed based on the geomechanical modeling results. Starting with a 3D restoration of the Split Mountain Anticline, three fold-fracture development models were generated, alternately assuming that the WNW-ESE fracture set is either present or absent prior to folding process. Depending on the initial fracture configuration, the calculated fracture patterns are markedly different, showing that assuming a WNW-ESE joint set to predate the fold best correlates with field observations. This study is a first step addressing the complex problem of identification of fold-related fracturing events using an elementary concept of rock mechanics. When tight to complementary field observations, including petrography, diagenesis and burial history, the approach can be used to better

  16. Predicting Folding Sequences Based on the Maximum Rock Strength and Mechanical Equilibrium

    NASA Astrophysics Data System (ADS)

    Cubas, N.; Souloumiac, P.; Maillot, B.; Leroy, Y. M.

    2007-12-01

    The objective is to propose and validate simple procedures, compared to the finite-element method, to select and optimize the dominant mode of folding in fold-and-thrust belts and accretionary wedges, and to determine its stress distribution. Mechanical equilibrium as well as the constraints due to the limited rock strength of the bulk material and of major discontinuities, such as décollements, are accounted for. The first part of the proposed procedure, which is at the core of the external approach of classical limit analysis, consists in estimating the least upper bound on the tectonic force by minimisation of the internal dissipation and part of the external work. The new twist to the method is that the optimization is also done with respect to the geometry of the evolving fold. If several folding events are possible, the dominant mode is the one leading to the least upper bound. The second part of the procedure is based on the Equilibrium Element Method, which is an application of the internal approach of limit analysis. The optimum stress field, obtained by spatial discretisation of the fold, provides the best lower bound on the tectonic force. The difference between the two bounds defines an error estimate of the exact unknown tectonic force. To show the merits of the proposed procedure, its first part is applied to predict the life span of a thrust within an accretionary prism, from its onset, its development with a relief build up and its arrest because of the onset of a more favorable new thrust (Cubas et al., 2007). This life span is sensitive to the friction angles over the ramp and the décollement. It is shown how the normal sequence of thrusting in a supercritical wedge is ended with the first out-of sequence event. The second part of the procedure provides the stress state over each thrust showing that the active back thrust is a narrow fan which dip is sensitive to the friction angle over the ramp and the amount of relief build up (Souloumiac et

  17. Detailed Chemical Kinetic Mechanisms for Combustion of Oxygenated Fuels

    SciTech Connect

    Fisher, E.M.; Pitz, W.J.; Curran, H.J.; Westbrook, C.K.

    2000-01-11

    Thermodynamic properties and detailed chemical kinetic models have been developed for the combustion of two oxygenates: methyl butanoate, a model compound for biodiesel fuels, and methyl formate, a related simpler molecule. Bond additivity methods and rules for estimating kinetic parameters were adopted from hydrocarbon combustion and extended. The resulting mechanisms have been tested against the limited combustion data available in the literature, which was obtained at low temperature, subatmospheric conditions in closed vessels, using pressure measurements as the main diagnostic. Some qualitative agreement was obtained, but the experimental data consistently indicated lower overall reactivities than the model, differing by factors of 10 to 50. This discrepancy, which occurs for species with well-established kinetic mechanisms as well as for methyl esters, is tentatively ascribed to the presence of wall reactions in the experiments. The model predicts a region of weak or negative dependence of overall reaction rate on temperature for each methyl ester. Examination of the reaction fluxes provides an explanation of this behavior, involving a temperature-dependent competition between chain-propagating unimolecular decomposition processes and chain-branching processes, similar to that accepted for hydrocarbons. There is an urgent need to obtain more complete experimental data under well-characterized conditions for thorough testing of the model.

  18. Microwave pyrolysis of rice straw: products, mechanism, and kinetics.

    PubMed

    Huang, Yu-Fong; Chiueh, Pei-Te; Kuan, Wen-Hui; Lo, Shang-Lien

    2013-08-01

    Rice straw is an abundant resource for the production of biofuels and bio-based products. How to convert the recalcitrant lignocellulose effectually is a critical issue. The objective of this study was to investigate the products, mechanism, and kinetics of rice straw pyrolysis by using microwave heating. The highest energy densification ratio of solid residues was achieved at the microwave power level of 300 W. The atomic H/C and O/C ratios of solid residues were much lower than those of rice straw. The primary components of gaseous product were CO, H2, CO2, and CH4, whose molecular fractions were 57%, 21%, 14%, and 8%, respectively. The more gaseous product and the less solid residues were obtained at higher microwave power levels, while the liquid production remained the same and showed a maximum of about 50 wt.%. The kinetic parameters of rice straw pyrolysis were increased with increasing microwave power level.

  19. Unraveling protein folding mechanism by analyzing the hierarchy of models with increasing level of detail

    NASA Astrophysics Data System (ADS)

    Hayashi, Tomohiko; Yasuda, Satoshi; Škrbić, Tatjana; Giacometti, Achille; Kinoshita, Masahiro

    2017-09-01

    Taking protein G with 56 residues for a case study, we investigate the mechanism of protein folding. In addition to its native structure possessing α-helix and β-sheet contents of 27% and 39%, respectively, we construct a number of misfolded decoys with a wide variety of α-helix and β-sheet contents. We then consider a hierarchy of 8 different models with increasing level of detail in terms of the number of entropic and energetic physical factors incorporated. The polyatomic structure is always taken into account, but the side chains are removed in half of the models. The solvent is formed by either neutral hard spheres or water molecules. Protein intramolecular hydrogen bonds (H-bonds) and protein-solvent H-bonds (the latter is present only in water) are accounted for or not, depending on the model considered. We then apply a physics-based free-energy function (FEF) corresponding to each model and investigate which structures are most stabilized. This special approach taken on a step-by-step basis enables us to clarify the role of each physical factor in contributing to the structural stability and separately elucidate its effect. Depending on the model employed, significantly different structures such as very compact configurations with no secondary structures and configurations of associated α-helices are optimally stabilized. The native structure can be identified as that with lowest FEF only when the most detailed model is employed. This result is significant for at least the two reasons: The most detailed model considered here is able to capture the fundamental aspects of protein folding notwithstanding its simplicity; and it is shown that the native structure is stabilized by a complex interplay of minimal multiple factors that must be all included in the description. In the absence of even a single of these factors, the protein is likely to be driven towards a different, more stable state.

  20. Folded Spring and Mechanically Switching SSHI for High Performance Miniature Piezoelectric Vibration Energy Harvester

    NASA Astrophysics Data System (ADS)

    Asanuma, H.; Okubo, H.; Komatsuzaki, T.; Iwata, Y.

    2016-11-01

    To downsize the clamp area and increase the output power of the harvester, we developed a miniature piezoelectric vibration energy harvester with combining a Z-shaped folded spring and a mechanically-switching SSHI (synchronized switch harvesting on inductor). The overall harvester size is 4×2×3 cm3. The FEM analysis revealed that the output power increases and the value of the 1st and 2nd resonance frequencies move closer as the angle of the Z-shaped spring decreases, therefore, the smaller angle would be more promising. The experimental results showed that the maximum output power of our harvester for the 1st (20.2 Hz) and 2nd (53.0 Hz) resonance frequencies at the applied acceleration of 4.9 m/s2 are 088 and 0.98 mW, respectively. The reason for a marked enhancement of the output power for the 2nd resonance frequency is attributed to the vertical movement of the 2nd vibrational mode which applies larger mechanical stress to the piezo ceramic and achieves better electrical contact between the tip of the Z-shaped spring and the spring plunger.

  1. Reaction route graphs. III. Non-minimal kinetic mechanisms.

    PubMed

    Fishtik, Ilie; Callaghan, Caitlin A; Datta, Ravindra

    2005-02-24

    The concept of reaction route (RR) graphs introduced recently by us for kinetic mechanisms that produce minimal graphs is extended to the problem of non-minimal kinetic mechanisms for the case of a single overall reaction (OR). A RR graph is said to be minimal if all of the stoichiometric numbers in all direct RRs of the mechanism are equal to +/-1 and non-minimal if at least one stoichiometric number in a direct RR is non-unity, e.g., equal to +/-2. For a given mechanism, four unique topological characteristics of RR graphs are defined and enumerated, namely, direct full routes (FRs), empty routes (ERs), intermediate nodes (INs), and terminal nodes (TNs). These are further utilized to construct the RR graphs. One algorithm involves viewing each IN as a central node in a RR sub-graph. As a result, the construction and enumeration of RR graphs are reduced to the problem of balancing the peripheral nodes in the RR sub-graphs according to the list of FRs, ERs, INs, and TNs. An alternate method involves using an independent set of RRs to draw the RR graph while satisfying the INs and TNs. Three examples are presented to illustrate the application of non-minimal RR graph theory.

  2. Atomimetic Mechanical Structures with Nonlinear Topological Domain Evolution Kinetics.

    PubMed

    Frazier, Michael J; Kochmann, Dennis M

    2017-03-21

    A mechanical metamaterial, a simple, periodic mechanical structure, is reported, which reproduces the nonlinear dynamic behavior of materials undergoing phase transitions and domain switching at the structural level. Tunable multistability is exploited to produce switching and transition phenomena whose kinetics are governed by the same Allen-Cahn law commonly used to describe material-level, structural-transition processes. The reported purely elastic mechanical system displays several key features commonly found in atomic- or mesoscale physics of solids. The rotating-mass network shows qualitatively analogous features as, e.g., ferroic ceramics or phase-transforming solids, and the discrete governing equation is shown to approach the phase field equation commonly used to simulate the above processes. This offers untapped opportunities for reproducing material-level, dissipative and diffusive kinetic phenomena at the structural level, which, in turn, invites experimental realization and paves the road for new active, intelligent, or phase-transforming mechanical metamaterials bringing small-scale processes to the macroscopically observable scale.

  3. Free energy landscape and folding mechanism of a beta-hairpin in explicit water: a replica exchange molecular dynamics study.

    PubMed

    Nguyen, Phuong H; Stock, Gerhard; Mittag, Emil; Hu, Chin-Kun; Li, Mai Suan

    2005-12-01

    The free energy landscape and the folding mechanism of the C-terminal beta-hairpin of protein G is studied by extensive replica exchange molecular dynamics simulations (40 replicas and 340 ns total simulation time), using the GROMOS96 force field and the SPC explicit water solvent. The study reveals that the system preferentially adopts a beta-hairpin structure at biologically important temperatures, and that the helix content is low at all temperatures studied. Representing the free energy landscape as a function of several types of reaction coordinates, four local minima corresponding to the folded, partially folded, molten globule, and unfolded states are identified. The findings suggest that the folding of the beta-hairpin occurs as the sequence: collapse of hydrophobic core --> formation of H-bond --> formation of the turn. Identifying the folded and molten globule states as the main conformations, the free energy landscape of the beta-hairpin is consistent with a two-state behavior with a broad transition state. The temperature dependence of the folding-unfolding transition is investigated in some detail. The enthalpy and entropy jumps at the folding transition temperature are found to be about three times lower than the experimental estimates, indicating that the folding-unfolding transition in silico is less cooperative than its in vitro counterpart. Proteins 2005. 2005 Wiley-Liss, Inc.

  4. Simplified jet-A kinetic mechanism for combustor application

    NASA Technical Reports Server (NTRS)

    Lee, Chi-Ming; Kundu, Krishna; Ghorashi, Bahman

    1993-01-01

    Successful modeling of combustion and emissions in gas turbine engine combustors requires an adequate description of the reaction mechanism. For hydrocarbon oxidation, detailed mechanisms are only available for the simplest types of hydrocarbons such as methane, ethane, acetylene, and propane. These detailed mechanisms contain a large number of chemical species participating simultaneously in many elementary kinetic steps. Current computational fluid dynamic (CFD) models must include fuel vaporization, fuel-air mixing, chemical reactions, and complicated boundary geometries. To simulate these conditions a very sophisticated computer model is required, which requires large computer memory capacity and long run times. Therefore, gas turbine combustion modeling has frequently been simplified by using global reaction mechanisms, which can predict only the quantities of interest: heat release rates, flame temperature, and emissions. Jet fuels are wide-boiling-range hydrocarbons with ranges extending through those of gasoline and kerosene. These fuels are chemically complex, often containing more than 300 components. Jet fuel typically can be characterized as containing 70 vol pct paraffin compounds and 25 vol pct aromatic compounds. A five-step Jet-A fuel mechanism which involves pyrolysis and subsequent oxidation of paraffin and aromatic compounds is presented here. This mechanism is verified by comparing with Jet-A fuel ignition delay time experimental data, and species concentrations obtained from flametube experiments. This five-step mechanism appears to be better than the current one- and two-step mechanisms.

  5. Network measures for protein folding state discrimination

    PubMed Central

    Menichetti, Giulia; Fariselli, Piero; Remondini, Daniel

    2016-01-01

    Proteins fold using a two-state or multi-state kinetic mechanisms, but up to now there is not a first-principle model to explain this different behavior. We exploit the network properties of protein structures by introducing novel observables to address the problem of classifying the different types of folding kinetics. These observables display a plain physical meaning, in terms of vibrational modes, possible configurations compatible with the native protein structure, and folding cooperativity. The relevance of these observables is supported by a classification performance up to 90%, even with simple classifiers such as discriminant analysis. PMID:27464796

  6. High temperature heterogeneous reaction kinetics and mechanisms of tungsten oxidation

    NASA Astrophysics Data System (ADS)

    Sabourin, Justin L.

    Tungsten, which is a material used in many high temperature applications, is limited by its susceptibility to oxidation at elevated temperatures. Although tungsten has the highest melting temperature of any metal, at much lower temperatures volatile oxides are formed during oxidation with oxygen containing species. This differs from many heterogeneous oxidation reactions involving metals since most reactions form very stable oxides that have higher melting or boiling points than the pure metal (e.g., aluminum, iron). Understanding heterogeneous oxidation and vaporization processes may allow for the expansion and improvement of high temperature tungsten applications. In order to increase understanding of the oxidation processes of tungsten, there is a need to develop reaction mechanisms and kinetics for oxidation processes involving oxidizers and environmental conditions of interest. Tungsten oxidation was thoroughly studied in the past, and today there is a good phenomenological understanding of these processes. However, as the design of large scale systems increasingly relies on computer modeling there becomes a need for improved descriptions of chemical reactions. With the increase in computing power over the last several decades, and the development of quantum chemistry and physics theories, heterogeneous systems can be modeled in detail at the molecular level. Thermochemical parameters that may not be measured experimentally may now be determined theoretically, a tool that was previously unavailable to scientists and engineers. Additionally, chemical kinetic modeling software is now available for both homogeneous and heterogeneous reactions. This study takes advantage of these new theoretical tools, as well as a thermogravimetric (TG) flow reactor developed as part of this study to learn about mechanisms and kinetics of tungsten oxidation. Oxidizers of interest are oxygen (O2), carbon dioxide (CO 2), water (H2O), and other oxidizers present in combustion and

  7. A new mechanism for producing cleavage in preexisting folds: The translation mechanism. An example in the Burela section (Variscan belt, NW Spain)

    NASA Astrophysics Data System (ADS)

    Bobillo-Ares, Nilo C.; Bastida, Fernando; Aller, Jesús; Lisle, Richard J.

    2017-01-01

    An outcrop on the Cantabrian coast (Burela section) shows a long train of tight meter-scale folds developed in Cambrian siliciclastic rocks. These folds have been shortened in the axial trace direction on the fold profile, developing a cleavage in the incompetent layers which obliterates the primary cleavage and crosscuts the folds. Several mechanisms have been analyzed to explain the development and attitude of this cleavage, some of them being the same as those that have previously been proposed to form folds but operating in a reverse sense. They are: anti-flexural flow, anti-reverse tangential longitudinal strain and homogeneous strain. The sole operation of these mechanisms cannot explain this cleavage and a new one has been defined with this aim. This mechanism consists of deformation of the incompetent layers by translation of the competent ones (translation mechanism), and it involves an area decrease within the incompetent layers in the fold profile plane and, if there is no important volume decrease, a stretching in the hinge direction that must affect both competent and incompetent layers. The geometrical properties of this mechanism have been analyzed in detail and it is concluded that, combined with a small amount of homogeneous flattening, this mechanism can explain the distribution of the cleavage through the folds.

  8. Kinetics and mechanism of the oxidation of amaranth with hypochlorite.

    PubMed

    Nadupalli, S; Koorbanally, N; Jonnalagadda, S B

    2011-07-14

    The reaction mechanism of the oxidation of Amaranth dye (2-hydroxy-1-(4-sulfonato-1-naphthylazo) naphthalene-3,6-disulfonate) with hypochlorite under varied pH conditions was elucidated by a kinetic approach. Under excess concentration of oxidant, the reaction followed pseudo-first-order kinetics with respect to Amaranth, and the oxidation was found to occur through two competitive reactions, initiated by hypochlorite and hypochlorous acid. The reaction order with respect to both OCl(-) ion and HOCl was unity. While the latter reaction was fast, the significance of the oxidation paths depended on the relative concentration of the two oxidizing species, which was dictated by the reaction pH. The role of the H(+) ion in the reaction was established. For the hypochlorite ion and hypochlorous acid facilitated reactions, the second-order rate coefficients were 1.9 and 23.2 M(-1) s(-1), respectively. The energy parameters were E(a) = 33.7 kJ mol(-1), ΔH(‡) = 31.2 kJ mol(-1) and ΔS(‡) = -190.6 J K(-1) mol(-1) for the OCl(-) ion-driven oxidation, and E(a) = 26.9 kJ mol(-1), ΔH(‡) = 24.3 kJ mol(-1) and ΔS(‡) = -222.8 J K(-1) mol(-1) for the reaction with HOCl-initiated oxidation. The major oxidation products for both the pathways were 3,4-dihydroxy naphthalene-2,7-disulfonic sodium salt (P(1)), dichloro-1,4-naphthoquione (P(2)) and naphtha(2,3)oxirene-2, 3-dione (P(3)). On the basis of the primary salt effect and other kinetic data, the rate law for the overall reaction and probable reaction mechanism was elucidated. The proposed mechanism was validated by simulations using Simkine-2.

  9. Kinetic mechanism of putrescine oxidase from Rhodococcus erythropolis.

    PubMed

    Kopacz, Malgorzata M; Heuts, Dominic P H M; Fraaije, Marco W

    2014-10-01

    Putrescine oxidase from Rhodococcus erythropolis (PuO) is a flavin-containing amine oxidase from the monoamine oxidase family that performs oxidative deamination of aliphatic diamines. In this study we report pre-steady-state kinetic analyses of the enzyme with the use of single- and double-mixing stopped-flow spectroscopy and putrescine as a substrate. During the fast and irreversible reductive half-reaction no radical intermediates were observed, suggesting a direct hydride transfer from the substrate to the FAD. The rate constant of flavin reoxidation depends on the ligand binding; when the imine product was bound to the enzyme the rate constant was higher than with free enzyme species. Similar results were obtained with product-mimicking ligands and this indicates that a ternary complex is formed during catalysis. The obtained kinetic data were used together with steady-state rate equations derived for ping-pong, ordered sequential and bifurcated mechanisms to explore which mechanism is operative. The integrated analysis revealed that PuO employs a bifurcated mechanism due to comparable rate constants of product release from the reduced enzyme and reoxidation of the reduced enzyme-product complex.

  10. Significance of first-order faults in folding mechanically isotropic layers: Evidence from the Sudbury Basin, Canada

    NASA Astrophysics Data System (ADS)

    Clark, Martin D.; Riller, Ulrich

    2017-02-01

    The Sudbury Basin is a non-cylindrical fold basin demarcated by the layered Sudbury Igneous Complex (SIC), the eastern part of which is transected by prominent curved faults. Folding of the SIC and adjacent rock units occurred in the brittle field and is peculiar due to its petrographically distinct, but initially mechanically similar layers. Overall, the layers are characterized by low levels of solid-state strain raising the question how the layer contacts acquired their curvature. We addressed this question by developing a G.I.S.-based workflow to analyze the orientation and slip vectors of the faults. Slip vectors form clusters of normal and reverse slip along a given fault. The clustering is best interpreted in terms of successive slip events during folding of the SIC. As the faults formed most likely as planar reverse faults prior to folding of the SIC they subsequently served as mechanically anisotropic elements to fold the SIC. The results contribute to (1) better understand the folding mechanisms of thick melt sheets in the upper crust, (2) explain apparently incompatible principal strain axes during progressive deformation, and (3) efficiently analyze the orientation and kinematics of fault zones close to the Earth's surface.

  11. Kinetic mechanism of the activation of human plasminogen by streptokinase.

    PubMed

    Kosow, D P

    1975-10-07

    A method of determining the initial rate of plasminogen activation has been developed. The method has been used to investigate the mechanism of activation of human plasminogen by streptokinase. Plasmin formation follows saturation kinetics. Inhibition of plasmin formation by epsilon-aminocaproic acid is uncompetitive with a Ki of 0.6 mM. A model consistent with the data is that streptokinase induces a conformational change in the plasminogen molecule, producing an active center which cleaves an internal peptide bond to produce plasmin. Thus, streptokinase functions as a catalytic allosteric effector.

  12. Kinetics and Mechanism of Bacterial Disinfection by Chlorine Dioxide1

    PubMed Central

    Benarde, Melvin A.; Snow, W. Brewster; Olivieri, Vincent P.; Davidson, Burton

    1967-01-01

    Survival data are presented for a fecal strain of Escherichia coli exposed to three concentrations of chlorine dioxide at four temperatures. Chick's first-order reaction equation is generalized to a pseudo nth-order model. Nonlinear least squares curve-fitting of the survival data to the nth order model was performed on an analogue computer. The data were observed to follow fractional order kinetics with respect to survival concentration, with an apparent activation energy of 12,000 cal/mole. Initial experiments support the thesis that the mechanism of chlorine dioxide kill occurs via disruption of protein synthesis. Images Fig. 1 Fig. 2 Fig. 3 PMID:5339839

  13. Subtle Differences in Virus Composition Affect Disinfection Kinetics and Mechanisms

    PubMed Central

    Sigstam, Thérèse; Gannon, Greg; Cascella, Michele; Pecson, Brian M.; Wigginton, Krista Rule

    2013-01-01

    Viral disinfection kinetics have been studied in depth, but the molecular-level inactivation mechanisms are not understood. Consequently, it is difficult to predict the disinfection behavior of nonculturable viruses, even when related, culturable viruses are available. The objective of this work was to determine how small differences in the composition of the viral genome and proteins impact disinfection. To this end, we investigated the inactivation of three related bacteriophages (MS2, fr, and GA) by UV254, singlet oxygen (1O2), free chlorine (FC), and chlorine dioxide (ClO2). Genome damage was quantified by PCR, and protein damage was assessed by quantitative matrix-assisted laser desorption ionization (MALDI) mass spectrometry. ClO2 caused great variability in the inactivation kinetics between viruses and was the only treatment that did not induce genome damage. The inactivation kinetics were similar for all viruses when treated with disinfectants possessing a genome-damaging component (FC, 1O2, and UV254). On the protein level, UV254 subtly damaged MS2 and fr capsid proteins, whereas GA's capsid remained intact. 1O2 oxidized a methionine residue in MS2 but did not affect the other two viruses. In contrast, FC and ClO2 rapidly degraded the capsid proteins of all three viruses. Protein composition alone could not explain the observed degradation trends; instead, molecular dynamics simulations indicated that degradation is dictated by the solvent-accessible surface area of individual amino acids. Finally, despite the similarities of the three viruses investigated, their mode of inactivation by a single disinfectant varied. This explains why closely related viruses can exhibit drastically different inactivation kinetics. PMID:23542618

  14. Kinetic analysis of the multistep aggregation mechanism of monoclonal antibodies.

    PubMed

    Nicoud, Lucrèce; Arosio, Paolo; Sozo, Margaux; Yates, Andrew; Norrant, Edith; Morbidelli, Massimo

    2014-09-11

    We investigate by kinetic analysis the aggregation mechanism of two monoclonal antibodies belonging to the IgG1 and IgG2 subclass under thermal stress. For each IgG, we apply a combination of size exclusion chromatography and light scattering techniques to resolve the time evolution of the monomer, dimer, and trimer concentrations, as well as the average molecular weight and the average hydrodynamic radius of the aggregate distribution. By combining the detailed experimental characterization with a theoretical kinetic model based on population balance equations, we extract relevant information on the contribution of the individual elementary steps on the global aggregation process. The analysis shows that the two molecules follow different aggregation pathways under the same operating conditions. In particular, while the monomer depletion of the IgG1 is found to be rate-limited by monomeric conformational changes, bimolecular collision is identified as the rate-limiting step in the IgG2 aggregation process. The measurement of the microscopic rate constants by kinetic analysis allows the quantification of the protein-protein interaction potentials expressed in terms of the Fuchs stability ratio (W). It is found that the antibody solutions exhibit large W values, which are several orders of magnitude larger than the values computed in the frame of the DLVO theory. This indicates that, besides net electrostatic repulsion, additional effects delay the aggregation kinetics of the antibody solutions with respect to diffusion-limited conditions. These effects likely include the limited efficiency of the collision events due to the presence of a limited number of specific aggregation-prone patches on the heterogeneous protein surface, and the contribution of additional repulsive non-DLVO forces to the protein-protein interaction potential, such as hydration forces.

  15. Mechanism-based corrector combination restores ΔF508-CFTR folding and function

    PubMed Central

    Okiyoneda, Tsukasa; Veit, Guido; Dekkers, Johanna F.; Bagdany, Miklos; Soya, Naoto; Xu, Haijin; Roldan, Ariel; Verkman, Alan S.; Kurth, Mark; Simon, Agnes; Hegedus, Tamas; Beekman, Jeffrey M.; Lukacs, Gergely L.

    2013-01-01

    The most common cystic fibrosis (CF) mutation, ΔF508 in the nucleotide binding domain-1 (NBD1), impairs CFTR coupled-domain folding, plasma membrane (PM) expression, function and stability. VX-809, a promising investigational corrector of ΔF508-CFTR misprocessing, has limited clinical benefit and incompletely understood mechanism, hampering drug development. Based on the effect of second site suppressor mutations, robust ΔF508-CFTR correction likely requires stabilization of NBD1 and the membrane spanning domains (MSDs)-NBD1 interface, both established primary conformational defects. Here, we elucidated the molecular targets of available correctors; class-I stabilizes the NBD1-MSD1/2 interface, class-II targets NBD2, and only chemical chaperones, surrogates of class-III correctors, stabilize the human ΔF508-NBD1. While VX-809 can correct missense mutations primarily destabilizing the NBD1-MSD1/2 interface, functional PM expression of ΔF508-CFTR also requires compounds that counteract the NBD1 and NBD2 stability defects in CF bronchial epithelial cells and intestinal organoids. Thus, structure-guided corrector combination represents an effective approach for CF therapy. PMID:23666117

  16. Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis

    PubMed Central

    Brás, Joana L. A.; Cartmell, Alan; Carvalho, Ana Luísa M.; Verzé, Genny; Bayer, Edward A.; Vazana, Yael; Correia, Márcia A. S.; Prates, José A. M.; Ratnaparkhe, Supriya; Boraston, Alisdair B.; Romão, Maria J.; Fontes, Carlos M. G. A.; Gilbert, Harry J.

    2011-01-01

    Clostridium thermocellum is a well-characterized cellulose-degrading microorganism. The genome sequence of C. thermocellum encodes a number of proteins that contain type I dockerin domains, which implies that they are components of the cellulose-degrading apparatus, but display no significant sequence similarity to known plant cell wall–degrading enzymes. Here, we report the biochemical properties and crystal structure of one of these proteins, designated CtCel124. The protein was shown to be an endo-acting cellulase that displays a single displacement mechanism and acts in synergy with Cel48S, the major cellulosomal exo-cellulase. The crystal structure of CtCel124 in complex with two cellotriose molecules, determined to 1.5 Å, displays a superhelical fold in which a constellation of α-helices encircle a central helix that houses the catalytic apparatus. The catalytic acid, Glu96, is located at the C-terminus of the central helix, but there is no candidate catalytic base. The substrate-binding cleft can be divided into two discrete topographical domains in which the bound cellotriose molecules display twisted and linear conformations, respectively, suggesting that the enzyme may target the interface between crystalline and disordered regions of cellulose. PMID:21393568

  17. Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis.

    PubMed

    Brás, Joana L A; Cartmell, Alan; Carvalho, Ana Luísa M; Verzé, Genny; Bayer, Edward A; Vazana, Yael; Correia, Márcia A S; Prates, José A M; Ratnaparkhe, Supriya; Boraston, Alisdair B; Romão, Maria J; Fontes, Carlos M G A; Gilbert, Harry J

    2011-03-29

    Clostridium thermocellum is a well-characterized cellulose-degrading microorganism. The genome sequence of C. thermocellum encodes a number of proteins that contain type I dockerin domains, which implies that they are components of the cellulose-degrading apparatus, but display no significant sequence similarity to known plant cell wall-degrading enzymes. Here, we report the biochemical properties and crystal structure of one of these proteins, designated CtCel124. The protein was shown to be an endo-acting cellulase that displays a single displacement mechanism and acts in synergy with Cel48S, the major cellulosomal exo-cellulase. The crystal structure of CtCel124 in complex with two cellotriose molecules, determined to 1.5 Å, displays a superhelical fold in which a constellation of α-helices encircle a central helix that houses the catalytic apparatus. The catalytic acid, Glu96, is located at the C-terminus of the central helix, but there is no candidate catalytic base. The substrate-binding cleft can be divided into two discrete topographical domains in which the bound cellotriose molecules display twisted and linear conformations, respectively, suggesting that the enzyme may target the interface between crystalline and disordered regions of cellulose.

  18. Identical folds used for distinct mechanical functions of the bacterial flagellar rod and hook

    PubMed Central

    Fujii, Takashi; Kato, Takayuki; Hiraoka, Koichi D.; Miyata, Tomoko; Minamino, Tohru; Chevance, Fabienne F. V.; Hughes, Kelly T.; Namba, Keiichi

    2017-01-01

    The bacterial flagellum is a motile organelle driven by a rotary motor, and its axial portions function as a drive shaft (rod), a universal joint (hook) and a helical propeller (filament). The rod and hook are directly connected to each other, with their subunit proteins FlgG and FlgE having 39% sequence identity, but show distinct mechanical properties; the rod is straight and rigid as a drive shaft whereas the hook is flexible in bending as a universal joint. Here we report the structure of the rod and comparison with that of the hook. While these two structures have the same helical symmetry and repeat distance and nearly identical folds of corresponding domains, the domain orientations differ by ∼7°, resulting in tight and loose axial subunit packing in the rod and hook, respectively, conferring the rigidity on the rod and flexibility on the hook. This provides a good example of versatile use of a protein structure in biological organisms. PMID:28120828

  19. Kinetic study of the inhibition mechanism of dehaloperoxidase-hemoglobin a by 4-bromophenol.

    PubMed

    Zhao, Jing; Franzen, Stefan

    2013-07-18

    The mechanism of dehaloperoxidase-hemoglobin (DHP) inhibition by 4-bromophenol (4-BP) was investigated using Michealis-Menten and transient-state kinetic analyses. Transient-state kinetics using the stopped-flow technique to mix DHP and H2O2 in the presence of inhibitor concentrations less than 10-fold greater than the enzyme concentration show that 4-BP does not fully impede H2O2 entering the distal pocket to activate DHP. It is not clear whether an oxoferryl intermediate is formed under these conditions and there may be alternative pathways for H2O2 reaction in the 4-BP bound form of DHP. Two new species have been identified during the reaction of 4-BP bound form of DHP in the transient-state kinetic experiment by using Singular Value Decomposition (SVD) and global-fitting analysis. Rather than forming Compound ES in the unbound form, an inhibitor bound intermediate that possesses blue-shifted Soret band and a double peaked Q-band is observed. This intermediate is subsequently converted to the end-point species that is distinguished from Compound RH formed in the uninhibited enzyme. Bench-top mixing kinetics of DHP were conducted in order to determine the inhibitor binding constant and to understand the enzyme inhibition mechanism from a thermodynamic perspective. It was found that the inhibition constant, Ki, decreased from 2.56 mM to 0.15 mM over the temperature range from 283 to 298 K, which permits determination of the enthalpy and entropy for inhibitor binding as -135.5 ± 20.9 kJ/mol and 526.1 ± 71.9 J/(mol·K), respectively, leading to the conclusion that inhibitor binding is entropically driven.

  20. Planarization mechanism of alkaline copper CMP slurry based on chemical mechanical kinetics

    NASA Astrophysics Data System (ADS)

    Shengli, Wang; Kangda, Yin; Xiang, Li; Hongwei, Yue; Yunling, Liu

    2013-08-01

    The planarization mechanism of alkaline copper slurry is studied in the chemical mechanical polishing (CMP) process from the perspective of chemical mechanical kinetics. Different from the international dominant acidic copper slurry, the copper slurry used in this research adopted the way of alkaline technology based on complexation. According to the passivation property of copper in alkaline conditions, the protection of copper film at the concave position on a copper pattern wafer surface can be achieved without the corrosion inhibitors such as benzotriazole (BTA), by which the problems caused by BTA can be avoided. Through the experiments and theories research, the chemical mechanical kinetics theory of copper removal in alkaline CMP conditions was proposed. Based on the chemical mechanical kinetics theory, the planarization mechanism of alkaline copper slurry was established. In alkaline CMP conditions, the complexation reaction between chelating agent and copper ions needs to break through the reaction barrier. The kinetic energy at the concave position should be lower than the complexation reaction barrier, which is the key to achieve planarization.

  1. Kinetics and mechanisms of reactions involving small aromatic reactive intermediates

    SciTech Connect

    Lin, M.C.

    1993-12-01

    Small aromatic radicals such as C{sub 6}H{sub 5}, C{sub 6}H{sub 5}O and C{sub 6}H{sub 4} are key prototype species of their homologs. C{sub 6}H{sub 5} and its oxidation product, C{sub 6}H{sub 5}O are believed to be important intermediates which play a pivotal role in hydrocarbon combustion, particularly with regard to soot formation. Despite their fundamental importance, experimental data on the reaction mechanisms and reactivities of these species are very limited. For C{sub 6}H{sub 5}, most kinetic data except its reactions with NO and NO{sub 2}, were obtained by relative rate measurements. For C{sub 6}H{sub 5}O, the authors have earlier measured its fragmentation reaction producing C{sub 5}H{sub 5} + CO in shock waves. For C{sub 6}H{sub 4}, the only rate constant measured in the gas phase is its recombination rate at room temperature. The authors have proposed to investigate systematically the kinetics and mechanisms of this important class of molecules using two parallel laser diagnostic techniques--laser resonance absorption (LRA) and resonance enhanced multiphoton ionization mass spectrometry (REMPI/MS). In the past two years, study has been focused on the development of a new multipass adsorption technique--the {open_quotes}cavity-ring-down{close_quotes} technique for kinetic applications. The preliminary results of this study appear to be quite good and the sensitivity of the technique is at least comparable to that of the laser-induced fluorescence method.

  2. Modeling the effect of codon translation rates on co-translational protein folding mechanisms of arbitrary complexity

    NASA Astrophysics Data System (ADS)

    Caniparoli, Luca; O'Brien, Edward P.

    2015-04-01

    In a cell, the folding of a protein molecule into tertiary structure can begin while it is synthesized by the ribosome. The rate at which individual amino acids are incorporated into the elongating nascent chain has been shown to affect the likelihood that proteins will populate their folded state, indicating that co-translational protein folding is a far from equilibrium process. Developing a theoretical framework to accurately describe this process is, therefore, crucial for advancing our understanding of how proteins acquire their functional conformation in living cells. Current state-of-the-art computational approaches, such as molecular dynamics simulations, are very demanding in terms of the required computer resources, making the simulation of co-translational protein folding difficult. Here, we overcome this limitation by introducing an efficient approach that predicts the effects that variable codon translation rates have on co-translational folding pathways. Our approach is based on Markov chains. By using as an input a relatively small number of molecular dynamics simulations, it allows for the computation of the probability that a nascent protein is in any state as a function of the translation rate of individual codons along a mRNA's open reading frame. Due to its computational efficiency and favorable scalability with the complexity of the folding mechanism, this approach could enable proteome-wide computational studies of the influence of translation dynamics on co-translational folding.

  3. Modeling the effect of codon translation rates on co-translational protein folding mechanisms of arbitrary complexity.

    PubMed

    Caniparoli, Luca; O'Brien, Edward P

    2015-04-14

    In a cell, the folding of a protein molecule into tertiary structure can begin while it is synthesized by the ribosome. The rate at which individual amino acids are incorporated into the elongating nascent chain has been shown to affect the likelihood that proteins will populate their folded state, indicating that co-translational protein folding is a far from equilibrium process. Developing a theoretical framework to accurately describe this process is, therefore, crucial for advancing our understanding of how proteins acquire their functional conformation in living cells. Current state-of-the-art computational approaches, such as molecular dynamics simulations, are very demanding in terms of the required computer resources, making the simulation of co-translational protein folding difficult. Here, we overcome this limitation by introducing an efficient approach that predicts the effects that variable codon translation rates have on co-translational folding pathways. Our approach is based on Markov chains. By using as an input a relatively small number of molecular dynamics simulations, it allows for the computation of the probability that a nascent protein is in any state as a function of the translation rate of individual codons along a mRNA's open reading frame. Due to its computational efficiency and favorable scalability with the complexity of the folding mechanism, this approach could enable proteome-wide computational studies of the influence of translation dynamics on co-translational folding.

  4. Kinetics and Mechanisms of Calcite Reactions with Saline Waters

    SciTech Connect

    Gorman, Brian P

    2015-09-02

    Project Description: The general objective of the proposed research is to determine the kinetics and mechanisms of calcite reactions with saline waters over a wide range of saline water composition, pCO2, and modest ranges in T and P. This will be accomplished by studying both reaction rates and solubility from changes in solution chemistry, and making nanoscale observations of calcite precipitate surface morphology and composition at the micro-to-nano-scale to provide an understanding of controlling reaction mechanisms and pathways. The specific objectives necessary to reach the general objective are: a) determination of how pCO2, Ca2+, ionic strength and “foreign” ions influence reaction rates; and b) investigate the influence of these parameters on apparent kinetic solubility from dissolution and precipitation reactions. This information will clearly be central to the construction of reliable reaction-transport models to predict reservoir and formation response to increased CO2 in saline waters. This program was initially collaborative with John Morse at Texas A&M, however his passing shortly after the beginning of this program resulted in abbreviated research time and effort. Summary of Results: Early studies using electron microscopy and spectroscopy indicated that carbonate precipitation from natural seawater (NSW) conditions onto aragonite substrates was mediated by a surface amorphous calcium carbonate layer. It was hypothesized that this ACC layer (observed after < 5days reaction time) was responsible for the abnormal reaction kinetics and also served as a metastable seed layer for growth of epitaxial aragonite. Further studies of the ACC formation mechanism indicated a strong dependence on the Mg concentration in solution. Subsequent studies at shorter times (10 hrs) on calcite substrates and in a wide range of supersaturation conditions did not indicate any ACC layer. Instead, an epitaxial layer by layer

  5. Mechanics of non-critical fold-thrust belts based on finite element models

    NASA Astrophysics Data System (ADS)

    Simpson, Guy

    2011-03-01

    The mechanics of fold-thrust belts and accretionary wedges is investigated using a two dimensional, plane strain, elastic-plastic (cohesive Mohr-Coulomb) mechanical model solved with the Finite Element Method. Results show that when a layer with an initially non-critical geometry is compressed from the rear, it does not form a wedge that is at failure throughout, as assumed in critical wedge theory. Rather, the wedge consists of narrow plastic shear zones that propagate sequentially outward with time, loading rocks ahead while unloading rocks behind. Not only are stress states within the wedge not everywhere at failure but principal stress orientations vary strongly in time and space, particularly across shear zones, near the basal detachment and in the hanging wall of active structures, where local surface extension may be observed. The reason the investigated wedges are not stressed to compressive failure throughout is related to strength reduction associated with strain localisation that enables material outside shear zones to unload and return to an elastic stress state. This mechanism is intrinsic to elastic-plastic materials and occurs regardless of any material degradation such as loss of cohesion. Even though the stress state of the investigated wedges is generally non-critical, the overall geometry may still be consistent with cohesionless critical wedge theory, since the local surface slope is created when a particular part of the wedge is at a limit state. Prowedge tapers display non self-similar growth through time but eventually evolve to the minimum critical taper. Retrowedges on the other hand, may get caught within this initial transient state and thus may have tapers anywhere between the minimum and maximum critical taper. However, if the basal detachment is such that lateral propagation is not kinematically inhibited, retrowedges are shown to also eventually evolve towards minimum critical tapers, resulting in a symmetrical doubly-vergent orogen

  6. Actin kinetics shapes cortical network structure and mechanics

    PubMed Central

    Fritzsche, Marco; Erlenkämper, Christoph; Moeendarbary, Emad; Charras, Guillaume; Kruse, Karsten

    2016-01-01

    The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs. PMID:27152338

  7. GeoFold: Topology-based protein unfolding pathways capture the effects of engineered disulfides on kinetic stability

    PubMed Central

    Ramakrishnan, Vibin; Srinivasan, Sai Praveen; Salem, Saeed M; Matthews, Suzanne J; Colón, Wilfredo; Zaki, Mohammed; Bystroff, Christopher

    2011-01-01

    Protein unfolding is modeled as an ensemble of pathways, where each step in each pathway is the addition of one topologically possible conformational degree of freedom. Starting with a known protein structure, GeoFold hierarchically partitions (cuts) the native structure into substructures using revolute joints and translations. The energy of each cut and its activation barrier are calculated using buried solvent accessible surface area, side chain entropy, hydrogen bonding, buried cavities, and backbone degrees of freedom. A directed acyclic graph is constructed from the cuts, representing a network of simultaneous equilibria. Finite difference simulations on this graph simulate native unfolding pathways. Experimentally observed changes in the unfolding rates for disulfide mutants of barnase, T4 lysozyme, dihydrofolate reductase, and factor for inversion stimulation were qualitatively reproduced in these simulations. Detailed unfolding pathways for each case explain the effects of changes in the chain topology on the folding energy landscape. GeoFold is a useful tool for the inference of the effects of disulfide engineering on the energy landscape of protein unfolding. PMID:22189917

  8. Kinetic mechanism of phenylacetone monooxygenase from Thermobifida fusca.

    PubMed

    Torres Pazmiño, Daniel E; Baas, Bert-Jan; Janssen, Dick B; Fraaije, Marco W

    2008-04-01

    Phenylacetone monooxygenase (PAMO) from Thermobifida fusca is a FAD-containing Baeyer-Villiger monooxygenase (BVMO). To elucidate the mechanism of conversion of phenylacetone by PAMO, we have performed a detailed steady-state and pre-steady-state kinetic analysis. In the catalytic cycle ( k cat = 3.1 s (-1)), rapid binding of NADPH ( K d = 0.7 microM) is followed by a transfer of the 4( R)-hydride from NADPH to the FAD cofactor ( k red = 12 s (-1)). The reduced PAMO is rapidly oxygenated by molecular oxygen ( k ox = 870 mM (-1) s (-1)), yielding a C4a-peroxyflavin. The peroxyflavin enzyme intermediate reacts with phenylacetone to form benzylacetate ( k 1 = 73 s (-1)). This latter kinetic event leads to an enzyme intermediate which we could not unequivocally assign and may represent a Criegee intermediate or a C4a-hydroxyflavin form. The relatively slow decay (4.1 s (-1)) of this intermediate yields fully reoxidized PAMO and limits the turnover rate. NADP (+) release is relatively fast and represents the final step of the catalytic cycle. This study shows that kinetic behavior of PAMO is significantly different when compared with that of sequence-related monooxygenases, e.g., cyclohexanone monooxygenase and liver microsomal flavin-containing monooxygenase. Inspection of the crystal structure of PAMO has revealed that residue R337, which is conserved in other BVMOs, is positioned close to the flavin cofactor. The analyzed R337A and R337K mutant enzymes were still able to form and stabilize the C4a-peroxyflavin intermediate. The mutants were unable to convert either phenylacetone or benzyl methyl sulfide. This demonstrates that R337 is crucially involved in assisting PAMO-mediated Baeyer-Villiger and sulfoxidation reactions.

  9. A Hooke׳s law-based approach to protein folding rate.

    PubMed

    Ruiz-Blanco, Yasser B; Marrero-Ponce, Yovani; Prieto, Pablo J; Salgado, Jesús; García, Yamila; Sotomayor-Torres, Clivia M

    2015-01-07

    Kinetics is a key aspect of the renowned protein folding problem. Here, we propose a comprehensive approach to folding kinetics where a polypeptide chain is assumed to behave as an elastic material described by the Hooke׳s law. A novel parameter called elastic-folding constant results from our model and is suggested to distinguish between protein with two-state and multi-state folding pathways. A contact-free descriptor, named folding degree, is introduced as a suitable structural feature to study protein-folding kinetics. This approach generalizes the observed correlations between varieties of structural descriptors with the folding rate constant. Additionally several comparisons among structural classes and folding mechanisms were carried out showing the good performance of our model with proteins of different types. The present model constitutes a simple rationale for the structural and energetic factors involved in protein folding kinetics.

  10. Mechanics, thermodynamics, and kinetics of ligand binding to biopolymers.

    PubMed

    Jarillo, Javier; Morín, José A; Beltrán-Heredia, Elena; Villaluenga, Juan P G; Ibarra, Borja; Cao, Francisco J

    2017-01-01

    Ligands binding to polymers regulate polymer functions by changing their physical and chemical properties. This ligand regulation plays a key role in many biological processes. We propose here a model to explain the mechanical, thermodynamic, and kinetic properties of the process of binding of small ligands to long biopolymers. These properties can now be measured at the single molecule level using force spectroscopy techniques. Our model performs an effective decomposition of the ligand-polymer system on its covered and uncovered regions, showing that the elastic properties of the ligand-polymer depend explicitly on the ligand coverage of the polymer (i.e., the fraction of the polymer covered by the ligand). The equilibrium coverage that minimizes the free energy of the ligand-polymer system is computed as a function of the applied force. We show how ligands tune the mechanical properties of a polymer, in particular its length and stiffness, in a force dependent manner. In addition, it is shown how ligand binding can be regulated applying mechanical tension on the polymer. Moreover, the binding kinetics study shows that, in the case where the ligand binds and organizes the polymer in different modes, the binding process can present transient shortening or lengthening of the polymer, caused by changes in the relative coverage by the different ligand modes. Our model will be useful to understand ligand-binding regulation of biological processes, such as the metabolism of nucleic acid. In particular, this model allows estimating the coverage fraction and the ligand mode characteristics from the force extension curves of a ligand-polymer system.

  11. A multipurpose reduced chemical-kinetic mechanism for methanol combustion

    NASA Astrophysics Data System (ADS)

    Fernández-Tarrazo, Eduardo; Sánchez-Sanz, Mario; Sánchez, Antonio L.; Williams, Forman A.

    2016-07-01

    A multipurpose reduced chemical-kinetic mechanism for methanol combustion comprising 8 overall reactions and 11 reacting chemical species is presented. The development starts by investigating the minimum set of elementary reactions needed to describe methanol combustion with reasonable accuracy over a range of conditions of temperature, pressure, and composition of interest in combustion. Starting from a 27-step mechanism that has been previously tested and found to give accurate predictions of ignition processes for these conditions, it is determined that the addition of 11 elementary reactions taken from its basis (San Diego) mechanism extends the validity of the description to premixed-flame propagation, strain-induced extinction of non-premixed flames, and equilibrium composition and temperatures, giving results that compare favourably with experimental measurements and also with computations using the 247-step detailed San Diego mechanism involving 50 reactive species. Specifically, premixed-flame propagation velocities and extinction strain rates for non-premixed counterflow flames calculated with the 38-step mechanism show departures from experimental measurements and detailed-chemistry computations that are roughly on the order of 10%, comparable with expected experimental uncertainties. Similar accuracy is found in comparisons of autoignition times over the range considered, except at very high temperatures, under which conditions the computations tend to overpredict induction times for all of the chemistry descriptions tested. From this 38-step mechanism, the simplification is continued by introducing steady-state approximations for the intermediate species CH3, CH4, HCO, CH3O, CH2OH, and O, leading to an 8-step reduced mechanism that provides satisfactory accuracy for all conditions tested. The flame computations indicate that thermal diffusion has a negligible influence on methanol combustion in all cases considered and that a mixture-average species

  12. Influence of Glu/Arg, Asp/Arg, and Glu/Lys Salt Bridges on α-Helical Stability and Folding Kinetics.

    PubMed

    Meuzelaar, Heleen; Vreede, Jocelyne; Woutersen, Sander

    2016-06-07

    Using a combination of ultraviolet circular dichroism, temperature-jump transient-infrared spectroscopy, and molecular dynamics simulations, we investigate the effect of salt bridges between different types of charged amino-acid residue pairs on α-helix folding. We determine the stability and the folding and unfolding rates of 12 alanine-based α-helical peptides, each of which has a nearly identical composition containing three pairs of positively and negatively charged residues (either Glu(-)/Arg(+), Asp(-)/Arg(+), or Glu(-)/Lys(+)). Within each set of peptides, the distance and order of the oppositely charged residues in the peptide sequence differ, such that they have different capabilities of forming salt bridges. Our results indicate that stabilizing salt bridges (in which the interacting residues are spaced and ordered such that they favor helix formation) speed up α-helix formation by up to 50% and slow down the unfolding of the α-helix, whereas salt bridges with an unfavorable geometry have the opposite effect. Comparing the peptides with different types of charge pairs, we observe that salt bridges between side chains of Glu(-) and Arg(+) are most favorable for the speed of folding, probably because of the larger conformational space of the salt-bridging Glu(-)/Arg(+) rotamer pairs compared to Asp(-)/Arg(+) and Glu(-)/Lys(+). We speculate that the observed impact of salt bridges on the folding kinetics might explain why some proteins contain salt bridges that do not stabilize the final, folded conformation. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  13. Precursory signatures of protein folding/unfolding: From time series correlation analysis to atomistic mechanisms

    SciTech Connect

    Hsu, P. J.; Lai, S. K.; Cheong, S. A.

    2014-05-28

    Folded conformations of proteins in thermodynamically stable states have long lifetimes. Before it folds into a stable conformation, or after unfolding from a stable conformation, the protein will generally stray from one random conformation to another leading thus to rapid fluctuations. Brief structural changes therefore occur before folding and unfolding events. These short-lived movements are easily overlooked in studies of folding/unfolding for they represent momentary excursions of the protein to explore conformations in the neighborhood of the stable conformation. The present study looks for precursory signatures of protein folding/unfolding within these rapid fluctuations through a combination of three techniques: (1) ultrafast shape recognition, (2) time series segmentation, and (3) time series correlation analysis. The first procedure measures the differences between statistical distance distributions of atoms in different conformations by calculating shape similarity indices from molecular dynamics simulation trajectories. The second procedure is used to discover the times at which the protein makes transitions from one conformation to another. Finally, we employ the third technique to exploit spatial fingerprints of the stable conformations; this procedure is to map out the sequences of changes preceding the actual folding and unfolding events, since strongly correlated atoms in different conformations are different due to bond and steric constraints. The aforementioned high-frequency fluctuations are therefore characterized by distinct correlational and structural changes that are associated with rate-limiting precursors that translate into brief segments. Guided by these technical procedures, we choose a model system, a fragment of the protein transthyretin, for identifying in this system not only the precursory signatures of transitions associated with α helix and β hairpin, but also the important role played by weaker correlations in such protein

  14. The kinetics of composite particle formation during mechanical alloying

    NASA Technical Reports Server (NTRS)

    Aikin, B. J. M.; Courtney, T. H.

    1993-01-01

    The kinetics of composite particle formation during attritor milling of insoluble binary elemental powders have been examined. The effects of processing conditions (i.e., mill power, temperature, and charge ratio) on these kinetics were studied. Particle size distributions and fractions of elemental and composite particles were determined as functions of milling time and processing conditions. This allowed the deduction of phenomenological rate constants describing the propensity for fracture and welding during processing. For the mill-operating conditions investigated, the number of particles in the mill generally decreased with milling time, indicating a greater tendency for particle welding than fracture. Moreover, a bimodal size distribution is often obtained as a result of preferential welding. Copper and chromium 'alloy' primarily by encapsulation of Cr particles within Cu. This form of alloying also occurs in Cu-Nb alloys processed at low mill power and/or for short milling times. For other conditions, however, Cu-Nb alloys develop a lamellar morphology characteristic of mechanically alloyed two-phase ductile metals. Increasing mill power or charge (ball-to-powder weight) ratio (CR) increases the rate of composite particle formation.

  15. Mechanisms and kinetics of granulated sewage sludge combustion.

    PubMed

    Kijo-Kleczkowska, Agnieszka; Środa, Katarzyna; Kosowska-Golachowska, Monika; Musiał, Tomasz; Wolski, Krzysztof

    2015-12-01

    This paper investigates sewage sludge disposal methods with particular emphasis on combustion as the priority disposal method. Sewage sludge incineration is an attractive option because it minimizes odour, significantly reduces the volume of the starting material and thermally destroys organic and toxic components of the off pads. Additionally, it is possible that ashes could be used. Currently, as many as 11 plants use sewage sludge as fuel in Poland; thus, this technology must be further developed in Poland while considering the benefits of co-combustion with other fuels. This paper presents the results of experimental studies aimed at determining the mechanisms (defining the fuel combustion region by studying the effects of process parameters, including the size of the fuel sample, temperature in the combustion chamber and air velocity, on combustion) and kinetics (measurement of fuel temperature and mass changes) of fuel combustion in an air stream under different thermal conditions and flow rates. The combustion of the sludge samples during air flow between temperatures of 800 and 900°C is a kinetic-diffusion process. This process determines the sample size, temperature of its environment, and air velocity. The adopted process parameters, the time and ignition temperature of the fuel by volatiles, combustion time of the volatiles, time to reach the maximum temperature of the fuel surface, maximum temperature of the fuel surface, char combustion time, and the total process time, had significant impacts. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Complete kinetic mechanism for recycling of the bacterial ribosome.

    PubMed

    Borg, Anneli; Pavlov, Michael; Ehrenberg, Måns

    2016-01-01

    How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec(-1), is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of ∼5 sec(-1), are discussed in the perspective of rapidly growing bacterial cells.

  17. Complete kinetic mechanism for recycling of the bacterial ribosome

    PubMed Central

    Borg, Anneli; Pavlov, Michael

    2016-01-01

    How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec−1, is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of ∼5 sec−1, are discussed in the perspective of rapidly growing bacterial cells. PMID:26527791

  18. Integration of kinetic isotope effect analyses to elucidate ribonuclease mechanism.

    PubMed

    Harris, Michael E; Piccirilli, Joseph A; York, Darrin M

    2015-11-01

    The well-studied mechanism of ribonuclease A is believed to involve concerted general acid-base catalysis by two histidine residues, His12 and His119. The basic features of this mechanism are often cited to explain rate enhancement by both protein and RNA enzymes that catalyze RNA 2'-O-transphosphorylation. Recent kinetic isotope effect analyses and computational studies are providing a more chemically detailed description of the mechanism of RNase A and the rate limiting transition state. Overall, the results support an asynchronous mechanism for both solution and ribonuclease catalyzed reactions in which breakdown of a transient dianoinic phosphorane intermediate by 5'OP bond cleavage is rate limiting. Relative to non-enzymatic reactions catalyzed by specific base, a smaller KIE on the 5'O leaving group and a less negative βLG are observed for RNase A catalysis. Quantum mechanical calculations consistent with these data support a model in which electrostatic and H-bonding interactions with the non-bridging oxygens and proton transfer from His119 render departure of the 5'O less advanced and stabilize charge buildup in the transition state. Both experiment and computation indicate advanced 2'OP bond formation in the rate limiting transition state. However, this feature makes it difficult to resolve the chemical steps involved in 2'O activation. Thus, modeling the transition state for RNase A catalysis underscores those elements of its chemical mechanism that are well resolved, as well as highlighting those where ambiguity remains. This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment. Published by Elsevier B.V.

  19. Sonolytic degradation of dimethoate: kinetics, mechanisms and toxic intermediates controlling.

    PubMed

    Yao, Juan-Juan; Hoffmann, Michael R; Gao, Nai-Yun; Zhang, Zhi; Li, Lei

    2011-11-15

    The sonolytic degradation of aqueous solutions of dimethoate, O,O-dimethyl S-[2-(methylamino)-2-oxoethyl]dithiophosphate, was examined. Optimal degradation rates were obtained at 619 kHz for continuous sonolysis and 406 kHz for pulse sonolysis. The primary pathways for degradation include hydroxyl radical oxidation, hydrolysis and pyrolysis on collapsing cavitation bubble interfaces. Reaction mechanisms coupled with the corresponding kinetic models are proposed to reproduce the observed concentration versus time profiles for dimethoate, omethoate and N-(methyl) mercaptoacetamide during sonolysis. The oxidation and hydrolysis of dimethoate and omethoate occurred at the water-bubble interface was the rate-determining step for sonolytic overall degradation of dimethoate. More than 90% toxicity of dimethoate was reduced within 45 min ultrasonic irradiation. Ferrous ion at micro molar level can significantly enhance the sonolytic degradation of dimethoate and effectively reduce the yields of toxic intermediate omethoate.

  20. Secondary Structure Propensities in Peptide Folding Simulations: A Systematic Comparison of Molecular Mechanics Interaction Schemes

    PubMed Central

    Matthes, Dirk; de Groot, Bert L.

    2009-01-01

    Abstract We present a systematic study directed toward the secondary structure propensity and sampling behavior in peptide folding simulations with eight different molecular dynamics force-field variants in explicit solvent. We report on the combinational result of force field, water model, and electrostatic interaction schemes and compare to available experimental characterization of five studied model peptides in terms of reproduced structure and dynamics. The total simulation time exceeded 18 μs and included simulations that started from both folded and extended conformations. Despite remaining sampling issues, a number of distinct trends in the folding behavior of the peptides emerged. Pronounced differences in the propensity of finding prominent secondary structure motifs in the different applied force fields suggest that problems point in particular to the balance of the relative stabilities of helical and extended conformations. PMID:19619475

  1. Significance of first-order faults in folding mechanically isotropic layers: evidence from the Sudbury Basin, Canada.

    NASA Astrophysics Data System (ADS)

    Clark, Martin; Riller, Ulrich

    2016-04-01

    The Sudbury Basin in Canada is a fold basin demarcated by the Sudbury Igneous Complex (SIC). Folding of the SIC is particularly notable due to its petrographically distinct but mechanically similar layers that are hardly strained when compared to folded strata in other deformed terranes. The Sudbury Basin has three ranges, the North Range, the South Range, and the East Range. The East Range differs from the other ranges by inclosing a remarkably shorter SIC segment with a strong concave curvature. Lacking significant mechanical anisotropy and solid-state strain within the SIC brings to question how the SIC in the East Range acquired its curvature. To address this question, we analyzed the orientation of prominent km-scale faults and their slip vectors. These faults transect the SIC at low angles and mimic its plan view curvature suggesting that the faults were folded along with the SIC. We have developed a G.I.S.-based workflow to address this problem that harnesses high-resolution LiDAR data to generate near surface fault geometries, and combines these geometries with local fault-slip inversions of slickensides to identify slip vectors of prominent curved faults. Analysis of slip vectors along curved faults yields clusters of slip vectors with normal and reverse slip motion in the northern and southern fault segments, respectively. The variation in slip vectors is interpreted to be non-primary and thus shows a temporal relationship between faulting and folding of the SIC. Therefore, prominent curved faults in the East Range must have occurred as a pre-folding brittle response to horizontal shortening. These faults later assumed the role of mechanical anisotropic elements necessary for folding of the SIC layers to occur. This interpretation is corroborated by two sets of principal strain axes inferred from fault-slip inversions. The first set is characterized by its principal axis of shortening oriented NW-SE, comparable in orientation to regional shortening as

  2. Distinguishing between sequential and nonsequentially folded proteins: implications for folding and misfolding.

    PubMed Central

    Tsai, C. J.; Maizel, J. V.; Nussinov, R.

    1999-01-01

    We describe here an algorithm for distinguishing sequential from nonsequentially folding proteins. Several experiments have recently suggested that most of the proteins that are synthesized in the eukaryotic cell may fold sequentially. This proposed folding mechanism in vivo is particularly advantageous to the organism. In the absence of chaperones, the probability that a sequentially folding protein will misfold is reduced significantly. The problem we address here is devising a procedure that would differentiate between the two types of folding patterns. Footprints of sequential folding may be found in structures where consecutive fragments of the chain interact with each other. In such cases, the folding complexity may be viewed as being lower. On the other hand, higher folding complexity suggests that at least a portion of the polypeptide backbone folds back upon itself to form three-dimensional (3D) interactions with noncontiguous portion(s) of the chain. Hence, we look at the mechanism of folding of the molecule via analysis of its complexity, that is, through the 3D interactions formed by contiguous segments on the polypeptide chain. To computationally splice the structure into consecutively interacting fragments, we either cut it into compact hydrophobic folding units or into a set of hypothetical, transient, highly populated, contiguous fragments ("building blocks" of the structure). In sequential folding, successive building blocks interact with each other from the amino to the carboxy terminus of the polypeptide chain. Consequently, the results of the parsing differentiate between sequentially vs. nonsequentially folded chains. The automated assessment of the folding complexity provides insight into both the likelihood of misfolding and the kinetic folding rate of the given protein. In terms of the funnel free energy landscape theory, a protein that truly follows the mechanism of sequential folding, in principle, encounters smoother free energy barriers

  3. Kinetics, products, and mechanisms of secondary organic aerosol formation.

    PubMed

    Ziemann, Paul J; Atkinson, Roger

    2012-10-07

    Secondary organic aerosol (SOA) is formed in the atmosphere when volatile organic compounds (VOCs) emitted from anthropogenic and biogenic sources are oxidized by reactions with OH radicals, O(3), NO(3) radicals, or Cl atoms to form less volatile products that subsequently partition into aerosol particles. Once in particles, these organic compounds can undergo heterogenous/multiphase reactions to form more highly oxidized or oligomeric products. SOA comprises a large fraction of atmospheric aerosol mass and can have significant effects on atmospheric chemistry, visibility, human health, and climate. Previous articles have reviewed the kinetics, products, and mechanisms of atmospheric VOC reactions and the general chemistry and physics involved in SOA formation. In this article we present a detailed review of VOC and heterogeneous/multiphase chemistry as they apply to SOA formation, with a focus on the effects of VOC molecular structure on the kinetics of initial reactions with the major atmospheric oxidants, the subsequent reactions of alkyl, alkyl peroxy, and alkoxy radical intermediates, and the composition of the resulting products. Structural features of reactants and products discussed include compound carbon number; linear, branched, and cyclic configurations; the presence of C[double bond, length as m-dash]C bonds and aromatic rings; and functional groups such as carbonyl, hydroxyl, ester, hydroxperoxy, carboxyl, peroxycarboxyl, nitrate, and peroxynitrate. The intention of this review is to provide atmospheric chemists with sufficient information to understand the dominant pathways by which the major classes of atmospheric VOCs react to form SOA products, and the further reactions of these products in particles. This will allow reasonable predictions to be made, based on molecular structure, about the kinetics, products, and mechanisms of VOC and heterogeneous/multiphase reactions, including the effects of important variables such as VOC, oxidant, and NO

  4. Kinetic mechanism of rabbit muscle glycogen synthase I.

    PubMed

    Gold, A M

    1980-08-05

    The kinetic mechanism of rabbit muscle glycogen synthase I was investigated by determining isotope-exchange rates at chemical equilibrium between uridine diphosphoglucose (UDPG) and glycogen and between UDPG and uridine 5'-diphosphate (UDP). The rates were followed simultaneously by use of UDPG labeled with 14C in the glucose moiety and with 3H in the uracil group. They were found to be independent of the concentrations of glycogen and the UDPG-UDP pair, averaging 6 X 10(-9) mol min-1 mg-1, with a ratio of UDPG-glycogen exchange to UDPG-UDP exchange of 0.85-0.95. The conclusion is that glycogen synthase has a rapid equilibrium random bi bi mechanism. The previously reported slow activation of glycogen-free synthase in the presence of glycogen was examined kinetically. The activation rate appears to be independent of glycogen concentration over a wide range, while the maximum activation is related to the third or fourth root of the glycogen concentration. This suggest that the slow bimolecular reaction mechanism proposed for human polymorphonuclear leucocyte glycogen synthase I [Sølling, H., & Esmann, V. (1977) Eur. J. Biochem. 81, 129] does not apply to rabbit muscle synthase I. The rate of exchange of glycogen molecules in the complex between glycogen and rabbit muscle synthase I under conditions where the enzyme is catalytically active was estimated by a novel method. The enzyme-glycogen complex was treated with [glucose-14C]UDPG and glycogen of different molecular weight. The distribution of isotope between the two forms of glycogen was determined after their separation by agarose gel chromatography. A rate constant of 0.3 min-1 was estimated for the exchange. It can be calculated, on the basis of the specific activity of the enzyme (20 mumol min-1 mg-1) and its action pattern, that hundreds of individual chains in the glycogen molecule must be available to the enzyme during the average lifetime of the complex. A mechanism is proposed for this process.

  5. Domain closure in the catalytic chains of Escherichia coli aspartate transcarbamoylase influences the kinetic mechanism.

    PubMed

    Lee, B H; Ley, B W; Kantrowitz, E R; O'Leary, M H; Wedler, F C

    1995-06-30

    The closure of the two domains of the catalytic chains of Escherichia coli aspartate transcarbamoylase, which is critical for completion of the T-->R transition, is stabilized by salt-bridges between Glu-50 and both Arg-167 and Arg-234. Mutation of Glu-50 to Ala shifts the enzyme toward a low activity, low affinity state (Newton, C. J., and Kantrowitz, E. R. (1990) Biochemistry, 29, 1444-1451). Kinetic isotope effects (KIE) and equilibrium isotope exchange kinetics (EIEK) have been used to probe the dynamic properties of the Glu-50-->Ala enzyme. Unlike the behavior of the wild-type enzyme, the observed kinetic isotope effect for 13C versus 12C at the carbonyl group of carbamoyl phosphate (CP) increased upon the binding of ligands which promote the formation of the R-state (Asp, N-phosphonacetyl-L-aspartate (PALA), or ATP). The maximum rate for the [14C]Asp<-->Carbamoyl aspartate (CAsp) exchange with the Glu-50-->Ala enzyme was 500-fold slower than for the wild-type enzyme; however, the rate for the [14C]CP<-->CAsp exchange was only 50-fold slower, reversing the relative rates observed with the wild-type enzyme. In addition, upon variation of substrate pairs involving Asp or CAsp, loss of inhibition effects in the CP<-->CAsp exchange indicated that the Glu-50-->Ala substitution caused the kinetic mechanism for the mutant enzyme to shift from ordered to random. Computer simulations of the EIEK data indicate that the Glu-50-->Ala mutation specifically causes strong decreases in the rates of catalysis and association-dissociation for Asp and CAsp, with minimal effects on the CP and Pi on-off rates. With substrates bound, the Glu-50-->Ala enzyme apparently does not attain a full R-state conformation. The PALA-activated Glu-50-->Ala enzyme, however, exhibits substrate affinities comparable to those for the wild-type enzyme, but fails to restore the preferred order substrate binding. Unlike the wild-type enzyme, both the T and R-states of the Glu-50-->Ala enzyme

  6. From the endoplasmic reticulum to the plasma membrane: mechanisms of CFTR folding and trafficking.

    PubMed

    Farinha, Carlos M; Canato, Sara

    2017-01-01

    CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.

  7. Structural heterogeneity of 6 M GdmCl-denatured proteins: implications for the mechanism of protein folding.

    PubMed

    Chang, Jui-Yoa

    2009-10-13

    An in vitro experiment with protein folding is typically initiated with 6 M GdmCl-denatured proteins, which are generally considered fully unfolded. However, studies conducted by various laboratories have shown that many 6 M GdmCl-denatured proteins are structurally heterogeneous and still retain nativelike residual structures. The extent of conformational heterogeneity of the 6 M GdmCl-denatured protein has significant implications for the folding landscape as well as the interpretation of the observed early stage folding mechanism. Using the method of disulfide scrambling, we are able to gain rough insight into the diverse structural properties of 6 M GdmCl-denatured proteins. It demonstrates that most 6 M GdmCl-denatured proteins are approximately fully denatured, but partially unfolded. Most of them comprise diverse conformational isomers. We review here the cumulative evidence obtained from various laboratories and also provide experimental data obtained in our laboratory.

  8. Kinetic mechanism of phenylalanine hydroxylase: intrinsic binding and rate constants from single-turnover experiments.

    PubMed

    Roberts, Kenneth M; Pavon, Jorge Alex; Fitzpatrick, Paul F

    2013-02-12

    Phenylalanine hydroxylase (PheH) catalyzes the key step in the catabolism of dietary phenylalanine, its hydroxylation to tyrosine using tetrahydrobiopterin (BH(4)) and O(2). A complete kinetic mechanism for PheH was determined by global analysis of single-turnover data in the reaction of PheHΔ117, a truncated form of the enzyme lacking the N-terminal regulatory domain. Formation of the productive PheHΔ117-BH(4)-phenylalanine complex begins with the rapid binding of BH(4) (K(d) = 65 μM). Subsequent addition of phenylalanine to the binary complex to form the productive ternary complex (K(d) = 130 μM) is approximately 10-fold slower. Both substrates can also bind to the free enzyme to form inhibitory binary complexes. O(2) rapidly binds to the productive ternary complex; this is followed by formation of an unidentified intermediate, which can be detected as a decrease in absorbance at 340 nm, with a rate constant of 140 s(-1). Formation of the 4a-hydroxypterin and Fe(IV)O intermediates is 10-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines k(cat). Similar reactions using 6-methyltetrahydropterin indicate a preference for the physiological pterin during hydroxylation.

  9. A bioreactor for the dynamic mechanical stimulation of vocal-fold fibroblasts based on vibro-acoustography

    NASA Astrophysics Data System (ADS)

    Chan, Roger W.; Rodriguez, Maritza

    2005-09-01

    During voice production, the vocal folds undergo airflow-induced self-sustained oscillation at a fundamental frequency of around 100-1000 Hz, with an amplitude of around 1-3 mm. The vocal-fold extracellular matrix (ECM), with appropriate tissue viscoelastic properties, is optimally tuned for such vibration. Vocal-fold fibroblasts regulate the gene expressions for key ECM proteins (e.g., collagen, fibronectin, fibromodulin, and hyaluronic acid), and these expressions are affected by the stress fields experi- enced by the fibroblasts. This study attempts to develop a bioreactor for cultivating cells under a micromechanical environment similar to that in vivo, based on the principle of vibro-acoustography. Vocal-fold fibroblasts from primary culture were grown in 3D, biodegradable scaffolds, and were excited dynamically by the radiation force generated by amplitude modulation of two confocal ultrasound beams of slightly different frequencies. Low-frequency acoustic radiation force was applied to the scaffold surface, and its vibratory response was imaged by videostroboscopy. A phantom tissue (standard viscoelastic material) with known elastic modulus was also excited and its vibratory frequency and amplitude were measured by videostroboscopy. Results showed that the bioreactor was capable of delivering mechanical stimuli to the tissue constructs in a physiological frequency range (100-1000 Hz), supporting its potential for vocal-fold tissue engineering applications. [Work supported by NIH Grant R01 DC006101.

  10. Investigation kinetics mechanisms of adsorption malachite green onto activated carbon.

    PubMed

    Onal, Y; Akmil-Başar, C; Sarici-Ozdemir, C

    2007-07-19

    Lignite was used to prepare activated carbon (T3K618) by chemical activation with KOH. Pore properties of the activated carbon such as BET surface area, pore volume, pore size distribution, and pore diameter were characterized by t-plot based on N2 adsorption isotherm. BET surface area of activated carbon is determined as 1000 m2/g. Adsorption capacity of malachite green (MG) onto T3K618 activated carbon was investigated in a batch system by considering the effects of various parameters like initial concentration (100, 150 and 200 mg/L) and temperature (25, 40 and 50 degrees C). The adsorption process was relatively fast and equilibrium was reached after about 20 min for 100, 150 mg/L at all adsorption temperature. Equilibrium time for 200 mg/L was determined as 20 min and 40 min at 298, 313 and 323 K, respectively. Simple mass and kinetic models were applied to the experimental data to examine the mechanisms of adsorption and potential rate controlling steps such as external mass transfer, intraparticle diffusion. Pseudo second-order model was found to explain the kinetics of MG adsorption most effectively. It was found that both mass transfer and pore diffusion are important in determining the adsorption rates. The intraparticle diffusion rate constant, external mass transfer coefficient, film and pore diffusion coefficient at various temperatures were evaluated. The activation energy (Ea) was determined as 48.56, 63.16, 67.93 kJ/mol for 100, 150, 200 mg/L, respectively. The Langmiur and Freundlich isotherm were used to describe the adsorption equilibrium studies at different temperatures. Langmiur isotherm shows better fit than Freundlich isotherm in the temperature range studied. The thermodynamic parameters, such as DeltaG degrees, DeltaS and DeltaH degrees were calculated. The thermodynamics of dyes-T3K618 system indicates endothermic process.

  11. A steady-state kinetic analysis of the prolyl-4-hydroxylase mechanism.

    PubMed

    Soskel, N T; Kuby, S A

    1981-01-01

    Published kinetic data by Kivirikko, et al. on the prolyl-4-hydroxylase reaction have been re-evaluated using the overall steady-state velocity equation in the forward and reverse directions for an ordered ter ter kinetic mechanism. Qualitatively, the published data for prolyl-4-hydroxylase appear to fit the predicted patterns for this kinetic mechanism. More kinetic data are needed to confirm these results and to quantitate the kinetic parameters but, tentatively, the order of substrate addition would appear to be alpha-ketoglutarate, oxygen, and peptide; and the order of product release would be hydroxylated peptide (or collagen), carbon dioxide, and succinate.

  12. Structural geometry and deformation mechanism of the Longquan anticline in the Longmen Shan fold-and-thrust belt, eastern Tibet

    NASA Astrophysics Data System (ADS)

    Li, Zhi-Gang; Jia, Dong; Chen, Wei

    2013-03-01

    The 2008 Mw 7.9 Wenchuan earthquake is a consequence of ongoing India-Tibet collision and reflects the growth of the Longmen Shan fold-and-thrust belt. In this paper, we present new constraints on the deformation mechanism of the Longmen Shan fold-and-thrust belt, by comparing the physical models to the example of the Longmen Shan fold-and-thrust belt. The result indicates that the deformation mechanism of the belt is mainly dominated by the pre-Sinian layer, whereas locally is controlled by the Lower Triassic layer, such as the Longquan anticline. In addition, we discuss the deformation style of the Longquan anticline various along strike, based on the seismic reflection data, interpretations of structural cross-sections and field observations, as well as physical modeling. The sandbox modeling suggests that the deformation of the central segment of Longquan anticline is likely controlled by higher displacement rate, higher erosion and lower sedimentation, which is in contrast with the southern and northern segment. Moreover, the structural geometry of the central segment of Longquan anticline is more complex than the end-member models of fault-related folds, which is mainly controlled by pure-shear wedge fault-bend fold and bounded by west-verging thrust fault.Combining the studies of the structural geometry, deformation mechanism, and previous studies, we infer that the Longquan anticline is active and potentially seismogenic. Therefore, a quantitative re-evaluation of seismic hazard in Longquan anticline and adjacent area directly beneath the densely populated Sichuan basin is urgently needed.

  13. Reaction Kinetics and Mechanism of Magnetic Field Effects in Cryptochrome

    PubMed Central

    2012-01-01

    Creatures as varied as mammals, fish, insects, reptiles, and birds have an intriguing ‘sixth’ sense that allows them to orient themselves in the Earth's magnetic field. Despite decades of study, the physical basis of this magnetic sense remains elusive. A likely mechanism is furnished by magnetically sensitive radical pair reactions occurring in the retina, the light-sensitive part of animal eyes. A photoreceptor, cryptochrome, has been suggested to endow birds with magnetoreceptive abilities as the protein has been shown to exhibit the biophysical properties required for an animal magnetoreceptor to operate properly. Here, we propose a theoretical analysis method for identifying cryptochrome's signaling reactions involving comparison of measured and calculated reaction kinetics in cryptochrome. Application of the method yields an exemplary light-driven reaction cycle, supported through transient absorption and electron-spin-resonance observations together with known facts on avian magnetoreception. The reaction cycle permits one to predict magnetic field effects on cryptochrome activation and deactivation. The suggested analysis method gives insight into structural and dynamic design features required for optimal detection of the geomagnetic field by cryptochrome and suggests further experimental and theoretical studies. PMID:22171949

  14. Mechanisms, kinetics, impurities and defects: consequences in macromolecular crystallization

    PubMed Central

    McPherson, Alexander; Kuznetsov, Yurii G.

    2014-01-01

    The nucleation and growth of protein, nucleic acid and virus crystals from solution are functions of underlying kinetic and thermodynamic parameters that govern the process, and these are all supersaturation-dependent. While the mechanisms of macromolecular crystal growth are essentially the same as for conventional crystals, the underlying parameters are vastly different, in some cases orders of magnitude lower, and this produces very different crystallization processes. Numerous physical features of macromolecular crystals are of serious interest to X-ray diffractionists; the resolution limit and mosaicity, for example, reflect the degree of molecular and lattice order. The defect structure of crystals has an impact on their response to flash-cooling, and terminal crystal size is dependent on impurity absorption and incorporation. The variety and extent of these issues are further unique to crystals of biological macromolecules. All of these features are amenable to study using atomic force microscopy, which provides direct images at the nanoscale level. Some of those images are presented here. PMID:24699728

  15. Kinetics, mechanism and thermodynamics of bisulfite-aldehyde adduct formation

    SciTech Connect

    Olson, T.M.; Boyce, S.D.; Hoffmann, M.R.

    1986-04-01

    The kinetics and mechanism of bisulfite addition to benzaldehyde were studied at low pH in order to assess the importance of this reaction in stabilizing S(IV) in fog-, cloud-, and rainwater. Previously, the authors established that appreciable concentrations of the formaldehyde-bisulfite adduct (HMSA) are often present in fogwater. Measured HMSA concentrations in fogwater often do not fully account for observed excess S(IV) concentrations, however, so that other S(IV)-aldehyde adducts may be present. Reaction rates were determined by monitoring the disappearance of benzaldehyde by U.V. spectrophotometry under pseudo-first order conditions, (S(IV))/sub T/ >>(phi-CHO)/sub T/, in the pH range 0 - 4.4 at 25/sup 0/C. The equilibrium constant was determined by dissolving the sodium salt of the addition compound in a solution adjusted to pH 3.9, and measuring the absorbance of the equilibrated solution at 250 nm. A literature value of the extinction coefficient for benzaldehyde was used to calculate the concentration of free benzaldehyde. All solutions were prepared under an N/sub 2/ atmosphere using deoxygenated, deionized water and ionic strength was maintained at 1.0 M with sodium chloride.

  16. Reaction kinetics and mechanism of magnetic field effects in cryptochrome.

    PubMed

    Solov'yov, Ilia A; Schulten, Klaus

    2012-01-26

    Creatures as varied as mammals, fish, insects, reptiles, and birds have an intriguing sixth sense that allows them to orient themselves in the Earth's magnetic field. Despite decades of study, the physical basis of this magnetic sense remains elusive. A likely mechanism is furnished by magnetically sensitive radical pair reactions occurring in the retina, the light-sensitive part of animal eyes. A photoreceptor, cryptochrome, has been suggested to endow birds with magnetoreceptive abilities as the protein has been shown to exhibit the biophysical properties required for an animal magnetoreceptor to operate properly. Here, we propose a theoretical analysis method for identifying cryptochrome's signaling reactions involving comparison of measured and calculated reaction kinetics in cryptochrome. Application of the method yields an exemplary light-driven reaction cycle, supported through transient absorption and electron-spin-resonance observations together with known facts on avian magnetoreception. The reaction cycle permits one to predict magnetic field effects on cryptochrome activation and deactivation. The suggested analysis method gives insight into structural and dynamic design features required for optimal detection of the geomagnetic field by cryptochrome and suggests further experimental and theoretical studies.

  17. Analysis of repeat-protein folding using nearest-neighbor statistical mechanical models

    PubMed Central

    Aksel, Tural; Barrick, Doug

    2010-01-01

    The linear “Ising” model, which has been around for nearly a century, treats the behavior of linear arrays of repetitive, interacting subunits. Linear “repeat-proteins” have only been described in the last decade or so, and their folding energies have only been characterized very recently. Owing to their repetitive structures, linear repeat-proteins are particularly well suited for analysis by the nearest-neighbor Ising formalism. After briefly describing the historical origins and applications of the Ising model to biopolymers, and introducing repeat protein structure, this chapter will focus on the application of the linear Ising model to repeat proteins. When applied to homopolymers, the model can be represented and applied in a fairly simplified form. When applied to heteropolymers, where differences in energies among individual subunits (i.e. repeats) must be included, some (but not all) of this simplicity is lost. Derivations of the linear Ising model for both homopolymer and heteropolymer repeat-proteins will be presented. With the increased complexity required for analysis of heteropolymeric repeat proteins, the ability to resolve different energy terms from experimental data can be compromised. Thus, a simple matrix approach will be developed to help inform on the degree to which different thermodynamic parameters can be extracted from a particular set of unfolding curves. Finally, we will describe the application of these models to analyze repeat-protein folding equilibria, focusing on simplified repeat proteins based on “consensus” sequence information. PMID:19289204

  18. Influences of heterogeneous native contact energy and many-body interactions on the prediction of protein folding mechanisms.

    PubMed

    Zhang, Zhuqing; Ouyang, Yanhua; Chen, Tao

    2016-11-16

    Since single-point mutant perturbation has been used to probe protein folding mechanisms in experiments, the ϕ-value has become a critical parameter to infer the transition state (TS) for two-state proteins. Experimentally, large scale analysis has shown a nearly single uniform ϕ-value with normally distributed error from 24 different proteins; moreover, in zero stability conditions, the intrinsic variable ϕ(0) is around 0.36. To explore how and to what extent theoretical models can capture experimental phenomena, we here use structure-based explicit chain coarse-grained models to investigate the influence of single-point mutant perturbation on protein folding for single domain two-state proteins. Our results indicate that uniform, additive contact energetic interactions cannot predict experimental Brønsted plots well. Those points deviate largely from the main data sets in Brønsted plots, are mostly hydrophobic, and are located in N- and C-terminal contacting regions. Heterogenous contact energy, which is dependent on sequence separation, can narrow the point dispersion in a Brønsted plot. Moreover, we demonstrate that combining many-body interactions with heterogeneous native contact energy can present mean ϕ-values consistent with experimental findings, with a comparable distributed error. This indicates that for more accurate elucidation of protein folding mechanisms by residue-level structure-based models, these elements should be considered.

  19. Role of folded anisotropic fabric in the failure mode of gneiss: new insights from mechanical, microseismic and microstructural laboratory data

    NASA Astrophysics Data System (ADS)

    Agliardi, Federico; Vinciguerra, Sergio; Dobbs, Marcus R.; Zanchetta, Stefano

    2015-04-01

    Fabric anisotropy is a key control of the mechanical behaviour of rocks in a variety of geological settings and on different timescales. However, the effects of inherited, tectonically folded anisotropic fabrics on the brittle strength and failure mode of foliated metamorphic rocks is yet to be fully understood. Data from laboratory uniaxial compression tests on folded gneiss (Agliardi et al., 2014, Tectonophysics) recently showed that the brittle failure mode of this rock type depends on the arrangement of two distinct anisotropies (i.e. foliation and fold axial plane anisotropy), and that rock strength correlates with failure mode. Here we investigate the effects of confining pressure on this behaviour by performing triaxial compression experiments with acoustic emission (AE) monitoring, and analyse resulting fracture mechanisms and their microfabric controls using high resolution microanalysis techniques. We tested the Monte Canale Gneiss (Austroalpine Bernina nappe, Central Italian Alps), characterized by low phyllosilicate content, compositional layering folded at the cm-scale, and absence of a well-developed axial plane foliation. We used a servo-controlled hydraulic loading system to test 19 air-dry cylindrical specimens (diameter: 54 mm) that were characterized both in terms of fold geometry and orientation of foliation and fold axial planes to the axial load direction. We instrumented the specimens with direct contact axial and circumferential strain gauges. We performed tests at confining pressures of 40 MPa and constant axial strain rates of 5*10-6 s-1, measuring acoustic emissions and P- and S-wave velocities by three wideband (350-1000 kHz) piezoelectric transceivers with 40 dB preamps, mounted in the compression platens. We carried out post-failure microscale observation of fracture mechanisms, microcrack patterns and related fabric controls on resin-impregnated samples, using X-ray MicroCT (resolution: 9 μm), optical microscopy and SEM. Samples

  20. Mechanics of Shale Detachments - insights from the Khao-Kwang fold-and-thrust belt, Thailand

    NASA Astrophysics Data System (ADS)

    von Hagke, C.; Morley, C. K.; Kanitpanyacharoen, W.

    2016-12-01

    Shale detachments are the key slip horizons in fold and thrust belts, accretionary prisms, gravity driven systems ranging from major deltas to mass transport complexes and hybrid gravity-lithospheric-stress driven systems. Despite our qualitative understanding of factors contributing to detachment weakness such as mineralogy, pore fluid pressure, or efficiency of structure localization, it is difficult to assess the contribution of the individual factors. Here we present multi-scale analysis of a clay detachment within carbonate rocks, where it is possible to study structures formed within a similar temperature and pressure regime, and thus only varying due to lithological contrasts. We mapped the detachment at outcrop scale, where it shows considerable variations in structural style, as well as localization within different clay and limestone horizons. Zones of low and high strain have been identified. We investigate these changes in macroscopic deformation style using Virtual Polarizing Microscopy, and the combined methods of Broad Ion Beam milling and Scanning Electron Microscopy. We characterize the detachment at all scales, from outcrop down to nano-meters. Results show strain localization in the detachment is heterogeneous, and shows strong variations along strike. Within the clay package, strain localizes along zones rich in organic matter. Highest strain does not only occur in the clay units, but partly is accommodated in the surrounding limestone. We speculate clays only form the weakest layer when sufficient organic matter is present.

  1. Mechanical design of the folded waveguide for PBX-M and TFTR

    SciTech Connect

    Fogelman, C.H.; Bigelow, T.S.; Yugo, J.J.

    1995-12-31

    The folded waveguide (FWG) antenna is an advanced Cyclotron Range of Frequencies launcher being designed at Oak Ridge National Laboratory in collaboration with Princeton Plasma Physics Laboratory. The FWG offers a drastic increase in radio frequency (RF) power density over typical loop antennas. It also results in internal electric fields of much lower magnitude near the plasma. It is scheduled for installation on either the Tokamak Fusion Test Reactor (TFTR) or the Princeton Beta Experiment-Modified (PBX-M) tokamak in January 1996. The design objective is to provide an FWG that can withstand the thermal loads and disruption scenarios and meet the space constants of both machines. The design is also intended to be prototypical for the International Thermonuclear Experimental Reactor (ITER). The FWG is fully retractable, and maintenance operations can be performed while the vessel remains under vacuum. The FWG can operate in fast-wave mode, or it can be retracted, rotated 90{degrees}, and reengaged for the ion-Bernstein wave launch. The polarizing plate completely covers the front of the antenna, except for slots cut at every other gap between with plates of other configurations such as a 0-{pi} dipole plate.

  2. Self-oscillating Vocal Fold Model Mechanics: Healthy, Diseased, and Aging

    NASA Astrophysics Data System (ADS)

    Hiubler, Elizabeth P.; Pollok, Lucas F. E.; Apostoli, Adam G.; Hancock, Adrienne B.; Plesniak, Michael W.

    2014-11-01

    Voice disorders have been estimated to have a substantial economic impact of 2.5 billion annually. Approximately 30% of people will suffer from a voice disorder at some point in their lives. Life-sized, self-oscillating, synthetic vocal fold (VF) models are fabricated to exhibit material properties representative of human VFs. These models are created both with and without a polyp-like structure, a pathology that has been shown to produce rich viscous flow structures not normally observed for healthy VFs during normal phonation. Pressure measurements are acquired upstream of the VFs and high-speed images are captured at varying flow rates during VF oscillation to facilitate an understanding of the characteristics of healthy and diseased VFs. The images are analyzed using a videokymography line-scan technique. Clinically-relevant parameters calculated from the volume-velocity output of a circumferentially-vented mask (Rothenberg mask) are compared to human data collected from two groups of males aged 18-30 and 60-80. This study extends the use of synthetic VF models by assessing their ability to replicate behaviors observed in human subject data to advance a means of investigating changes associated with normal, pathological, and the aging voice. Supported by the GWU Institute for Biomedical Engineering (GWIBE) and GWU Center for Biomimetics and Bioinspired Engineering (COBRE).

  3. Modulating bilayer mechanical properties to promote the coupled folding and insertion of an integral membrane protein.

    PubMed

    Herrmann, Michaela; Danielczak, Bartholomäus; Textor, Martin; Klement, Jessica; Keller, Sandro

    2015-10-01

    Bilayer mechanical properties are not only of crucial importance to the mechanism of action of mechanosensation in lipid membranes but also affect preparative laboratory tasks such as membrane-protein refolding. We report this for coupled refolding and bilayer insertion of outer membrane phospholipase A (OmpLA), an integral membrane enzyme that catalyses the hydrolytic cleavage of glycerophospholipids. OmpLA can be refolded into a variety of detergent micelles and unilamellar vesicles composed of short-chain phospholipids but, in the absence of chemical or molecular chaperones, not into thicker membranes. Controlled modulation of bilayer mechanical properties by judicious use of subsolubilising concentrations of detergents induces monolayer curvature strain, acyl chain fluidisation, membrane thinning, and transient aqueous bilayer defects. This enables quantitative and functional refolding of OmpLA even into bilayer membranes composed of long-chain phospholipids to yield enzymatically active proteoliposomes without requiring membrane solubilisation.

  4. Identification of enzyme inhibitory mechanisms from steady-state kinetics.

    PubMed

    Fange, David; Lovmar, Martin; Pavlov, Michael Y; Ehrenberg, Måns

    2011-09-01

    Enzyme inhibitors are used in many areas of the life sciences, ranging from basic research to the combat of disease in the clinic. Inhibitors are traditionally characterized by how they affect the steady-state kinetics of enzymes, commonly analyzed on the assumption that enzyme-bound and free substrate molecules are in equilibrium. This assumption, implying that an enzyme-bound substrate molecule has near zero probability to form a product rather than dissociate, is valid only for very inefficient enzymes. When it is relaxed, more complex but also more information-rich steady-state kinetics emerges. Although solutions to the general steady-state kinetics problem exist, they are opaque and have been of limited help to experimentalists. Here we reformulate the steady-state kinetics of enzyme inhibition in terms of new parameters. These allow for assessment of ambiguities of interpretation due to kinetic scheme degeneracy and provide an intuitively simple way to analyze experimental data. We illustrate the method by concrete examples of how to assess scheme degeneracy and obtain experimental estimates of all available rate and equilibrium constants. We suggest simple, complementary experiments that can remove ambiguities and greatly enhance the accuracy of parameter estimation.

  5. Divalent Metal Ion-Induced Folding Mechanism of RNase H1 from Extreme Halophilic Archaeon Halobacterium sp. NRC-1

    PubMed Central

    Tannous, Elias; Kanaya, Shigenori

    2014-01-01

    RNase H1 from Halobacterium sp. NRC-1 (Halo-RNase H1) is characterized by the abundance of acidic residues on the surface, including bi/quad-aspartate site residues. Halo-RNase H1 exists in partially folded (I) and native (N) states in low-salt and high-salt conditions respectively. Its folding is also induced by divalent metal ions. To understand this unique folding mechanism of Halo-RNase H1, the active site mutant (2A-RNase H1), the bi/quad-aspartate site mutant (6A-RNase H1), and the mutant at both sites (8A-RNase H1) were constructed. The far-UV CD spectra of these mutants suggest that 2A-RNase H1 mainly exists in the I state, 6A-RNase H1 exists both in the I and N states, and 8A-RNase H1 mainly exists in the N state in a low salt-condition. These results suggest that folding of Halo-RNase H1 is induced by binding of divalent metal ions to the bi/quad-aspartate site. To examine whether metal-induced folding is unique to Halo-RNase H1, RNase H2 from the same organism (Halo-RNase H2) was overproduced and purified. Halo-RNase H2 exists in the I and N states in low-salt and high-salt conditions respectively, as does Halo-RNase H1. However, this protein exists in the I state even in the presence of divalent metal ions. Halo-RNase H2 exhibits junction ribonuclease activity only in a high-salt condition. A tertiary model of this protein suggests that this protein does not have a quad-aspartate site. We propose that folding of Halo-RNase H1 is induced by binding of divalent metal ion to the quad-aspartate site in a low-salt condition. PMID:25268753

  6. Divalent metal ion-induced folding mechanism of RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1.

    PubMed

    Tannous, Elias; Kanaya, Shigenori

    2014-01-01

    RNase H1 from Halobacterium sp. NRC-1 (Halo-RNase H1) is characterized by the abundance of acidic residues on the surface, including bi/quad-aspartate site residues. Halo-RNase H1 exists in partially folded (I) and native (N) states in low-salt and high-salt conditions respectively. Its folding is also induced by divalent metal ions. To understand this unique folding mechanism of Halo-RNase H1, the active site mutant (2A-RNase H1), the bi/quad-aspartate site mutant (6A-RNase H1), and the mutant at both sites (8A-RNase H1) were constructed. The far-UV CD spectra of these mutants suggest that 2A-RNase H1 mainly exists in the I state, 6A-RNase H1 exists both in the I and N states, and 8A-RNase H1 mainly exists in the N state in a low salt-condition. These results suggest that folding of Halo-RNase H1 is induced by binding of divalent metal ions to the bi/quad-aspartate site. To examine whether metal-induced folding is unique to Halo-RNase H1, RNase H2 from the same organism (Halo-RNase H2) was overproduced and purified. Halo-RNase H2 exists in the I and N states in low-salt and high-salt conditions respectively, as does Halo-RNase H1. However, this protein exists in the I state even in the presence of divalent metal ions. Halo-RNase H2 exhibits junction ribonuclease activity only in a high-salt condition. A tertiary model of this protein suggests that this protein does not have a quad-aspartate site. We propose that folding of Halo-RNase H1 is induced by binding of divalent metal ion to the quad-aspartate site in a low-salt condition.

  7. Determination of the elastic properties of short ssDNA molecules by mechanically folding and unfolding DNA hairpins.

    PubMed

    Alemany, Anna; Ritort, Felix

    2014-12-01

    The characterization of elastic properties of biopolymers is crucial to understand many molecular reactions determined by conformational bending fluctuations of the polymer. Direct measurement of such elastic properties using single-molecule methods is usually hindered by the intrinsic tendency of such biopolymers to form high-order molecular structures. For example, single-stranded deoxyribonucleic acids (ssDNA) tend to form secondary structures such as local double helices that prevent the direct measurement of the ideal elastic response of the ssDNA. In this work, we show how to extract the ideal elastic response in the entropic regime of short ssDNA molecules by mechanically pulling two-state DNA hairpins of different contour lengths. This is achieved by measuring the force dependence of the molecular extension and stiffness on mechanically folding and unfolding the DNA hairpin. Both quantities are fit to the worm-like chain elastic model giving values for the persistence length and the interphosphate distance. This method can be used to unravel the elastic properties of short ssDNA and RNA sequences and, more generally, any biopolymer that can exhibit a cooperative two-state transition between mechanically folded and unfolded states (such as proteins).

  8. Kinetics and mechanisms of chlorine dioxide oxidation of tryptophan.

    PubMed

    Stewart, David J; Napolitano, Michael J; Bakhmutova-Albert, Ekaterina V; Margerum, Dale W

    2008-03-03

    The reactions of aqueous ClO2 (*) and tryptophan (Trp) are investigated by stopped-flow kinetics, and the products are identified by high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry and by ion chromatography. The rates of ClO2 (*) loss increase from pH 3 to 5, are essentially constant from pH 5 to 7, and increase from pH 7 to 10. The reactions are first-order in Trp with variable order in ClO2 (*). Below pH 5.0, the reactions are second- or mixed-order in [ClO2 (*)], depending on the chlorite concentration. Above pH 5.0, the reactions are first-order in [ClO2 (*)] in the absence of added chlorite. At pH 7.0, the Trp reaction with ClO2 (*) is first-order in each reactant with a second-order rate constant of 3.4 x 10(4) M(-1) s(-1) at 25.0 degrees C. In the proposed mechanism, the initial reaction is a one-electron oxidation to form a tryptophyl radical cation and chlorite ion. The radical cation deprotonates to form a neutral tryptophyl radical that combines rapidly with a second ClO 2 (*) to give an observable, short-lived adduct ( k obs = 48 s(-1)) with proposed C(H)-OClO bonding. This adduct decays to give HOCl in a three-electron oxidation. The overall reaction consumes two ClO2 (*) per Trp and forms ClO2- and HOCl. This corresponds to a four-electron oxidation. Decay of the tryptophyl-OClO adduct at pH 6.4 gives five initial products that are observed after 2 min and are separated by HPLC with elution times that vary from 4 to 17 min (with an eluent of 6.3% CH 3OH and 0.1% CH 3COOH). Each of these products is characterized by mass spectrometry and UV-vis spectroscopy. One initial product with a molecular weight of 236 decays within 47 min to yield the most stable product, N-formylkynurenine (NFK), which also has a molecular weight of 236. Other products also are observed and examined.

  9. [Kinetics and mechanism of removing Microcystis aeruginosa using clay flocculation].

    PubMed

    Pan, Gang; Zhang, Mingming; Yan, Hai; Zou, Hua; Chen, Hao

    2003-09-01

    Twenty-six natural clays were studied for their kinetics of flocculating and removing algal cells of Microcystis aeruginosa. According to the 8 h equilibrium removal efficiencies and removal rates at a clay-loading of 0.7 g.L-1, all the 26 clays were classified into three categories. Type-I clay, which includes talc, ferric oxide, sepiolite, ferroferric oxide, and kaolinite, has an equilibrium removal efficiency greater than 90%, a t50 (time needed to remove 50% of the algae) of less than 30 min, and a t80 (time needed to remove 80% of the algae) of less than 2.5 h. Type-II clay, which includes argillanceous rocks, attapulgite, rectorite, illite, and argil, etc., has an equilibrium removal efficiency of 50%-80%, a t50 of less than 2.5 h, and a t80 of more than 5 h. Type-III clay consists of 14 minerals, including laterite, zeolite, mica, clinoptilolite, pumice, tripoli, feldspar and quartz, etc. with the removal efficiency less than 50%, and t50 > > 8 h. When the clay loading was decreased to 0.1-0.2 g.L-1, the 8 h equilibrium removal efficiencies for 25 clays declined to below 60%, except for sepiolite, a Type-I clay, which maintained around 90%. After the sepiolite was modified with Fe3+ to increase its surface charge (Zeta potential from -24.0 mV to +0.43 mV at pH 7.4), the initial removal rate was increased remarkably although its 8 h equilibrium removal efficiency was not improved substantially. As a comparison, the 8 h equilibrium removal efficiency of PAC was no greater than 40% at loadings of 0.02-0.2 g.L-1. Following the analysis of the flocculation mechanism it was concluded that the effect of bridging and netting may play a key role in the clay-algae flocculation processes, which may be important for selecting and modifying clays to improve significantly the removal efficiency.

  10. Sedimentary iron monosulfides: Kinetics and mechanism of formation

    NASA Astrophysics Data System (ADS)

    Pyzik, Albert J.; Sommer, Sheldon E.

    1981-05-01

    The reaction between hydrous iron oxides and aqueous sulfide species was studied at estuarine conditions of pH, total sulfide, and ionic strength to determine the kinetics and formation mechanism of the initial iron sulfide. Total, dissolved and acid extractable sulfide, thiosulfate, sulfate, and elemental sulfur were determined by spectrophotometric methods. Polysulfides, S 42- and S 52-, were determined from ultraviolet absorbance measurements and equilibrium calculations, while product hydroxyl ion was determined from pH measurements and solution buffer capacity. Elemental sulfur, as free and polysulfide sulfur, was 86% of the sulfide oxidation products; the remainder was thiosulfate. Rate expressions for the reduction and precipitation reactions were determined from analysis of electron balance and acid extractable iron monosulfide vs time, respectively, by the initial rate method. The rate of iron reduction in moles/liter/minute was given by d( reduction Fe) /dt = kS t0.5(J +) 0.5 A FeOOH1 where St was the total dissolved sulfide concentration, (H +) the hydrogen ion activity, both in moles/ liter; and AFeOOH the goethite specific surface area in square meters/liter. The rate constant, k, was 0.017 ± 0.002m -2 min -1. The rate of reduction was apparently determined by the rate of dissolution of the surface layer of ferrous hydroxide. The rate expression for the precipitation reaction was d(FeS)/dt = kS t1(H +) 1 A FeOOH1 where d(FeS)/dt was the rate of precipitation of acid extractable iron monosulfide in moles/liter/minute, and k = 82 ± 18 mol -1l2m-2 min -1. A model is proposed with the following steps: protonation of goethite surface layer; exchange of bisulfide for hydroxide in the mobile layer; reduction of surface ferric ions of goethite by dissolved bisulfide species which produces ferrous hydroxide surface layer elemental sulfur and thiosulfate; dissolution of surface layer of ferrous hydroxide; and precipitation of dissolved ferrous specie and

  11. How Does Your Protein Fold? Elucidating the Apomyoglobin Folding Pathway.

    PubMed

    Dyson, H Jane; Wright, Peter E

    2017-01-17

    Although each type of protein fold and in some cases individual proteins within a fold classification can have very different mechanisms of folding, the underlying biophysical and biochemical principles that operate to cause a linear polypeptide chain to fold into a globular structure must be the same. In an aqueous solution, the protein takes up the thermodynamically most stable structure, but the pathway along which the polypeptide proceeds in order to reach that structure is a function of the amino acid sequence, which must be the final determining factor, not only in shaping the final folded structure, but in dictating the folding pathway. A number of groups have focused on a single protein or group of proteins, to determine in detail the factors that influence the rate and mechanism of folding in a defined system, with the hope that hypothesis-driven experiments can elucidate the underlying principles governing the folding process. Our research group has focused on the folding of the globin family of proteins, and in particular on the monomeric protein apomyoglobin. Apomyoglobin (apoMb) folds relatively slowly (∼2 s) via an ensemble of obligatory intermediates that form rapidly after the initiation of folding. The folding pathway can be dissected using rapid-mixing techniques, which can probe processes in the millisecond time range. Stopped-flow measurements detected by circular dichroism (CD) or fluorescence spectroscopy give information on the rates of folding events. Quench-flow experiments utilize the differential rates of hydrogen-deuterium exchange of amide protons protected in parts of the structure that are folded early; protection of amides can be detected by mass spectrometry or proton nuclear magnetic resonance spectroscopy (NMR). In addition, apoMb forms an intermediate at equilibrium at pH ∼ 4, which is sufficiently stable for it to be structurally characterized by solution methods such as CD, fluorescence and NMR spectroscopies, and the

  12. Kinetic Mechanism of Human Histidine Triad Nucleotide Binding Protein 1 (Hint1)

    PubMed Central

    Zhou, Xin; Chou, Tsui-Fen; Aubol, Brandon E; Park, Chin Ju; Wolfenden, Richard; Adams, Joseph; Wagner, Carston R.

    2013-01-01

    Human histidine triad nucleotide binding protein 1 (hHint1) is a member of a ubiquitous and ancient branch of the histidine triad (HIT) protein superfamily. hHint1 is a homodimeric protein that catalyzes the hydrolysis of model substrates, phosphoramidate (TpAd) and acyl adenylate (AIPA), with a high efficiency. Recently, catalytically inactive hHint1 has been identified as the cause of inherited peripheral neuropathy (1). We have carried out the first detailed kinetic mechanistic studies of hHint1 and have found that the reaction mechanism is consistent with a double displacement mechanism, in which the active site nucleophile His112 is first adenylylated by the substrate, followed by hydrolysis of the AMP-enzyme intermediate. A transient burst phase followed by a linear phase from the stopped-flow fluorescence assay indicated that enzyme adenylylation was faster than the subsequent intermediate hydrolysis and product release. Solvent viscosity experiments suggested that both chemical transformation and diffusion-sensitive events (product release or protein conformational change) limit the overall turnover. The catalytic trapping experiments and data simulation indicated that the true koff rate of the final product AMP is unlikely to control the overall kcat. Therefore, a protein conformational change associated with product release is likely rate limiting. In addition, the rate of Hint1 adenylylation was found to be dependent on two residues with pKas of 6.5 and 8, with the former pKa agreeing well with the NMR titration results for the pKa of the active site nucleophile His112. When compared to the uncatalyzed rates, hHint1 was shown to enhance acyl-AMP and AMP phosphoramidate hydrolysis by 106 to 108- fold. Taken together, our analysis indicates that hHint1 catalyzes the hydrolysis of phosphoramidate and acyl adenylate with high efficiency, through a mechanism that relies on rapid adenylylation of the active residue, His-112, while being partially rate limited

  13. A Comparison of Kinetic Energy and Momentum in Special Relativity and Classical Mechanics

    ERIC Educational Resources Information Center

    Riggs, Peter J.

    2016-01-01

    Kinetic energy and momentum are indispensable dynamical quantities in both the special theory of relativity and in classical mechanics. Although momentum and kinetic energy are central to understanding dynamics, the differences between their relativistic and classical notions have not always received adequate treatment in undergraduate teaching.…

  14. A Comparison of Kinetic Energy and Momentum in Special Relativity and Classical Mechanics

    ERIC Educational Resources Information Center

    Riggs, Peter J.

    2016-01-01

    Kinetic energy and momentum are indispensable dynamical quantities in both the special theory of relativity and in classical mechanics. Although momentum and kinetic energy are central to understanding dynamics, the differences between their relativistic and classical notions have not always received adequate treatment in undergraduate teaching.…

  15. Kinetic isotope effects on the noncovalent flavin mutant protein of pyranose 2-oxidase reveal insights into the flavin reduction mechanism.

    PubMed

    Sucharitakul, Jeerus; Wongnate, Thanyaporn; Chaiyen, Pimchai

    2010-05-04

    Pyranose 2-oxidase (P2O) from Trametes multicolor contains a flavin adenine dinucleotide (FAD) cofactor covalently linked to the N(3) atom of His167. The enzyme catalyzes the oxidation of aldopyranoses by molecular oxygen to generate 2-keto-aldoses and H(2)O(2) as products. In this study, the transient kinetics and primary and solvent kinetic isotope effects of the mutant in which His167 has been replaced with Ala (H167A) were investigated, to elucidate the functional role of the 8a-N(3)-histidyl FAD linkage and to gain insights into the reaction mechanism of P2O. The results indicate that the covalent linkage is mainly important for a reductive half-reaction in which the FAD cofactor is reduced by d-glucose, while it is not important for an oxidative half-reaction in which oxygen reacts with the reduced FAD to generate H(2)O(2). d-Glucose binds to H167A via multiple binding modes before the formation of the active Michaelis complex, and the rate constant of flavin reduction decreases approximately 22-fold compared to that of the wild-type enzyme. The reduction of H167A using d-glucose isotopes (2-d-d-glucose, 3-d-d-glucose, and 1,2,3,4,5,6,6-d(7)-d-glucose) as substrates indicates that the primary isotope effect results only from substitution at the C2 position, implying that H167A catalyzes the oxidation of d-glucose regiospecifically at this position. No solvent kinetic isotope effect was detected during the reductive half-reaction of the wild-type or H167A enzyme, implying that the deprotonation of the d-glucose C2-OH group may occur readily upon the binding to P2O and is not synchronized with the cleavage of the d-glucose C2-H bond. The mutation has no drastic effect on the oxidative half-reaction of P2O, as H167A is very similar to the wild-type enzyme with respect to the kinetic constants and the formation of the C4a-hydroperoxyflavin intermediate. Kinetic mechanisms for both half-reactions of H167A were proposed on the basis of transient kinetic data and

  16. Renormalizing the Kinetic Energy Operator in Elementary Quantum Mechanics

    ERIC Educational Resources Information Center

    Coutinho, F. A. B.; Amaku, M.

    2009-01-01

    In this paper, we consider solutions to the three-dimensional Schrodinger equation of the form [psi](r) = u(r)/r, where u(0) [is not equal to] 0. The expectation value of the kinetic energy operator for such wavefunctions diverges. We show that it is possible to introduce a potential energy with an expectation value that also diverges, exactly…

  17. Renormalizing the Kinetic Energy Operator in Elementary Quantum Mechanics

    ERIC Educational Resources Information Center

    Coutinho, F. A. B.; Amaku, M.

    2009-01-01

    In this paper, we consider solutions to the three-dimensional Schrodinger equation of the form [psi](r) = u(r)/r, where u(0) [is not equal to] 0. The expectation value of the kinetic energy operator for such wavefunctions diverges. We show that it is possible to introduce a potential energy with an expectation value that also diverges, exactly…

  18. Kinetics and mechanism of soot formation in hydrocarbon combustion

    NASA Technical Reports Server (NTRS)

    Frenklach, Michael

    1990-01-01

    The focus of this work was on kinetic modeling. The specific objectives were: detailed modeling of soot formation in premixed flames, elucidation of the effects of fuel structure on the pathway to soot, and the development of a numerical technique for accurate modeling of soot particle coagulation and surface growth. Those tasks were successfully completed and are briefly summarized.

  19. NMR of α-synuclein–polyamine complexes elucidates the mechanism and kinetics of induced aggregation

    PubMed Central

    Fernández, Claudio O; Hoyer, Wolfgang; Zweckstetter, Markus; Jares-Erijman, Elizabeth A; Subramaniam, Vinod; Griesinger, Christian; Jovin, Thomas M

    2004-01-01

    The aggregation of α-synuclein is characteristic of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. The 140-aa protein is natively unstructured; thus, ligands binding to the monomeric form are of therapeutic interest. Biogenic polyamines promote the aggregation of α-synuclein and may constitute endogenous agents modulating the pathogenesis of PD. We characterized the complexes of natural and synthetic polyamines with α-synuclein by NMR and assigned the binding site to C-terminal residues 109–140. Dissociation constants were derived from chemical shift perturbations. Greater polyamine charge (+2 → +5) correlated with increased affinity and enhancement of fibrillation, for which we propose a simple kinetic mechanism involving a dimeric nucleation center. According to the analysis, polyamines increase the extent of nucleation by ∼104 and the rate of monomer addition ∼40-fold. Significant secondary structure is not induced in monomeric α-synuclein by polyamines at 15°C. Instead, NMR reveals changes in a region (aa 22–93) far removed from the polyamine binding site and presumed to adopt the β-sheet conformation characteristic of fibrillar α-synuclein. We conclude that the C-terminal domain acts as a regulator of α-synuclein aggregation. PMID:15103328

  20. Vitamin B2-sensitised photooxidation of the ophthalmic drugs Timolol and Pindolol: kinetics and mechanism.

    PubMed

    Criado, Susana; García, Norman A

    2004-01-01

    The kinetics and mechanistic aspects of the riboflavin-photosensitised oxidation of the topically administrable ophthalmic drugs Timolol (Tim) and Pindolol (Pin) were investigated in water-MeOH (9:1, v/v) solution employing light of wavelength > 400 nm. riboflavin, belonging to the vitamin B(2) complex, is a known human endogenous photosensitiser. The irradiation of riboflavin in the presence of ophthalmic drugs triggers a complex picture of competitive reactions which produces the photodegradation of both the drugs and the pigment itself. The mechanism was elucidated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Ophthalmic drugs quench riboflavin-excited singlet and triplet states. From the quenching of excited triplet riboflavin, the semireduced form of the pigment is generated, through an electron transfer process from the drug, with the subsequent production of superoxide anion radical (O(2)(*-)) by reaction with dissolved molecular oxygen. Through the interaction of dissolved oxygen with excited triplet riboflavin, the species singlet oxygen (O(2)((1)Delta(g))) is also generated to a lesser extent. Both O(2)(*-) and O(2)((1)Delta(g)) induce photodegradation of ophthalmic drugs, Tim being approximately 3-fold more easily photooxidisable than Pin, as estimated by oxygen consumption experiments.

  1. Analysis of flow-structure coupling in a mechanical model of the vocal folds and the subglottal system

    NASA Astrophysics Data System (ADS)

    Howe, M. S.; McGowan, R. S.

    2009-11-01

    An analysis is made of the nonlinear interactions between flow in the subglottal vocal tract and glottis, sound waves in the subglottal system and a mechanical model of the vocal folds. The mean flow through the system is produced by a nominally steady contraction of the lungs, and mechanical experiments frequently involve a ‘lung cavity’ coupled to an experimental subglottal tube of arbitrary or ill-defined effective length L, on the basis that the actual value of L has little or no influence on excitation of the vocal folds. A simple, self-exciting single-mass mathematical model of the vocal folds is used to investigate the sound generated within the subglottal domain and the unsteady volume flux from the glottis for experiments where it is required to suppress feedback of sound from the supraglottal vocal tract. In experiments where the assumed absorption of sound within the sponge-like interior of the lungs is small, the influence of changes in L can be very significant: when the subglottal tube behaves as an open-ended resonator (when L is as large as half the acoustic wavelength) there is predicted to be a mild increase in volume flux magnitude and a small change in waveform. However, the strong appearance of second harmonics of the acoustic field is predicted at intermediate lengths, when L is roughly one quarter of the acoustic wavelength. In cases of large lung damping, however, only modest changes in the volume flux are predicted to occur with variations in L.

  2. Constraints on deformation mechanisms during folding provided by rock physical properties: a case study at Sheep Mountain anticline (Wyoming, USA)

    NASA Astrophysics Data System (ADS)

    Amrouch, K.; Robion, P.; Callot, J.-P.; Lacombe, O.; Daniel, J.-M.; Bellahsen, N.; Faure, J.-L.

    2010-09-01

    The Sheep Mountain anticline (Wyoming, USA) is a well-exposed asymmetric, basement-cored anticline that formed during the Laramide orogeny in the early Tertiary. In order to unravel the history of strain during folding, we carried out combined anisotropy of magnetic susceptibility (AMS), anisotropy of P-wave velocity (APWV) and Fry strain analyses. The results are compared to previously published stress-strain data from calcite twins at the microscopic scale and from fracture sets at the mesoscopic scale, and are used to discuss the kinematics and mechanics of forced folding. The results obtained in sandstone and carbonate lithologies demonstrate a good agreement between (1) the principal axes of the AMS and APWV tensors, (2) stress-strain tensors derived from calcite twins, (3) Fry strain axes and mesoscopic fracture sets. Furthermore, these tensors are coaxial with the main structural trends of the anticline. The differences between AMS and APWV fabrics on one hand, and the differential stress values of the forelimb and the backlimb on the other hand, emphasize how the macroscopic asymmetry of Sheep Mountain anticline affects the strain pattern at the microscopic scale. The data set presented in this paper offers a consistent mechanical scenario for the development of Sheep Mountain anticline.

  3. Intramolecular Charge Transfer Dynamics of 4-(DIMETHYLAMINO)BENZONITRILE: Ultrafast Branching Followed by a Two-Fold Decay Mechanism

    NASA Astrophysics Data System (ADS)

    Coto, Pedro B.; Serrano-Andrés, Luis; Gustavsson, Thomas; Fujiwara, Takashige; Lim, Edward C.

    2010-06-01

    4-(Dimethylamino)benzonitrile (DMABN) is a paradigm molecule system that exhibits dual fluorescence and intramolecular charge transfer (ICT) in polar solvents. Although numbers of different experimental and theoretical methods have been carried out to date for elucidating the basic mechanism of its energy relaxation, there are still some crucial problems that remain unanswered. The time-resolved transient absorption and time-resolved fluorescence upconversion will be presented, as combined with ab initio CASPT2//CASSCF calculations, which indicate that a more complex mechanism may be suggested in the ICT reaction in a polar environment. A scheme of ultrafast branching relaxation followed by two-fold decay is proposed in which, whereas the fully twisted ICT (TICT) state is responsible for the transient absorption, a distinct partially twisted ICT (pTICT) structure is for the fluorescent ICT state, both displaying clearly different decay rates.

  4. Mechanics of neurulation: From classical to current perspectives on the physical mechanics that shape, fold, and form the neural tube.

    PubMed

    Vijayraghavan, Deepthi S; Davidson, Lance A

    2016-09-13

    Neural tube defects arise from mechanical failures in the process of neurulation. At the most fundamental level, formation of the neural tube relies on coordinated, complex tissue movements that mechanically transform the flat neural epithelium into a lumenized epithelial tube (Davidson, 2012). The nature of this mechanical transformation has mystified embryologists, geneticists, and clinicians for more than 100 years. Early embryologists pondered the physical mechanisms that guide this transformation. Detailed observations of cell and tissue movements as well as experimental embryological manipulations allowed researchers to generate and test elementary hypotheses of the intrinsic and extrinsic forces acting on the neural tissue. Current research has turned toward understanding the molecular mechanisms underlying neurulation. Genetic and molecular perturbation have identified a multitude of subcellular components that correlate with cell behaviors and tissue movements during neural tube formation. In this review, we focus on methods and conceptual frameworks that have been applied to the study of amphibian neurulation that can be used to determine how molecular and physical mechanisms are integrated and responsible for neurulation. We will describe how qualitative descriptions and quantitative measurements of strain, force generation, and tissue material properties as well as simulations can be used to understand how embryos use morphogenetic programs to drive neurulation. Birth Defects Research (Part A), 2016. © 2016 Wiley Periodicals, Inc.

  5. From Mechanism and Kinetics to Precise ATRP Synthesis

    NASA Astrophysics Data System (ADS)

    Mueller, Laura; Golas, Patricia; Matyjaszewski, Krzysztof

    Controlled/living radical polymerizations (CRP) give access to polymers with precisely controlled molecular weight, narrow molecular weight distribution, well-defined architecture and composition. CRP can be applied to a wide range of monomers and are tolerant to impurities and functional groups. Atom transfer radical polymerization (ATRP) is one of the most widely used CRP techniques. The mechanistic and kinetic studies of ATRP are of fundamental importance since they give access to polymers with various functionalities, compositions, and topologies.

  6. Kinetics and Mechanism in the Oxidation of Metal Vapors

    DTIC Science & Technology

    1974-10-01

    cooled induction coil enters the furnace chamber from the bottom flange. The crucible and its holder are supported on three tantalum rods on the inlet...In an indium furnace , heated by the same induction field as the crucible containing the thorium. When thorium was allowed to react with partially...identify by block number) Kinetics Reproduced by Uranium NATtONAL TECHNICALThorium INFORMATIOtM SERVICE US Depermeht of Cm’,ner’e Springfield, VA. 22151

  7. Kinetics and mechanism of the chlorine dioxide-trithionate reaction.

    PubMed

    Cseko, György; Horváth, Attila K

    2012-03-22

    The trithionate-chlorine dioxide reaction has been studied spectrophotometrically in a slightly acidic medium at 25.0 ± 0.1 °C in acetate/acetic acid buffer monitoring the decay of chlorine dioxide at constant ionic strength (I = 0.5 M) adjusted by sodium perchlorate. We found that under our experimental conditions two limiting stoichiometries exist and the pH, the concentration of the reactants, and even the concentration of chloride ion affects the actual stoichiometry of the reaction that can be augmented by an appropriate linear combination of these limiting processes. It is also shown that although the formal kinetic order of trithionate is strictly one that of chlorine dioxide varies between 1 and 2, depending on the actual chlorine dioxide excess and the pH. Moreover, the otherwise sluggish chloride ion, which is also a product of the reaction, slightly accelerates the initial rate of chlorine dioxide consumption and may therefore act as an autocatalyst. In addition to that, overshoot-undershoot behavior is also observed in the [(·)ClO(2)]-time curves in the presence of chloride ion at chlorine dioxide excess. On the basis of the experiments, a 13-step kinetic model with 6 fitted kinetic parameter is proposed by nonlinear parameter estimation.

  8. Mechanisms of kinetic trapping in self-assembly and phase transformation

    PubMed Central

    Hagan, Michael F.; Elrad, Oren M.; Jack, Robert L.

    2011-01-01

    In self-assembly processes, kinetic trapping effects often hinder the formation of thermodynamically stable ordered states. In a model of viral capsid assembly and in the phase transformation of a lattice gas, we show how simulations in a self-assembling steady state can be used to identify two distinct mechanisms of kinetic trapping. We argue that one of these mechanisms can be adequately captured by kinetic rate equations, while the other involves a breakdown of theories that rely on cluster size as a reaction coordinate. We discuss how these observations might be useful in designing and optimising self-assembly reactions. PMID:21932884

  9. The functioning neuronal transporter for dopamine: kinetic mechanisms and effects of amphetamines, cocaine and methylphenidate.

    PubMed

    Schenk, James O

    2002-01-01

    The dopamine transporter (DAT) is a transmembrane spanning protein that catalyzes the transport of dopamine across the neuronal membrane to concentrate the neurotransmitter inside the cell. Although the uptake of dopamine has been studied since the 1960s, more recent advances in knowledge of the protein itself and in making kinetically resolved measurements of its action have led to more insights into its mechanism and pharmacology. The literature of the kinetics of transporters and kinetic measurements of DAT activity is reviewed to provide an overview of the multisubstrate mechanism of DAT activity, its pharmacology with regard to amphetamine, cocaine and methylphenidate, and correlations of DAT activity with some behavioral outputs.

  10. Kinetics and mechanism of the synthesis of a novel protein-based plastic using subcritical water.

    PubMed

    Abdelmoez, Wael; Yoshida, Hiroyuki

    2008-01-01

    We investigated the intermolecular mechanism and kinetics of the synthesis of a novel biodegradable protein-based plastic from bovine serum albumin under subcritical water conditions using batch reactors. The reaction mechanism could be viewed as a chain reaction stabilized by the formation of intermolecular disulfide bonds. The kinetic analysis was based on non-steady-state kinetics using a theoretical model developed in one of our previous works. The activation energy and pre-exponential factor were found to be 7.2 kJ/mol and 0.9 s-1, respectively. These low values signify that the reaction is relatively temperature-insensitive with some diffusion limitation.

  11. Folding Beauties

    ERIC Educational Resources Information Center

    Berman, Leah Wrenn

    2006-01-01

    This article has its genesis in an MAA mini-course on origami, where a way to get a parabola by folding paper was presented. This article discusses the methods and mathematics of other curves obtained by paper-folding.

  12. Folding Beauties

    ERIC Educational Resources Information Center

    Berman, Leah Wrenn

    2006-01-01

    This article has its genesis in an MAA mini-course on origami, where a way to get a parabola by folding paper was presented. This article discusses the methods and mathematics of other curves obtained by paper-folding.

  13. The effects of pK(a) tuning on the thermodynamics and kinetics of folding: design of a solvent-shielded carboxylate pair at the a-position of a coiled-coil.

    PubMed

    Lau, Wai Leung; Degrado, William F; Roder, Heinrich

    2010-10-06

    The tuning of the pK(a) of ionizable residues plays a critical role in various protein functions, such as ligand-binding, catalysis, and allostery. Proteins harness the free energy of folding to position ionizable groups in highly specific environments that strongly affect their pK(a) values. To investigate the interplay among protein folding kinetics, thermodynamics, and pK(a) modulation, we introduced a pair of Asp residues at neighboring interior positions of a coiled-coil. A single Asp residue was replaced for an Asn side chain at the a-position of the coiled-coil from GCN4, which was also crosslinked at the C-terminus via a flexible disulfide bond. The thermodynamic and kinetic stability of the system was measured by circular dichroism and stopped-flow fluorescence as a function of pH and concentration of guanidine HCl. Both sets of data are consistent with a two-state equilibrium between fully folded and unfolded forms. Distinct pK(a) values of 6.3 and 5.35 are assigned to the first and second protonation of the Asp pair; together they represent an energetic difference of 5 kcal/mol relative to the protonation of two Asp residues with unperturbed pK(a) values. Analysis of the rate data as a function of pH and denaturant concentration allowed calculation of the kinetic constants for the conformational transitions of the peptide with the Asp residues in the doubly protonated, singly protonated, and unprotonated forms. The doubly and singly protonated forms fold rapidly, and a ϕ-value analysis shows that their contribution to folding occurs subsequent to the transition state ensemble for folding. By contrast, the doubly charged state shows a reduced rate of folding and a ϕ-value near 0.5 indicative of a repulsive interaction, and possibly also heterogeneity in the transition state ensemble.

  14. How the folding rates of two- and multistate proteins depend on the amino acid properties.

    PubMed

    Huang, Jitao T; Huang, Wei; Huang, Shanran R; Li, Xin

    2014-10-01

    Proteins fold by either two-state or multistate kinetic mechanism. We observe that amino acids play different roles in different mechanism. Many residues that are easy to form regular secondary structures (α helices, β sheets and turns) can promote the two-state folding reactions of small proteins. Most of hydrophilic residues can speed up the multistate folding reactions of large proteins. Folding rates of large proteins are equally responsive to the flexibility of partial amino acids. Other properties of amino acids (including volume, polarity, accessible surface, exposure degree, isoelectric point, and phase transfer energy) have contributed little to folding kinetics of the proteins. Cysteine is a special residue, it triggers two-state folding reaction and but inhibits multistate folding reaction. These findings not only provide a new insight into protein structure prediction, but also could be used to direct the point mutations that can change folding rate. © 2014 Wiley Periodicals, Inc.

  15. Detailed Chemical Kinetic Reaction Mechanisms for Incineration of Organophosphorus and Fluoro-Organophosphorus Compounds

    SciTech Connect

    Glaude, P A; Melius, C; Pitz, W J; Westbrook, C K

    2001-12-13

    A detailed chemical kinetic reaction mechanism is developed to describe incineration of the chemical warfare nerve agent sarin (GB), based on commonly used principles of bond additivity and hierarchical reaction mechanisms. The mechanism is based on previous kinetic models of organophosphorus compounds such as TMP, DMMP and DIMP that are often used as surrogates to predict incineration of GB. Kinetic models of the three surrogates and GB are then used to predict their consumption in a perfectly stirred reactor fueled by natural gas to simulate incineration of these chemicals. Computed results indicate that DIMP is the only one of these surrogates that adequately describes combustion of GB under comparable conditions. The kinetic pathways responsible for these differences in reactivity are identified and discussed. The most important reaction in GB and DIMP that makes them more reactive than TMP or DMMP is found to be a six-center molecular elimination reaction producing propene.

  16. Fracture mechanics at a large scale: Splay-fault origin for the Yakima fold-and-thrust belt, Washington State

    NASA Astrophysics Data System (ADS)

    Pratt, T. L.

    2011-12-01

    The Yakima fold-and-thrust belt (YFTB) is a series of ridges formed by reverse faults cutting the extensive Miocene Columbia River Basalts (CRB) of central Washington State. The YFTB is bisected by the ~1100-km-long Olympic-Wallowa geomorphic lineament (OWL). There is considerable debate about the origin and earthquake potential of the YFTB and OWL, which lie in a region with six major dams and the largest nuclear waste storage site in the United States. The YFTB structures have been compared to "wrinkle ridges" formed by contraction during cooling on other planets and have been interpreted as a simple fold-and-thrust belt formed under N-S compression. The YFTB ridges are cored by faults whose depth of penetration has been debated, with a "thick-skin" model in which the faults penetrate to basement rocks being more recently favored over a "thin-skin" model in which the faults extend only to the base of the ~4-km-thick basalt flows. The OWL may be a major strike-slip fault zone, but lacks definitive evidence for or against substantial horizontal offset. Here I argue that the YFTB are splay faults formed by an abrupt lateral decrease in the amount of strike-slip motion along the OWL. The argument is based on the remarkable similarity between the trends of the YFTB in relation to the OWL, to trends of splay faults predicted by Linear Elastic Fracture Mechanics (LEFM) at the end of a vertical strike-slip fault. The YFTB also lacks a root zone for a fold-and-thrust belt, and instead seems to be rooted along the OWL. The comparison with the LEFM model implies that the YFTB is a spectacular example of splay faults in a large "damage zone" near the tip of a strike-slip fault. The OWL and the associated YFTB should be deeply-rooted structures in this model, which is confirmed by a key seismic reflection profile across one of the YFTB structures that indeed shows folding of strata well below the CRB.

  17. Solution growth kinetics and mechanism: Prismatic face of ADP

    NASA Astrophysics Data System (ADS)

    Chernov, A. A.; Rashkovich, L. N.; Mkrtchan, A. A.

    1986-01-01

    Laser Michelson interferometry has been applied to in situ study the (001) ADP growth kinetics in aqueous solution in the kinetic regime. The technique allows one to simultaneously measure the slope p of a growth hillock and normal growth rate R provided by this hillock. From these data, the average step growth rate v=R/p has been determined as a function of relative supersaturation σ. The dependencev(σ) is found to be linear, demonstrating the unimportance of surface and bulk diffusion. The direct incorporation at steps is characterized by the step kinetic coefficient βl=(5.1-6.4)X10-3 cm/s. The specific step free energy αl=(1.2-1.9) X10-6 erg/cm was determined from the measured linear dependence of the hillock slope on supersaturation for the hillock around presumably single elementary dislocation. For complex dislocation sources with large total Burgers vectors, the tendency to saturationin the hillock slope-supersaturation curves has been found. The curve perfectly fits the BCF expression which takes into account the perimeter 2L of the region occupied by the points in which the dislocation of the complex step source cross the growing face. For two dislocation sources,L=0.92 μm andL=0.31 μm and total Burgers vectors ⋍12h and 6h (h=7.53Å) have been found. The supersaturation dependence of activities for various complex dislocation sources have been directly demonstrated.

  18. Kinetics and Mechanism of Electron Transfer in Proteins

    NASA Astrophysics Data System (ADS)

    Kulys, J.

    1986-10-01

    The results of studies on the kinetics of the oxidation-reduction reactions of individual proteins (electron transfer agents and enzymes) are described. Attention has been concentrated on the effect of the nature of the active centres in the protein molecules and of the modification of individual aminoacid residues on the rate of electron transfer in a homogeneous medium. Questions associated with the electrochemical reactions of proteins and with the effect of the state of the interface on the rate of this process are considered in detail. Ideas concerning the theoretical calculation of the rate constants for electron transfer in proteins are described. The bibliography includes 154 references.

  19. Extreme Folding

    NASA Astrophysics Data System (ADS)

    Demaine, Erik

    2012-02-01

    Our understanding of the mathematics and algorithms behind paper folding, and geometric folding in general, has increased dramatically over the past several years. These developments have found a surprisingly broad range of applications. In the art of origami, it has helped spur the technical origami revolution. In engineering and science, it has helped solve problems in areas such as manufacturing, robotics, graphics, and protein folding. On the recreational side, it has led to new kinds of folding puzzles and magic. I will give an overview of the mathematics and algorithms of folding, with a focus on new mathematics and sculpture.

  20. Protein folding by distributed computing and the denatured state ensemble.

    PubMed

    Marianayagam, Neelan J; Fawzi, Nicolas L; Head-Gordon, Teresa

    2005-11-15

    The distributed computing (DC) paradigm in conjunction with the folding@home (FH) client server has been used to study the folding kinetics of small peptides and proteins, giving excellent agreement with experimentally measured folding rates, although pathways sampled in these simulations are not always consistent with the folding mechanism. In this study, we use a coarse-grain model of protein L, whose two-state kinetics have been characterized in detail by using long-time equilibrium simulations, to rigorously test a FH protocol using approximately 10,000 short-time, uncoupled folding simulations starting from an extended state of the protein. We show that the FH results give non-Poisson distributions and early folding events that are unphysical, whereas longer folding events experience a correct barrier to folding but are not representative of the equilibrium folding ensemble. Using short-time, uncoupled folding simulations started from an equilibrated denatured state ensemble (DSE), we also do not get agreement with the equilibrium two-state kinetics because of overrepresented folding events arising from higher energy subpopulations in the DSE. The DC approach using uncoupled short trajectories can make contact with traditionally measured experimental rates and folding mechanism when starting from an equilibrated DSE, when the simulation time is long enough to sample the lowest energy states of the unfolded basin and the simulated free-energy surface is correct. However, the DC paradigm, together with faster time-resolved and single-molecule experiments, can also reveal the breakdown in the two-state approximation due to observation of folding events from higher energy subpopulations in the DSE.

  1. Impact of kinetic beds on the incidence of atelectasis in mechanically ventilated patients.

    PubMed

    Chandy, Dipak; Sahityani, Rachna; Aronow, Wilbert S; Khan, Safdar; DeLorenzo, Lawrence J

    2007-01-01

    We investigated the impact of kinetic beds on the incidence of atelectasis in mechanically ventilated patients in an intensive care unit (ICU). All bronchoscopies performed for atelectasis on mechanically ventilated patients between July 2000 and June 2001 and between July 2002 and June 2003 were reviewed. On July 26, 2001, 50 kinetic beds, 20 continuous lateral rotation therapy modules, and 20 percussion and vibration modules were introduced to our institution. Of the 3399 ICU admissions between July 2000 and June 2001, 71 patients developed atelectasis while being mechanically ventilated. Of the 3065 ICU admissions between July 2002 and June 2003, 83 patients developed atelectasis while being mechanically ventilated. Of these, 48 (58%) patients had left-sided atelectasis, 30 (36%) had right-sided atelectasis, and 5 (6%) had bilateral atelectasis. There was no decrease in the incidence of atelectasis in mechanically ventilated patients at our institution after the introduction of kinetic beds and vibration, percussion, and rotation modules despite their widespread availability.

  2. A study of the molecular mechanism of binding kinetics and long residence times of human CCR5 receptor small molecule allosteric ligands

    PubMed Central

    Swinney, David C; Beavis, Paul; Chuang, Kai-Ting; Zheng, Yue; Lee, Ina; Gee, Peter; Deval, Jerome; Rotstein, David M; Dioszegi, Marianna; Ravendran, Palani; Zhang, Jun; Sankuratri, Surya; Kondru, Rama; Vauquelin, Georges

    2014-01-01

    BACKGROUND AND PURPOSE The human CCR5 receptor is a co-receptor for HIV-1 infection and a target for anti-viral therapy. A greater understanding of the binding kinetics of small molecule allosteric ligand interactions with CCR5 will lead to a better understanding of the binding process and may help discover new molecules that avoid resistance. EXPERIMENTAL APPROACH Using [3H] maraviroc as a radioligand, a number of different binding protocols were employed in conjunction with simulations to determine rate constants, kinetic mechanism and mutant kinetic fingerprints for wild-type and mutant human CCR5 with maraviroc, aplaviroc and vicriviroc. KEY RESULTS Kinetic characterization of maraviroc binding to the wild-type CCR5 was consistent with a two-step kinetic mechanism that involved an initial receptor–ligand complex (RA), which transitioned to a more stable complex, R'A, with at least a 13-fold increase in affinity. The dissociation rate from R'A, k−2, was 1.2 × 10−3 min−1. The maraviroc time-dependent transition was influenced by F85L, W86A, Y108A, I198A and Y251A mutations of CCR5. CONCLUSIONS AND IMPLICATIONS The interaction between maraviroc and CCR5 proceeded according to a multi-step kinetic mechanism, whereby initial mass action binding and later reorganizations of the initial maraviroc–receptor complex lead to a complex with longer residence time. Site-directed mutagenesis identified a kinetic fingerprint of residues that affected the binding kinetics, leading to the conclusion that allosteric ligand binding to CCR5 involved the rearrangement of the binding site in a manner specific to each allosteric ligand. PMID:24628038

  3. The effect of surface tethering on the folding of the src-SH3 protein domain

    NASA Astrophysics Data System (ADS)

    Zhuang, Zhuoyun; Jewett, Andrew I.; Soto, Patricia; Shea, Joan-Emma

    2009-03-01

    The effect of surface tethering on the folding mechanism of the src-SH3 protein domain was investigated using a coarse-grained Gō-type protein model. The protein was tethered at various locations along the protein chain and the thermodynamics and kinetics of folding were studied using replica exchange and constant temperature Langevin dynamics. Our simulations reveal that tethering in a structured part of the transition state can dramatically alter the folding mechanism, while tethering in an unstructured part leaves the folding mechanism unaltered as compared to bulk folding. Interestingly, there is only modest correlation between the tethering effect on the folding mechanism and its effect on thermodynamic stability and folding rates. We suggest locations on the protein at which tethering could be performed in single-molecule experiments so as to leave the folding mechanism unaltered from the bulk.

  4. Kinetic Ballooning Instability as a Substorm Onset Mechanism

    SciTech Connect

    C.Z.Cheng

    1999-10-01

    A new scenario of substorm onset and current disruption and the corresponding physical processes are presented based on the AMPTE/CCE spacecraft observation and a kinetic ballooning instability theory. During the growth phase of substorms the plasma beta is larger than unity (20 greater than or equal to beta greater than or equal to 1). Toward the end of the late growth phase the plasma beta increases from 20 to greater than or equal to 50 in approximately 3 minutes and a low-frequency instability with a wave period of 50 - 75 sec is excited and grows exponentially to a large amplitude at the current disruption onset. At the onset, higher-frequency instabilities are excited so that the plasma and electromagnetic field form a turbulent state. Plasma transport takes place to modify the ambient pressure profile so that the ambient magnetic field recovers from a tail-like geometry to a dipole-like geometry. A kinetic ballooning instability (KBI) theory is proposed to explain the low-frequency instability (frequency and growth rate) and its observed high beta threshold (beta subscript c is greater than or equal to 50). Based on the ideal-MHD theory beta subscript c, superscript MHD approximately equals 1 and the ballooning modes are predicted to be unstable during the growth phase, which is inconsistent with observation that no appreciable magnetic field fluctuation is observed. The enhancement beta subscript c over beta subscript c, superscript MHD is due to the kinetic effects of trapped electrons and finite ion-Larmor radii which provide a large stabilizing effect by producing a large parallel electric field and hence a parallel current that greatly enhances the stabilizing effect of field line tension. As a result, beta subscript c is greatly increased over beta subscript c, superscript MHD by a factor proportional to the ratio of the total electron density to the untrapped electron density (n subscript e divided by n subscript eu) which is greater than or equal to

  5. Transformation of the mechanism of triple-helix peptide folding in the absence of a C-terminal nucleation domain and its implications for mutations in collagen disorders.

    PubMed

    Buevich, Alexei V; Silva, Teresita; Brodsky, Barbara; Baum, Jean

    2004-11-05

    Folding abnormalities of the triple helix have been demonstrated in collagen diseases such as osteogenesis imperfecta in which the mutation leads to the substitution of a single Gly in the (Gly-X-Y)n sequence pattern by a larger residue. Model peptides can be used to clarify the details of normal collagen folding and the consequences of the interruption of that folding by a Gly substitution. NMR and CD studies show that placement of a (GPO)4 nucleation domain at the N terminus rather than the C terminus of a native collagen sequence allows the formation of a stable triple helix but alters the folding mechanism. Although C- to N-terminal directional folding occurs when the nucleation domain is at the C terminus, there is no preferential folding direction when the nucleation domain is at the N terminus. The lack of zipper-like directional folding does not interfere with triple-helix formation, and when a Gly residue is replaced by Ser to model an osteogenesis imperfecta mutation, the peptide with the N-terminal (GPO)4 domain can still form a good triple helix N-terminal to the mutation site. These peptide studies raise the possibility that mutant collagen could fold in a C to N direction in a zipper-like manner up to the mutation site and that completion of the triple helix N-terminal to the mutation would involve an alternative mechanism.

  6. Kinetics and mechanism of HO2 and DO2 disproportionations

    NASA Technical Reports Server (NTRS)

    Kircher, C. C.; Sander, S. P.

    1984-01-01

    A flash photolysis/UV abosrption technique was used to study the HO2 + HO2 and DO2 + DO2 reactions in the gas phase. Rate constants were measured at pressures between 100 and 700 torr of Ar and N2, and at temperatures between 230 and 420 K with up to 10 torr of added water vapor. The overall disproportionation rate constants for the reaction is given as the sum of pressure-independent and pressure-dependent terms. A kinetic analysis shows that both reactions have a zero-pressure bimolecular component and a termolecular component which is linearly dependent on pressure up to 700 torr. A priori estimates of the vibrational frequencies of the product of the HO2 + HO2 reaction suggest binding energies of 12-20 kcal per mol (for the initial association).

  7. Solid State Kinetic Parameters and Chemical Mechanism of the Dehydration of CoCl2.6H2O.

    ERIC Educational Resources Information Center

    Ribas, Joan; And Others

    1988-01-01

    Presents an experimental example illustrating the most common methods for the determination of kinetic parameters. Discusses the different theories and equations to be applied and the mechanism derived from the kinetic results. (CW)

  8. Solid State Kinetic Parameters and Chemical Mechanism of the Dehydration of CoCl2.6H2O.

    ERIC Educational Resources Information Center

    Ribas, Joan; And Others

    1988-01-01

    Presents an experimental example illustrating the most common methods for the determination of kinetic parameters. Discusses the different theories and equations to be applied and the mechanism derived from the kinetic results. (CW)

  9. Mechanics of the Salt Range-Potwar Plateau, Pakistan: A fold-and-thrust belt underlain by evaporites

    NASA Astrophysics Data System (ADS)

    Jaumé, Steven C.; Lillie, Robert J.

    1988-02-01

    The Salt Range and Potwar Plateau are part of the active foreland fold-and-thrust belt of the Himalaya in northern Pakistan. In this region the distance from the Main Boundary Thrust (MBT) to the front of the fold-and-thrust belt is very wide (100-150 km) because a thick evaporite sequence forms the zone of décollement. Recent studies have combined seismic reflection profiles, petroleum exploration wells, Bouguer gravity anomalies, and surface geology to construct cross sections in the eastern, central, and western Salt Range-Potwar Plateau areas. In this study the sections are compared with a model that considers the mechanics of a fold-and-thrust belt to be analogous to that of a wedge of snow or soil pushed in front of a bulldozer (Chapple, 1978; Davis et al., 1983; Dahlen et al., 1984; Dahlen, 1984). Models which include the effects of evaporites at the base (Chapple, 1978; Davis and Engelder, 1985) suggest that these thrust belts will have (1) narrow (< 1°) cross-sectional tapers, (2) larger widths than areas not underlain by evaporites, (3) symmetrical structures, and (4) changes in deformational style at the edge of the evaporite basin. The section across the eastern Potwar Plateau most closely resembles this latter model, having (1) a taper of 0.8° ± 0.1°, (2) a width of 100-150 km, (3) thrust faults that verge both to the north and south, and (4) structures rotated 30° counterclockwise with respect to the Salt Range. From the observed taper and pore fluid pressures of the eastern Potwar Plateau, estimates of the values for the yield strength of the evaporites (τo) and the coefficient of internal friction of the overlying wedge (μ) are calculated as τo = 1.33-1.50 MPa and μ = 0.95-1.04, which are then applied to the other cross sections. In the central and western sections a basement uplift, the Sargodha High, interferes with the front of the fold-and-thrust belt. This feature causes the ramping of the Salt Range Thrust and produces a relatively

  10. Remote toehold: a mechanism for flexible control of DNA hybridization kinetics.

    PubMed

    Genot, Anthony J; Zhang, David Yu; Bath, Jonathan; Turberfield, Andrew J

    2011-02-23

    Hybridization of DNA strands can be used to build molecular devices, and control of the kinetics of DNA hybridization is a crucial element in the design and construction of functional and autonomous devices. Toehold-mediated strand displacement has proved to be a powerful mechanism that allows programmable control of DNA hybridization. So far, attempts to control hybridization kinetics have mainly focused on the length and binding strength of toehold sequences. Here we show that insertion of a spacer between the toehold and displacement domains provides additional control: modulation of the nature and length of the spacer can be used to control strand-displacement rates over at least 3 orders of magnitude. We apply this mechanism to operate displacement reactions in potentially useful kinetic regimes: the kinetic proofreading and concentration-robust regimes.

  11. Kinetic analysis of a Michaelis-Menten mechanism in which the enzyme is unstable.

    PubMed Central

    Garrido-del Solo, C; García-Cánovas, F; Havsteen, B H; Varón-Castellanos, R

    1993-01-01

    A kinetic analysis of the Michaelis-Menten mechanism is made for the cases in which the free enzyme, or the enzyme-substrate complex, or both, are unstable, either spontaneously or as a result of the addition of a reagent. The explicit time-course equations of all of the species involved has been derived under conditions of limiting enzyme concentration. The validity of these equations has been checked by using numerical simulations. An experimental design and a kinetic data analysis allowing the evaluation of the parameters and kinetic constants are recommended. PMID:8373361

  12. Reduced and Validated Kinetic Mechanisms for Hydrogen-CO-sir Combustion in Gas Turbines

    SciTech Connect

    Yiguang Ju; Frederick Dryer

    2009-02-07

    Rigorous experimental, theoretical, and numerical investigation of various issues relevant to the development of reduced, validated kinetic mechanisms for synthetic gas combustion in gas turbines was carried out - including the construction of new radiation models for combusting flows, improvement of flame speed measurement techniques, measurements and chemical kinetic analysis of H{sub 2}/CO/CO{sub 2}/O{sub 2}/diluent mixtures, revision of the H{sub 2}/O{sub 2} kinetic model to improve flame speed prediction capabilities, and development of a multi-time scale algorithm to improve computational efficiency in reacting flow simulations.

  13. Transition-path sampling of -hairpin folding

    NASA Astrophysics Data System (ADS)

    Bolhuis, Peter G.

    2003-10-01

    We examine the dynamical folding pathways of the C-terminal -hairpin of protein G-B1 in explicit solvent at room temperature by means of a transition-path sampling algorithm. In agreement with previous free-energy calculations, the resulting path ensembles reveal a folding mechanism in which the hydrophobic residues collapse first followed by backbone hydrogen-bond formation, starting with the hydrogen bonds inside the hydrophobic core. In addition, the path ensembles contain information on the folding kinetics, including solvent motion. Using the recently developed transition interface sampling technique, we calculate the rate constant for unfolding of the protein fragment and find it to be in reasonable agreement with experiments. The results support the validation of using all-atom force fields to study protein folding.

  14. [Changes in the kinetics of quanta secretion-effective mechanism of synaptic transmission modulation].

    PubMed

    Bukharaeva, É A; Nikol'skiĭ, E E

    2010-08-01

    It is widely accepted that the leading presynaptic mechanisms underlying the synaptic plasticity involve changes of the number of neurotransmitter quanta released by one nerve pulse (the quantal content of postsynaptic response) and of the size of a single quantum. In addition, the existence of one more effective though previously ignored mechanism of modulation of synaptic plasticity was suggested related to the change in the time course (kinetics) of secretion of single neurotransmitter quanta forming the multiquantal response. This article reviews current data (including the authors' own results) on the kinetics of evoked neurotransmitter quanta secretion from motor nerve endings in peripheral synapses, mechanisms of their modulation and methods of quantitative analysis.

  15. Biofilm Formation Mechanisms of Pseudomonas aeruginosa Predicted via Genome-Scale Kinetic Models of Bacterial Metabolism

    DTIC Science & Technology

    2016-03-15

    RESEARCH ARTICLE Biofilm Formation Mechanisms of Pseudomonas aeruginosa Predicted via Genome-Scale Kinetic Models of Bacterial Metabolism Francisco G...we addressed questions regarding biofilm metabolism using a genome-scale kinetic model of the P. aeruginosametabolic network and gene expression...synthesis pathway, but also through the biofilm-specific expression of genes in pathways competing for precursors to these molecules. Finally, we

  16. Kinetic studies of the mechanism of direct chlorination of methane in the presence of porous fillers

    SciTech Connect

    Aglulin, A.G.; Bakshi, Yu.M.; Gel'bshtein, A.I.

    1983-05-01

    The kinetics of methane chlorination were studied in the gas phase and in the presence of various kinds of porous fillers. The rate of initiation by chlorine atoms on the filler surface was estimated, the activation energy of the heterogeneous dissociation of chlorine was calculated, and an initiation mechanism was proposed. It was concluded that homogeneous chain scission predominates. A kinetic equation that corresponds to the experimental data was obtained. 3 figures, 2 tables.

  17. Protein Folding: Then and Now

    PubMed Central

    Chen, Yiwen; Ding, Feng; Nie, Huifen; Serohijos, Adrian W.; Sharma, Shantanu; Wilcox, Kyle C.; Yin, Shuangye; Dokholyan, Nikolay V.

    2007-01-01

    Over the past three decades the protein folding field has undergone monumental changes. Originally a purely academic question, how a protein folds has now become vital in understanding diseases and our abilities to rationally manipulate cellular life by engineering protein folding pathways. We review and contrast past and recent developments in the protein folding field. Specifically, we discuss the progress in our understanding of protein folding thermodynamics and kinetics, the properties of evasive intermediates, and unfolded states. We also discuss how some abnormalities in protein folding lead to protein aggregation and human diseases. PMID:17585870

  18. Exact results in nonequilibrium statistical mechanics: Formalism and applications in chemical kinetics and single-molecule free energy estimation

    NASA Astrophysics Data System (ADS)

    Adib, Artur B.

    In the last two decades or so, a collection of results in nonequilibrium statistical mechanics that departs from the traditional near-equilibrium framework introduced by Lars Onsager in 1931 has been derived, yielding new fundamental insights into far-from-equilibrium processes in general. Apart from offering a more quantitative statement of the second law of thermodynamics, some of these results---typified by the so-called "Jarzynski equality"---have also offered novel means of estimating equilibrium quantities from nonequilibrium processes, such as free energy differences from single-molecule "pulling" experiments. This thesis contributes to such efforts by offering three novel results in nonequilibrium statistical mechanics: (a) The entropic analog of the Jarzynski equality; (b) A methodology for estimating free energies from "clamp-and-release" nonequilibrium processes; and (c) A directly measurable symmetry relation in chemical kinetics similar to (but more general than) chemical detailed balance. These results share in common the feature of remaining valid outside Onsager's near-equilibrium regime, and bear direct applicability in protein folding kinetics as well as in single-molecule free energy estimation.

  19. Kinetic mechanism of L-α-glycerophosphate oxidase from Mycoplasma pneumoniae.

    PubMed

    Maenpuen, Somchart; Watthaisong, Pratchaya; Supon, Pacharee; Sucharitakul, Jeerus; Parsonage, Derek; Karplus, P Andrew; Claiborne, Al; Chaiyen, Pimchai

    2015-08-01

    L-α-glycerophosphate oxidase is an FAD-dependent enzyme that catalyzes the oxidation of L-α-glycerophosphate (Glp) by molecular oxygen to generate dihydroxyacetone phosphate (DHAP) and hydrogen peroxide (H2O2). The catalytic properties of recombinant His6-GlpO from Mycoplasma pneumoniae (His6-MpGlpO) were investigated through transient and steady-state kinetics and ligand binding studies. The results indicate that the reaction mechanism of His6-MpGlpO follows a ping-pong model. Double-mixing mode stopped-flow experiments show that, after flavin-mediated substrate oxidation, DHAP leaves rapidly prior to the oxygen reaction. The values determined for the individual rate constants and kcat (4.2 s(-1) at 4 °C), in addition to the finding that H2 O2 binds to the oxidized enzyme, suggest that H2O2 release is the rate-limiting step for the overall reaction. The results indicate that His6 -MpGlpO contains mixed populations of fast- and slow-reacting species. It is predominantly the fast-reacting species that participates in turnover. In contrast to other GlpO enzymes previously described, His6-MpGlpO is able to catalyze the reverse reaction of reduced enzyme and DHAP. This result may be explained by the standard reduction potential value of His6-MpGlpO (-167 ± 1 mV), which is lower than those of GlpO from other species. We found that D,L-glyceraldehyde 3-phosphate (GAP) may be used as a substrate in the His6-MpGlpO reaction, although it exhibited an approximately 100-fold lower kcat value in comparison with the reaction of Glp. These results also imply involvement of GlpO in glycolysis, as well as in lipid and glycerol metabolism. The kinetic models and distinctive properties of His6-MpGlpO reported here should be useful for future drug development against Mycoplasma pneumoniae infection.

  20. Increase of ion kinetic temperature across a collisionless shock. I - A new mechanism

    NASA Technical Reports Server (NTRS)

    Lee, L. C.; Wu, C. S.; Hu, X. W.

    1986-01-01

    A simple but effective mechanism is proposed to account for the increase of ion kinetic temperature across an oblique or perpendicular shock. This mechanism is based on the nonadiabatic motion of the transmitted ions across the shock ramp, which can lead to an ion gyrophase-bunching behind the shock.

  1. Study of the kinetics and mechanism of heterogeneous catalytic reactions by the relaxation method

    SciTech Connect

    Bal`zhinimaev, B.S.; Pinaeva, L.G.

    1995-01-01

    The main results of studying reaction mechanisms by relaxation methods are presented for SO{sub 2} oxidation, o-xylene oxidation, and ethylene epoxidation. It is shown that reaction mechanisms can be efficiently investigated, and qualitative correlations between the characteristics of catalytic activity and the composition and structure of the adsorption layer can be found by combining kinetic methods with physical methods.

  2. Microsecond subdomain folding in dihydrofolate reductase.

    PubMed

    Arai, Munehito; Iwakura, Masahiro; Matthews, C Robert; Bilsel, Osman

    2011-07-08

    The characterization of microsecond dynamics in the folding of multisubdomain proteins has been a major challenge in understanding their often complex folding mechanisms. Using a continuous-flow mixing device coupled with fluorescence lifetime detection, we report the microsecond folding dynamics of dihydrofolate reductase (DHFR), a two-subdomain α/β/α sandwich protein known to begin folding in this time range. The global dimensions of early intermediates were monitored by Förster resonance energy transfer, and the dynamic properties of the local Trp environments were monitored by fluorescence lifetime detection. We found that substantial collapse occurs in both the locally connected adenosine binding subdomain and the discontinuous loop subdomain within 35 μs of initiation of folding from the urea unfolded state. During the fastest observable ∼550 μs phase, the discontinuous loop subdomain further contracts, concomitant with the burial of Trp residue(s), as both subdomains achieve a similar degree of compactness. Taken together with previous studies in the millisecond time range, a hierarchical assembly of DHFR--in which each subdomain independently folds, subsequently docks, and then anneals into the native conformation after an initial heterogeneous global collapse--emerges. The progressive acquisition of structure, beginning with a continuously connected subdomain and spreading to distal regions, shows that chain entropy is a significant organizing principle in the folding of multisubdomain proteins and single-domain proteins. Subdomain folding also provides a rationale for the complex kinetics often observed.

  3. Pleistocene northward fold propagation of the Jura within the southern URG: Insight from the coupling of mechanical and geomorphological modelling.

    NASA Astrophysics Data System (ADS)

    Cornu, T.; Cartier, S.; Niviere, B.; Winter, T.

    2003-04-01

    On the basis of geophysical, geomorphological and geological data, the modelling addresses the Plio-Pleistocene northward propagation of the Jura fold and thrust belt and its impact on the geomorphology. The structural domain comprises the area up to Mulhouse proceeding from a succession of four ramps (from north to south: Ferrette, Muespach, Magstatt and Rixheim) rooted within the late triassic, acting as a decollement. The mechanical modelling addresses the tectonics evolution of the domain and focuses onto the fault reactivation and its link with the triassic decollement level. This model accounts for 3D elastic deformation of the first kilometers in depth. The geomorphological modelling addresses the landscape evolution according to the tectonic movements, predicted with the mechanical modelling, and highlights the capture of the hydrographic network. Coupling between erosion and tectonic movements are examined through simple erosion power laws depending on local gradient and water discharge. The combination of the two approaches provides different scenarii for the faults reactivation history and the influence of the tectonics onto the observed large scale morphological features such as river profiles, river captures or diversion.

  4. Effect of experimental and sample factors on dehydration kinetics of mildronate dihydrate: mechanism of dehydration and determination of kinetic parameters.

    PubMed

    Bērziņš, Agris; Actiņš, Andris

    2014-06-01

    The dehydration kinetics of mildronate dihydrate [3-(1,1,1-trimethylhydrazin-1-ium-2-yl)propionate dihydrate] was analyzed in isothermal and nonisothermal modes. The particle size, sample preparation and storage, sample weight, nitrogen flow rate, relative humidity, and sample history were varied in order to evaluate the effect of these factors and to more accurately interpret the data obtained from such analysis. It was determined that comparable kinetic parameters can be obtained in both isothermal and nonisothermal mode. However, dehydration activation energy values obtained in nonisothermal mode showed variation with conversion degree because of different rate-limiting step energy at higher temperature. Moreover, carrying out experiments in this mode required consideration of additional experimental complications. Our study of the different sample and experimental factor effect revealed information about changes of the dehydration rate-limiting step energy, variable contribution from different rate limiting steps, as well as clarified the dehydration mechanism. Procedures for convenient and fast determination of dehydration kinetic parameters were offered.

  5. The Kinetic Chain Revisited: New Concepts on Throwing Mechanics and Injury.

    PubMed

    Chu, Samuel K; Jayabalan, Prakash; Kibler, W Ben; Press, Joel

    2016-03-01

    The overhead throwing motion is a complex activity that is achieved through activation of the kinetic chain. The kinetic chain refers to the linkage of multiple segments of the body that allows for transfer of forces and motion. The lower extremities and core provide a base of support, generating energy that is transferred eventually through the throwing arm and hand, resulting in release of the ball. The kinetic chain requires optimal anatomy, physiology, and mechanics and is involved in all 6 phases of overhead throwing: windup, stride, arm cocking, acceleration, deceleration, and follow-through. Breaks or deficits in the kinetic chain can lead to injury or decreased performance. Through an understanding of the mechanics and pathomechanics seen in each phase of throwing, the clinician can better evaluate and screen for potential kinetic chain deficits in the overhead throwing athlete. The purpose of this article is to review the biomechanics of the overhead throwing motion, the role of the kinetic chain in throwing, and the clinical evaluation and management of abnormal throwing mechanics and related injuries.

  6. Kinetics and mechanism of dimethoate chlorination during drinking water treatment.

    PubMed

    Tian, Fang; Liu, Wenjun; Guo, Guang; Qiang, Zhimin; Zhang, Can

    2014-05-01

    Dimethoate (DMT), a commonly used organophosphorus pesticide, is of great concern because of its toxicity and potentially harmful effects on water sources. The elimination of DMT as well as the toxicity and persistence of the byproducts formed during DMT degradation is most important for the safety of drinking water. This study first determined the reaction kinetics of DMT with free chlorine (FC) under typical water treatment conditions. The reaction between DMT and FC proceeded rapidly, exhibiting first-order with respect to each reactant. The degradation of DMT by FC was highly pH dependent, and the pseudo-first-order rate constant decreased obviously from 0.13 to 0.02 s(-1) with an increase in pH from 7.0 to 8.3. Bromide ion accelerated the reaction by acting as a catalyst, and the accelerated reaction rate was linearly proportional to the bromide concentration. As a ubiquitous component in natural waters, humic acid also increased the reaction rate. However, the presence of ammonium inhibited the degradation of DMT due to its rapid converting FC to chloramines. Omethoate (OMT) was identified as an important byproduct of DMT chlorination, but only accounted for ca. 28% of the DMT degraded; and other two organic byproducts were also identified. The acute toxicity of DMT solution increased after treatment with FC due to the formation of more toxic byproducts (e.g. OMT). Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Mild hydrolysis of 2-trifluoromethylphenol: kinetics, mechanism and environmental relevance.

    PubMed

    Reinscheid, Uwe M; Vervoort, Jacques; Zuilhof, Han

    2006-10-01

    2-Trifluoromethylphenol was hydrolysed in a phosphate buffer at neutral pH. At mild temperatures ranging from 34 degrees C to 69 degrees C this compound liberates consecutively fluorine anions to form salicylic acid. This process is energetically driven by the hydration of the fluorine anions. No intermediates have been detected by HPLC and (19)F-NMR and this was confirmed by computer calculations which favor the first step in the whole reaction sequence being rate-limiting. Accordingly, the reaction energy of the first dehalogenation of the trifluoromethyl anion is 28.4 kcal mol(-1) higher than for the second dehalogenation. The pseudo-first-order kinetic was determined and from an Arrhenius diagram an activation energy of E(a)=25.1 kcal mol(-1) has been estimated. At 37 degrees C and a pH of 7.4 the half-life was 6.9 h. The rate of hydrolysis was favored at higher pH and it was not influenced by oxygen, sunlight or trace elements found in natural water. The latter was shown by incubations with lake water instead of distilled water.

  8. Kinetics and mechanism of Dionaea muscipula trap closing.

    PubMed

    Volkov, Alexander G; Adesina, Tejumade; Markin, Vladislav S; Jovanov, Emil

    2008-02-01

    The Venus flytrap (Dionaea muscipula) possesses an active trapping mechanism to capture insects with one of the most rapid movements in the plant kingdom, as described by Darwin. This article presents a detailed experimental investigation of trap closure by mechanical and electrical stimuli and the mechanism of this process. Trap closure consists of three distinctive phases: a silent phase with no observable movement; an accelerated movement of the lobes; and the relaxation of the lobes in their closed state, resulting in a new equilibrium. Uncouplers and blockers of membrane channels were used to investigate the mechanisms of different phases of closing. Uncouplers increased trap closure delay and significantly decreased the speed of trap closure. Ion channel blockers and aquaporin inhibitors increased time of closing. Transmission of a single electrical charge between a lobe and the midrib causes closure of the trap and induces an electrical signal propagating between both lobes and midrib. The Venus flytrap can accumulate small subthreshold charges, and when the threshold value is reached, the trap closes. Repeated application of smaller charges demonstrates the summation of stimuli. The cumulative character of electrical stimuli points to the existence of electrical memory in the Venus flytrap. The observed fast movement can be explained by the hydroelastic curvature model without invoking buckling instability. The new hydroelastic curvature mechanism provides an accurate description of the authors' experimental data.

  9. Transtensional folding

    NASA Astrophysics Data System (ADS)

    Fossen, Haakon; Teyssier, Christian; Whitney, Donna L.

    2014-05-01

    For now three decades transpression has dominated the concepts that underlie oblique tectonics, but in more recent years transtension has garnered much interest as a simple model that can be applied to shallow and deep crustal tectonics. One fundamental aspect that distinguishes transtension from transpression is that material lines in transtension rotate toward the direction of oblique divergence. Another point that may be less intuitive when thinking of transtension is that while transtensional strain involves shortening in the vertical direction, one of the horizontal axes is also a shortening axis, whatever the angle of divergence. It is the combination of these two shortening axes that leads to constrictional finite strain in transtension. The existence of a horizontal shortening strain axis implies that transtension offers the potential for folds of horizontal layers to form and then rotate toward the direction of oblique divergence. An investigation of transtensional folding using 3D strain modeling reveals that folding is more likely for simple shear dominated transtension (large wrench component). Transtensional folds can only accumulate a fixed amount of horizontal shortening and tightness that are prescribed by the angle of oblique divergence, regardless of finite strain. Transtensional folds are characterized by hinge-parallel stretching that exceeds that expected from pure wrenching. In addition, the magnitude of hinge-parallel stretching always exceeds hinge-perpendicular shortening, causing constrictional fabrics and hinge-parallel boudinage to develop. Because the dominant vertical strain axis is shortening, transtensional fold growth is generally suppressed, but when folds do develop their limbs enter the field of shortening, resulting in possible fold interference patterns akin to cascading folds. Application of these transtensional folding principles to regions of oblique rifting (i.e. Gulf of California) or exhumation of deep crust (i.e. Western

  10. Kinetic-Dominated Charging Mechanism within Representative Aqueous Electrolyte-based Electric Double-Layer Capacitors.

    PubMed

    Yang, Huachao; Yang, Jinyuan; Bo, Zheng; Chen, Xia; Shuai, Xiaorui; Kong, Jing; Yan, Jianhua; Cen, Kefa

    2017-08-03

    The chemical nature of electrolytes has been demonstrated to play a pivotal role in the charge storage of electric double-layer capacitors (EDLCs), whereas primary mechanisms are still partially resolved but controversial. In this work, a systematic exploration into EDL structures and kinetics of representative aqueous electrolytes is performed with numerical simulation and experimental research. Unusually, a novel charging mechanism exclusively predominated by kinetics is recognized, going beyond traditional views of manipulating capacitances preferentially via interfacial structural variations. Specifically, strikingly distinctive EDL structures stimulated by diverse ion sizes, valences, and mixtures manifest a virtually identical EDL capacitance, where the dielectric nature of solvents attenuates ionic effects on electrolyte redistributions, in stark contradiction with solvent-free counterpart and traditional Helmholtz theory. Meanwhile, corresponding kinetics evolve conspicuously with ionic species, intimately correlated with ion-solvent interactions. The achieved mechanisms are subsequently illuminated by electrochemical measurements, highlighting the crucial interplay between ions and solvents in regulating EDLC performances.

  11. Suitable combination of promoter and micellar catalyst for kilo fold rate acceleration on benzaldehyde to benzoic acid conversion in aqueous media at room temperature: a kinetic approach.

    PubMed

    Ghosh, Aniruddha; Saha, Rumpa; Ghosh, Sumanta K; Mukherjee, Kakali; Saha, Bidyut

    2013-05-15

    The kinetics of oxidation of benzaldehyde by chromic acid in aqueous and aqueous surfactant (sodium dodecyl sulfate, SDS, alkyl phenyl polyethylene glycol, Triton X-100 and N-cetylpyridinium chloride, CPC) media have been investigated in the presence of promoter at 303 K. The pseudo-first-order rate constants (kobs) were determined from a logarithmic plot of absorbance as a function time. The rate constants were found to increase with introduction of heteroaromatic nitrogen base promoters such as Picolinic acid (PA), 2,2'-bipyridine (bipy) and 1,10-phenanthroline (phen). The product benzoic acid has been characterized by conventional melting point experiment, NMR, HRMS and FTIR spectral analysis. The mechanism of both unpromoted and promoted reaction path has been proposed for the reaction. In presence of the anionic surfactant SDS, cationic surfactant CPC and neutral surfactant TX-100 the reaction can undergo simultaneously in both aqueous and micellar phase with an enhanced rate of oxidation in the micellar phase. Both SDS and TX-100 produce normal micellar effect whereas CPC produce reverse micellar effect in the presence of benzaldehyde. The observed net enhancement of rate effects has been explained by considering the hydrophobic and electrostatic interaction between the surfactants and reactants. SDS and bipy combination is the suitable one for benzaldehyde oxidation. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Students' Misconceptions about Heat Transfer Mechanisms and Elementary Kinetic Theory

    ERIC Educational Resources Information Center

    Pathare, S. R.; Pradhan, H. C.

    2010-01-01

    Heat and thermodynamics is a conceptually rich area of undergraduate physics. In the Indian context in particular there has been little work done in this area from the point of view of misconceptions. This prompted us to undertake a study in this area. We present a study of students' misconceptions about heat transfer mechanisms, i.e. conduction,…

  13. [Mechanism of injury--trauma kinetics. What happend? How?].

    PubMed

    Beuran, M; Negoi, I; Paun, S; Runcanu, A; Gaspar, B

    2012-01-01

    Understanding the mechanism of injuries represents a key element in blunt and penetrating trauma management. Systematic review of the main types of the modem trauma mechanisms, using Medline, Cochrane Library and Embase databases. To properly understand the road car accident injuries, trauma surgeon should know as many details from the scene: the speed of cars, impact direction, if the car rolled over, if occupants were restrained, if airbags exploded, vehicle telemetry, extrication time. Motorcyclists are 20 to 30 times more at risk for severe injuries or death than the four-wheel vehicle occupants. Current evidence shows a significant decrease in injuries severity by increasing use of seat-belts, motorcycle helmets, childrestrains and speed limit. Despite this, few countries around the world have road safety laws relating to key factors that can be considered sufficiently comprehensive in scope. Many modern trauma systems use for prehospital triage mechanism of injury criteria. The trauma surgeon should know the mechanism of injury. This allows a high suspicion for potential injuries, their early diagnosis and increased quality in the care of trauma patients.

  14. Students' Misconceptions about Heat Transfer Mechanisms and Elementary Kinetic Theory

    ERIC Educational Resources Information Center

    Pathare, S. R.; Pradhan, H. C.

    2010-01-01

    Heat and thermodynamics is a conceptually rich area of undergraduate physics. In the Indian context in particular there has been little work done in this area from the point of view of misconceptions. This prompted us to undertake a study in this area. We present a study of students' misconceptions about heat transfer mechanisms, i.e. conduction,…

  15. Development of a surrogate kinetic mechanism for photochemical smog. Final report

    SciTech Connect

    Leone, J.A.; Seinfeld, J.H.

    1985-01-01

    A surrogate condensed chemical reaction mechanism for photochemical smog containing the latest available kinetic and mechanistic data is developed here and is extensively tested against experimental data from two smog-chamber facilities. In addition, a counter species analysis has shown that the prediction of the individual portions of the surrogate mechanism are in good agreement with those of a detailed explicit mechanism for photochemical smog. The new mechanism contains 20 non-steady-state species, making it compact enough for use in multidimensional grid models of urban air pollution. A major advantage of the new mechanism is the ease with which it can be modified to incorporate new kinetic or mechanistic information because of the straightforward manner in which the mechanism was formulated from the detailed explicit chemistry.

  16. Kinetic mechanism for the excision of hypoxanthine by Escherichia coli AlkA and evidence for binding to DNA ends.

    PubMed

    Zhao, Boyang; O'Brien, Patrick J

    2011-05-24

    The Escherichia coli 3-methyladenine DNA glycosylase II protein (AlkA) recognizes a broad range of oxidized and alkylated base lesions and catalyzes the hydrolysis of the N-glycosidic bond to initiate the base excision repair pathway. Although the enzyme was one of the first DNA repair glycosylases to be discovered more than 25 years ago and there are multiple crystal structures, the mechanism is poorly understood. Therefore, we have characterized the kinetic mechanism for the AlkA-catalyzed excision of the deaminated purine, hypoxanthine. The multiple-turnover glycosylase assays are consistent with Michaelis-Menten kinetics. However, under single-turnover conditions that are commonly employed for studying other DNA glycosylases, we observe an unusual biphasic protein saturation curve. Initially, the observed rate constant for excision increases with an increasing level of AlkA protein, but at higher protein concentrations, the rate constant decreases. This behavior can be most easily explained by tight binding to DNA ends and by crowding of multiple AlkA protamers on the DNA. Consistent with this model, crystal structures have shown the preferential binding of AlkA to DNA ends. By varying the position of the lesion, we identified an asymmetric substrate that does not show inhibition at higher concentrations of AlkA, and we performed pre-steady state and steady state kinetic analysis. Unlike the situation in other glycosylases, release of the abasic product is faster than N-glycosidic bond cleavage. Nevertheless, AlkA exhibits significant product inhibition under multiple-turnover conditions, and it binds approximately 10-fold more tightly to an abasic site than to a hypoxanthine lesion site. This tight binding could help protect abasic sites when the adaptive response to DNA alkylation is activated and very high levels of AlkA protein are present.

  17. Folding mechanisms of individual beta-hairpins in a Go model of Pin1 WW domain by all-atom molecular dynamics simulations.

    PubMed

    Luo, Zhonglin; Ding, Jiandong; Zhou, Yaoqi

    2008-06-14

    This paper examines the folding mechanism of an individual beta-hairpin in the presence of other hairpins by using an off-lattice model of a small triple-stranded antiparallel beta-sheet protein, Pin1 WW domain. The turn zipper model and the hydrophobic collapse model originally developed for a single beta-hairpin in literature is confirmed to be useful in describing beta-hairpins in model Pin1 WW domain. We find that the mechanism for folding a specific hairpin is independent of whether it folds first or second, but the formation process are significantly dependent on temperature. More specifically, beta1-beta2 hairpin folds via the turn zipper model at a low temperature and the hydrophobic collapse model at a high temperature, while the folding of beta2-beta3 hairpin follows the turn zipper model at both temperatures. The change in folding mechanisms is interpreted by the interplay between contact stability (enthalpy) and loop lengths (entropy), the effect of which is temperature dependent.

  18. Chemical Kinetics Mechanism Reduction Based on Principal Component Analysis: Development and Testing of Some New Implementations

    DTIC Science & Technology

    2013-05-01

    prediction of propulsion system performance. In addition, programs employed in this study for screening the merit of reduced mechanisms were...development of system -specific gas-phase finite-rate chemical kinetics mechanisms is a significant part of these efforts (Anderson et al., 2010; Chen and...employed to model other combustion systems . The final step involves producing a “reduced” (or skeletal) mechanism from the detailed/full one

  19. Reaction Mechanism and Kinetics of Enargite Oxidation at Roasting Temperatures

    NASA Astrophysics Data System (ADS)

    Padilla, Rafael; Aracena, Alvaro; Ruiz, Maria C.

    2012-10-01

    Roasting of enargite (Cu3AsS4) in the temperature range of 648 K to 898 K (375 °C to 625 °C) in atmospheres containing variable amounts of oxygen has been studied by thermogravimetric methods. From the experimental results of weight loss/gain data and X-ray diffraction (XRD) analysis of partially reacted samples, the reaction mechanism of the enargite oxidation was determined, which occurred in three sequential stages:

  20. Remaining uncertainties in the kinetic mechanism of hydrogen combustion

    SciTech Connect

    Konnov, Alexander A.

    2008-03-15

    An analysis of the performance of an updated hydrogen combustion mechanism is presented. Particular attention was paid to different channels of reaction between H atoms and HO{sub 2} radicals, to pressure dependence of the recombination of HO{sub 2} radicals, and to the anomalous rate constant of reaction between OH and HO{sub 2} radicals. The contemporary choice of the reaction rate constants is presented with the emphasis on their uncertainties. Then the predictions of ignition, oxidation, flame burning velocities, and flame structure of hydrogen-oxygen-inert mixtures are shown. The modeling range covers ignition experiments from 950 to 2700 K and from subatmospheric pressures up to 87 atm; hydrogen oxidation in a flow reactor at temperatures around 900 K from 0.3 up to 15.7 atm; flame burning velocities in hydrogen-oxygen-inert mixtures from 0.35 up to 4 atm; and hydrogen flame structure at 1 and 10 atm. Comparison of the modeling and experiments is discussed in terms of the range of applicability of the present detailed mechanism. The necessity for analysis of the mechanism to have an exhaustive list of reactions is emphasized. (author)

  1. Effects of cohesion on the structural and mechanical evolution of fold and thrust belts and contractional wedges: Discrete element simulations

    NASA Astrophysics Data System (ADS)

    Morgan, Julia K.

    2015-05-01

    Particle-based numerical simulations of cohesive contractional wedges can yield important perspectives on the formation and evolution of fold and thrust belts, offering particular insights into the mechanical evolution of the systems. Results of several discrete element method simulations are presented here, demonstrating the stress and strain evolution of systems with different initial cohesive strengths. Particle assemblages consolidated under gravity, and bonded to impart cohesion, are pushed from the left at a constant velocity above a weak, unbonded décollement surface. Internal thrusting causes horizontal shortening and vertical thickening, forming wedge geometries. The mean wedge taper is similar for all simulations, consistent with their similar residual and basal sliding friction values. In all examples presented here, both forethrusts and back thrusts occur, but forethrusts accommodate most of the shortening. Fault spacing and offset increase with increasing cohesion. Significant tectonic volume strain also occurs, with the greatest incremental volume strain occurring just outboard of the deformation front. This diffuse shortening serves to strengthen the unfaulted domain in front of the deformed wedge, preconditioning these materials for brittle (dilative) failure. The reach of this volumetric strain and extent of décollement slip increase with cohesive strength, defining the extent of stress transmission. Stress paths for elements tracked through the simulations demonstrate systematic variations in shear stress in response to episodes of both décollement slip and thrust fault activity, providing a direct explanation for stress fluctuations during convergence.

  2. Microstructures, deformation mechanisms and strain patterns in a vertical profile, inner appalachian fold-thrust belt, Alabama

    NASA Astrophysics Data System (ADS)

    Wu, Schuman

    1993-02-01

    A core from the American Anniston No. 1 well, drilled in the Pell City thrust sheet, Calhoun County, Alabama, provides an excellent scientific opportunity to study in a vertical profile the deformation mechanisms and strain patterns associated with large-scale structures. The core contains stratigraphic units from Cambrian to Mississippian in age and two major thrust faults. A detailed structural analysis revealed two deformation episodes, a pre-orogenic deformation and Alleghanian orogenic deformation. In the pre-orogenic deformation, synsedimentary folds and growth faults are the characteristic early structures, and normal faults formed in lithifield rocks later. During Alleghanian deformation, earlier fractures are overprinted by later S1 penetrative structures. In limestone, shale and siltstone within thrust sheets, S1 is solution cleavage. Dolomites in the thrust sheets were deformed by fracturing, and no penetrative cleavages formed. In major fault zones, S1 mylonitic foliation formed in limestone, shale and siltstone. Fault-related dolomites were deformed cataclastically and no S surfaces formed. Strain magnitude increases towards major thrust faults in both the hanging walls and footwalls. For the typical fault configuration in the core with limestone-dolomite in the hanging wall and shale-siltstone in the footwall, strain is mainly caused by pressure solution and cataclasis in the hanging wall and by plastic deformation in the footwall.

  3. Interplay among tertiary contacts, secondary structure formation and side-chain packing in the protein folding mechanism: all-atom representation study of protein L.

    PubMed

    Clementi, Cecilia; García, Angel E; Onuchic, José N

    2003-02-21

    Experimental and theoretical results suggest that, since proteins are energetically minimally frustrated, the native fold, or topology, plays a primary role in determining the structure of the transition state ensemble and on-pathway intermediate states in protein folding. Although the central role of native state topology in determining the folding mechanism is thought to be a quite general result-at least for small two-state folding proteins-there are remarkable exceptions. Recent experimental findings have shown that topology alone cannot always determine the folding mechanism, and demonstrated that the balance between topology and energetics is very delicate. This balance seems to be particularly critical in proteins with a highly symmetrical native structure, such as proteins L and G, which have similar native structure topology but fold by different mechanisms. Simplified, C(alpha)-atom only protein models have shown not be sufficient to differentiate these mechanisms. An all-atom Gō model provides a valuable intermediate model between structurally simplified protein representations and all-atom protein simulations with explicit/implicit solvent descriptions. We present here a detailed study of an all-atom Gō-like representation of protein L, in close comparison with the experimental results and with the results obtained from a simple C(alpha)-atom representation of the same protein. We also perform simulations for protein G, where we obtain a folding mechanism in which the protein symmetry is broken exactly in the opposite way to protein L as has been observed experimentally. A detailed analysis for protein L also shows that the role of specific residues is correctly and quantitatively reproduced by the all-atom Gō model over almost the entire protein.

  4. Characterization of unfolding mechanism of human lamin A Ig fold by single-molecule force spectroscopy-implications in EDMD.

    PubMed

    Bera, Manindra; Kotamarthi, Hema Chandra; Dutta, Subarna; Ray, Angana; Ghosh, Saptaparni; Bhattacharyya, Dhananjay; Ainavarapu, Sri Rama Koti; Sengupta, Kaushik

    2014-11-25

    A- and B-type lamins are intermediate filament proteins constituting the nuclear lamina underneath the nuclear envelope thereby conferring proper shape and mechanical rigidity to the nucleus. Lamin proteins are also shown to be related diversely to basic nuclear processes. More than 400 mutations in human lamin A protein alone have been reported to produce at least 11 different disease conditions jointly termed as laminopathies. These mutations in lamin A are scattered throughout its helical rod domain, as well as the C-terminal domain containing the conserved Ig-fold region. The commonality of phenotypes in all these diseases is characterized by misshapen nuclei of the affected tissues which might stem from altered rigidity of the supporting lamina hence lamins. Here we have focused on autosomal dominant Emery-Dreifuss Muscular Dystrophy, one such laminopathy where R453W is the causative mutation located in the Ig domain of lamin A. We have investigated by single-molecule force spectroscopy how a stretching mechanical perturbation senses the destabilizing effect of the mutation in the lamin A Ig domain and compared the mechanoelastic properties of the mutant R453W with that of the wild-type in conjunction with steered molecular dynamics. Furthermore, we have shown the interaction of Ig domain with emerin, another key player and interacting partner in the pathogenesis of EDMD, is disrupted in the R453W mutant. This altered mechanoresistance of Ig domain itself and consequent uncoupling of lamin A-emerin interaction might underlie the altered mechanotransduction properties of EDMD affected nuclei.

  5. Mechanism and kinetics of COS-induced diethanolamine degradation

    SciTech Connect

    Dawodu, O.F.; Meisen, A. . Dept. of Chemical Engineering)

    1994-03-01

    The degradation of aqueous diethanolamine (DEA) solutions by carbonyl sulfide was examined by using a 600-mL well-stirred reactor operating under the following conditions: DEA concentration 20--40 wt %, temperature 120--180 C, COS partial pressure 0.3--1.17 MPa. The reaction products were identified by GC/MS, and reaction mechanisms are developed which conform with experimental observations. The reaction rate constants are determined, and a mathematical model for estimating DEA degradation by COS is presented.

  6. Kinetics and mechanism of dye adsorption on WO3 nanoparticles

    NASA Astrophysics Data System (ADS)

    Adhikari, Sangeeta; Mandal, Sandip; Sarkar, Debasish; Kim, Do-Heyoung; Madras, Giridhar

    2017-10-01

    Monoclinic WO3 nanoparticles were synthesized by a simple acid catalyzed co-precipitation reaction. Spherical particles with average size ∼55 nm were confirmed from electron microscopy followed by functional, structural and optical characterizations. The adsorption of methylene blue was examined by using WO3 nanoparticles and the capacity was higher than most of the reported studies. The effect of pH and material loading on adsorption was determined. The mechanism of adsorption was examined by XPS and a detailed explanation of surface phenomena was proposed. Regeneration study was carried and a high stability of heat treated WO3 towards adsorption of methylene blue was observed.

  7. Kinetic method for the large-scale analysis of the binding mechanism of histone deacetylase inhibitors.

    PubMed

    Meyners, Christian; Baud, Matthias G J; Fuchter, Matthew J; Meyer-Almes, Franz-Josef

    2014-09-01

    Performing kinetic studies on protein ligand interactions provides important information on complex formation and dissociation. Beside kinetic parameters such as association rates and residence times, kinetic experiments also reveal insights into reaction mechanisms. Exploiting intrinsic tryptophan fluorescence a parallelized high-throughput Förster resonance energy transfer (FRET)-based reporter displacement assay with very low protein consumption was developed to enable the large-scale kinetic characterization of the binding of ligands to recombinant human histone deacetylases (HDACs) and a bacterial histone deacetylase-like amidohydrolase (HDAH) from Bordetella/Alcaligenes. For the binding of trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), and two other SAHA derivatives to HDAH, two different modes of action, simple one-step binding and a two-step mechanism comprising initial binding and induced fit, were verified. In contrast to HDAH, all compounds bound to human HDAC1, HDAC6, and HDAC8 through a two-step mechanism. A quantitative view on the inhibitor-HDAC systems revealed two types of interaction, fast binding and slow dissociation. We provide arguments for the thesis that the relationship between quantitative kinetic and mechanistic information and chemical structures of compounds will serve as a valuable tool for drug optimization. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Shape, Thermodynamics, Kinetics and Growth Mechanisms of Metal and Bimetallic Nanoparticles

    NASA Astrophysics Data System (ADS)

    Peng, Lingxuan

    Metal and bimetallic nanoparticles are of interest and are widely used in various applications because of their unique optical, electronic, and catalytic properties, which differ from those of their bulk counterparts. Better understanding of the thermodynamic and kinetic properties of nanoparticles and their underlying growth mechanisms can serve as a basis for improving reproducibility and rational design of nanoparticle syntheses. The primary objective of this dissertation was to study the structural-related thermodynamic and kinetic properties of nanoparticles via the combination of experimental and theoretical techniques and to further unravel their underlying growth mechanisms. In this dissertation, the structure and elemental distribution of colloidally-synthesized bimetallic nanoparticles were characterized via scanning/transmission electron microscopy (S/TEM) and energy dispersive X-ray spectroscopy (EDX). In colloidally-synthesized bimetallic Pt/Pd nanoparticles, smooth composition gradients from the particle centers to their surfaces and corner enrichment of Pt were observed experimentally. A growth model was developed to demonstrate that the smooth composition gradients within the particles were the result of the difference in the deposition rate constants of Pd and Pt, causing Pd to deposit faster than Pt. The deposition rate constant ratio between Pd and Pt increased with total Pd and Pt precursor concentration. The corner Pt enrichment was a result of local thermodynamic control at the corners. At the nanoparticle corner, a Lyapunov stable solution could be achieved when the chemical potential at the corner equals the external chemical potential in the solution. This stable solution leads to size-independent corner rounding in colloidal synthesized nanoparticles. Strain-induced segregation in bimetallic multiply twinned particles, namely decahedral (Dh) and icosahedral (Ic) particles, was analyzed by an analytic first-order expansion within a

  9. Kinetics and mechanism of photopromoted oxidative dissolution of antimony trioxide.

    PubMed

    Hu, Xingyun; Kong, Linghao; He, Mengchang

    2014-12-16

    Light (sunlight, ultraviolet, simulated sunlight) irradiation was used to initiate the dissolution of antimony trioxide (Sb2O3). Dissolution rate of Sb2O3 was accelerated and dissolved trivalent antimony (Sb(III)) was oxidized in the irradiation of light. The photopromoted oxidative dissolution mechanism of Sb2O3 was studied through experiments investigating the effects of pH, free radicals scavengers, dissolved oxygen removal and Sb2O3 dosage on the release rate of antimony from Sb2O3 under simulated sunlight irradiation. The key oxidative components were hydroxyl free radicals, photogenerated holes and superoxide free radicals; their contribution ratios were roughly estimated. In addition, a conceptual model of the photocatalytic oxidation dissolution of Sb2O3 was proposed. The overall pH-dependent dissolution rate of Sb2O3 and the oxidation of Sb(III) under light irradiation were expressed by r = 0.08 ·[OH(-)](0.63) and rox = 0.10 ·[OH(-)](0.79). The present study on the mechanism of the photo-oxidation dissolution of Sb2O3 could help clarify the geochemical cycle and fate of Sb in the environment.

  10. Photochemistry of dipenylketyl radicals: spectroscopy, kinetics, and mechanisms

    SciTech Connect

    Johnston, L.J.; Lougnot, D.J.; Wintgens, V.; Scaiano, J.C.

    1988-01-20

    The photochemistry of the diphenylketyl radical has been examined in nonpolar solutions. Transient studies using two-laser techniques yield an excited-state lifetime of 3.9 ns in toluene at room temperature, while for diphenylketyl-O-d the lifetime is 8.7 ns. Dye laser irradiation (515 nm) in the ketyl's visible absorption band leads to efficient photobleaching with Phi/sub bleach/ = 0.27 +/- 0.06 for the parent radical and 0.39 and 0.26 for the 4-methyl and 4-chloro derivatives, respectively. The photobleaching reaction involves the cleavage of the O-H ketyl bond to yield benzophenone and hydrogen atoms; in cyclohexane the latter abstract hydrogen from the solvent to produce molecular hydrogen which was characterized by Raman spectroscopy. In accordance with this mechanism, two-laser experiments produce lower yields of photoreduction products than the one-laser experiments in which the ketyls are not photobleached. When the ketyl radicals are generated by reaction of tert-butoxy radicals with benzhydrol, dye laser irradiation leads to a large increase in the yield of benzophenone (now a product), although the mechanism here is somewhat more complex due to the quenching of excited ketyl radicals by di-tert-butyl peroxide (k/sub q/ = 1.9 x 10/sup 9/ M/sup -1/ s/sup -1/). Detailed studies of the fluorescence, isotope effects, temperature effects, and products are also included.

  11. Pleural liquid and kinetic friction coefficient of mesothelium after mechanical ventilation.

    PubMed

    Bodega, Francesca; Sironi, Chiara; Porta, Cristina; Zocchi, Luciano; Agostoni, Emilio

    2015-01-15

    Volume and protein concentration of pleural liquid in anesthetized rabbits after 1 or 3h of mechanical ventilation, with alveolar pressure equal to atmospheric at end expiration, were compared to those occurring after spontaneous breathing. Moreover, coefficient of kinetic friction between samples of visceral and parietal pleura, obtained after spontaneous or mechanical ventilation, sliding in vitro at physiological velocity under physiological load, was determined. Volume of pleural liquid after mechanical ventilation was similar to that previously found during spontaneous ventilation. This finding is contrary to expectation of Moriondo et al. (2005), based on measurement of lymphatic and interstitial pressure. Protein concentration of pleural liquid after mechanical ventilation was also similar to that occurring after spontaneous ventilation. Coefficient of kinetic friction after mechanical ventilation was 0.023±0.001, similar to that obtained after spontaneous breathing.

  12. Reduced kinetic mechanism of ignition for nonpremixed hydrogen/air in a supersonic mixing layer

    SciTech Connect

    Ju, Y.; Niioka, T. . Inst. of Fluid Science)

    1994-11-01

    Transient ignition processes in a two-dimensional spatially evolving supersonic mixing layer consisting of a parallel nonpremixed airstream and a hydrogen stream both with temperatures higher than 1,000 K were investigated numerically by using the full chemistry and its reduced chemistry. A phenomenon different from that examined in previous studies, in which ignition of hydrogen/oxygen mixtures was considered, was found in the nonpremixed case examined here. It was shown that the concentration of O was greater than that of OH before ignition, but became smaller with the development of ignition process. Fourteen important reactions for ignition were obtained and verified using sensitivity analyses of ignition delay time and radical concentrations. Several different four-step and three-step reduced kinetic mechanisms were then deduced by introducing the steady-state approximation to different species. Comparison of these reduced kinetic mechanisms with the full chemistry showed that the steady-state approximation of O used in previous studies caused serious errors in the prediction of ignition delay time in supersonic flow, in which nonpremixed character is predominant and the transport phenomenon is important. Ignition locations predicted with the proper four-step and three-step reduced kinetic mechanisms were within 5% and 20% of those predicted with the full chemistry. Finally, these two reduced mechanisms were used to evaluate the effect of viscous dissipation on ignition in the supersonic shear layer. Good agreements between the results of the present reduced kinetic mechanisms and those of the full chemistry were obtained.

  13. The kinetics and mechanism of catstrophic oxidation of metals

    SciTech Connect

    Belousov, V.V.

    1994-12-01

    A set of independent methods has been used to study the catastrophic oxidation of copper in the system Cu-Me{sub x}O{sub y} (where Me is Bi, W, Mo, or V). Two stages of the catastrophic oxidation have been revealed: a rapid stage (K {approximately} 10{sup -4} kg{sup 2} m{sub -4} sec{sup -1}) and a {open_quotes}super rapid{close_quotes} stage when the metal is oxidized within 1-5 sec. The weight ratios of metal to oxidizer and the partial oxygen pressure for the superrapid copper oxidation have been established. The mechanism of the catastrophic oxidation of metals is considered.

  14. Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR.

    PubMed

    Uzawa, Takanori; Nishimura, Chiaki; Akiyama, Shuji; Ishimori, Koichiro; Takahashi, Satoshi; Dyson, H Jane; Wright, Peter E

    2008-09-16

    The earliest steps in the folding of proteins are complete on an extremely rapid time scale that is difficult to access experimentally. We have used rapid-mixing quench-flow methods to extend the time resolution of folding studies on apomyoglobin and elucidate the structural and dynamic features of members of the ensemble of intermediate states that are populated on a submillisecond time scale during this process. The picture that emerges is of a continuum of rapidly interconverting states. Even after only 0.4 ms of refolding time a compact state is formed that contains major parts of the A, G, and H helices, which are sufficiently well folded to protect amides from exchange. The B, C, and E helix regions fold more slowly and fluctuate rapidly between open and closed states as they search docking sites on this core; the secondary structure in these regions becomes stabilized as the refolding time is increased from 0.4 to 6 ms. No further stabilization occurs in the A, G, H core at 6 ms of folding time. These studies begin to time-resolve a progression of compact states between the fully unfolded and native folded states and confirm the presence an ensemble of intermediates that interconvert in a hierarchical sequence as the protein searches conformational space on its folding trajectory.

  15. G2 chromatid aberrations: Kinetics and possible mechanisms

    SciTech Connect

    Bryant, P.E.; Slijepcevic, P. )

    1993-01-01

    Chromatid breaks and exchanges are induced by radiation in G2 mammalian cells. Breaks are at a maximum number at about 30 min after irradiation and decrease apparently exponentially with time between irradiation and sampling. Few breaks are observed immediately following exposure, probably as a result of selection of mitotic cells where chromosomes are condensed and there is consequently a lack of time for expression of damage. The change in frequency of breaks with time, from 30 min after radiation exposure and onwards, can be interpreted in two possible ways: either in terms of a repair process or in terms of a change in radiosensitivity through G2. However, the results with an inhibitor of repair of DNA double-strand breaks (ara A) and with [open quotes]transient hypothermia[close quotes] which extends the G2 phase, argue for an interpretation based on rejoining of chromatid breaks, possibly reflecting the repair of a subclass of dsb. Data from experiments with irradiated and restriction endonuclease treated radiosensitive mutant rodent lines indicate that enhanced levels of conversion of dsb into chromosomal aberrations may be largely independent of repair rates of bulk dsb. In CHO cells and in human lymphocytes exchanges initially increase rapidly with time and then remain at a constant frequency, supporting the notion of a uniform chromosomal radiosensitivity throughout most of G2 and providing further evidence that the mechanism for misjoining broken chromatids (leading to exchanges) is different from that for rejoining of chromatid breaks. Ratios of breaks to exchanges were found to vary in different cell lines and at different times during treatment with inhibitors or at altered temperatures, possibly (in different cell lines) indicating different levels of enzymes involved in misjoining, but suggesting that the mechanisms of chromosomal rejoining and misjoining are independent, at least to some degree. 19 refs., 11 figs., 1 tab.

  16. GroEL-mediated protein folding.

    PubMed Central

    Fenton, W. A.; Horwich, A. L.

    1997-01-01

    I. Architecture of GroEL and GroES and the reaction pathway A. Architecture of the chaperonins B. Reaction pathway of GroEL-GroES-mediated folding II. Polypeptide binding A. A parallel network of chaperones binding polypeptides in vivo B. Polypeptide binding in vitro 1. Role of hydrophobicity in recognition 2. Homologous proteins with differing recognition-differences in primary structure versus effects on folding pathway 3. Conformations recognized by GroEL a. Refolding studies b. Binding of metastable intermediates c. Conformations while stably bound at GroEL 4. Binding constants and rates of association 5. Conformational changes in the substrate protein associated with binding by GroEL a. Observations b. Kinetic versus thermodynamic action of GroEL in mediating unfolding c. Crossing the energy landscape in the presence of GroEL III. ATP binding and hydrolysis-driving the reaction cycle IV. GroEL-GroES-polypeptide ternary complexes-the folding-active cis complex A. Cis and trans ternary complexes B. Symmetric complexes C. The folding-active intermediate of a chaperonin reaction-cis ternary complex D. The role of the cis space in the folding reaction E. Folding governed by a "timer" mechanism F. Release of nonnative polypeptides during the GroEL-GroES reaction G. Release of both native and nonnative forms under physiologic conditions H. A role for ATP binding, as well as hydrolysis, in the folding cycle V. Concluding remarks. PMID:9098884

  17. Molecular mechanisms of protein aggregation from global fitting of kinetic models.

    PubMed

    Meisl, Georg; Kirkegaard, Julius B; Arosio, Paolo; Michaels, Thomas C T; Vendruscolo, Michele; Dobson, Christopher M; Linse, Sara; Knowles, Tuomas P J

    2016-02-01

    The elucidation of the molecular mechanisms by which soluble proteins convert into their amyloid forms is a fundamental prerequisite for understanding and controlling disorders that are linked to protein aggregation, such as Alzheimer's and Parkinson's diseases. However, because of the complexity associated with aggregation reaction networks, the analysis of kinetic data of protein aggregation to obtain the underlying mechanisms represents a complex task. Here we describe a framework, using quantitative kinetic assays and global fitting, to determine and to verify a molecular mechanism for aggregation reactions that is compatible with experimental kinetic data. We implement this approach in a web-based software, AmyloFit. Our procedure starts from the results of kinetic experiments that measure the concentration of aggregate mass as a function of time. We illustrate the approach with results from the aggregation of the β-amyloid (Aβ) peptides measured using thioflavin T, but the method is suitable for data from any similar kinetic experiment measuring the accumulation of aggregate mass as a function of time; the input data are in the form of a tab-separated text file. We also outline general experimental strategies and practical considerations for obtaining kinetic data of sufficient quality to draw detailed mechanistic conclusions, and the procedure starts with instructions for extensive data quality control. For the core part of the analysis, we provide an online platform (http://www.amylofit.ch.cam.ac.uk) that enables robust global analysis of kinetic data without the need for extensive programming or detailed mathematical knowledge. The software automates repetitive tasks and guides users through the key steps of kinetic analysis: determination of constraints to be placed on the aggregation mechanism based on the concentration dependence of the aggregation reaction, choosing from several fundamental models describing assembly into linear aggregates and

  18. On the estimation of cooperativity in ion channel kinetics: activation free energy and kinetic mechanism of Shaker K+ channel.

    PubMed

    Banerjee, Kinshuk; Das, Biswajit; Gangopadhyay, Gautam

    2013-04-28

    In this paper, we have explored generic criteria of cooperative behavior in ion channel kinetics treating it on the same footing with multistate receptor-ligand binding in a compact theoretical framework. We have shown that the characterization of cooperativity of ion channels in terms of the Hill coefficient violates the standard Hill criteria defined for allosteric cooperativity of ligand binding. To resolve the issue, an alternative measure of cooperativity is proposed here in terms of the cooperativity index that sets a unified criteria for both the systems. More importantly, for ion channel this index can be very useful to describe the cooperative kinetics as it can be readily determined from the experimentally measured ionic current combined with theoretical modelling. We have analyzed the correlation between the voltage value and slope of the voltage-activation curve at the half-activation point and consequently determined the standard free energy of activation of the ion channel using two well-established mechanisms of cooperativity, namely, Koshland-Nemethy-Filmer (KNF) and Monod-Wyman-Changeux (MWC) models. Comparison of the theoretical results for both the models with appropriate experimental data of mutational perturbation of Shaker K(+) channel supports the experimental fact that the KNF model is more suitable to describe the cooperative behavior of this class of ion channels, whereas the performance of the MWC model is unsatisfactory. We have also estimated the mechanistic performance through standard free energy of channel activation for both the models and proposed a possible functional disadvantage in the MWC scheme.

  19. Kinetics and Mechanism of Iodide Oxidation by Iron(III): A Clock Reaction Approach

    ERIC Educational Resources Information Center

    Bauer, Jurica; Tomisic, Vladislav; Vrkljan, Petar B. A.

    2008-01-01

    A simple method for studying the kinetics of a chemical reaction is described and the significance of reaction orders in deducing reaction mechanisms is demonstrated. In this student laboratory experiment, oxidation of iodide by iron(III) ions in an acidic medium is transformed into a clock reaction. By means of the initial rates method, it is…

  20. Kinetics and Mechanism of Iodide Oxidation by Iron(III): A Clock Reaction Approach

    ERIC Educational Resources Information Center

    Bauer, Jurica; Tomisic, Vladislav; Vrkljan, Petar B. A.

    2008-01-01

    A simple method for studying the kinetics of a chemical reaction is described and the significance of reaction orders in deducing reaction mechanisms is demonstrated. In this student laboratory experiment, oxidation of iodide by iron(III) ions in an acidic medium is transformed into a clock reaction. By means of the initial rates method, it is…

  1. Kinetic mechanism and fidelity of nick sealing by Escherichia coli NAD+-dependent DNA ligase (LigA)

    PubMed Central

    Chauleau, Mathieu; Shuman, Stewart

    2016-01-01

    Escherichia coli DNA ligase (EcoLigA) repairs 3′-OH/5′-PO4 nicks in duplex DNA via reaction of LigA with NAD+ to form a covalent LigA-(lysyl-Nζ)–AMP intermediate (step 1); transfer of AMP to the nick 5′-PO4 to form an AppDNA intermediate (step 2); and attack of the nick 3′-OH on AppDNA to form a 3′-5′ phosphodiester (step 3). A distinctive feature of EcoLigA is its stimulation by ammonium ion. Here we used rapid mix-quench methods to analyze the kinetic mechanism of single-turnover nick sealing by EcoLigA–AMP. For substrates with correctly base-paired 3′-OH/5′-PO4 nicks, kstep2 was fast (6.8–27 s−1) and similar to kstep3 (8.3–42 s−1). Absent ammonium, kstep2 and kstep3 were 48-fold and 16-fold slower, respectively. EcoLigA was exquisitely sensitive to 3′-OH base mispairs and 3′ N:abasic lesions, which elicited 1000- to >20000-fold decrements in kstep2. The exception was the non-canonical 3′ A:oxoG configuration, which EcoLigA accepted as correctly paired for rapid sealing. These results underscore: (i) how EcoLigA requires proper positioning of the nick 3′ nucleoside for catalysis of 5′ adenylylation; and (ii) EcoLigA's potential to embed mutations during the repair of oxidative damage. EcoLigA was relatively tolerant of 5′-phosphate base mispairs and 5′ N:abasic lesions. PMID:26857547

  2. Kinetics and Mechanisms of Ciprofloxacin Oxidation on Hematite Surfaces.

    PubMed

    Martin, Sébastien; Shchukarev, Andrey; Hanna, Khalil; Boily, Jean-François

    2015-10-20

    Adsorption of antibiotics at mineral surfaces has been extensively studied over the past 20 years, yet much remains to be learned on their interfacial properties and transformation mechanisms. In this study, interactions of Ciprofloxacin (CIP), a fluoroquinolone antibiotic with two sets of synthetic nanosized hematite particles, with relatively smooth (H10, 10-20 nm in diameter) and roughened (H80, 80-90 nm in diameter) surfaces, were studied by means of liquid chromatography (LC), mass spectrometry (MS), and spectroscopy (vibration and X-ray photoelectron). Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy provides evidence for inner-sphere bidentate complex formation of CIP at hematite surfaces in 0.01 M NaCl, irrespective of pH and particle size. ATR-FTIR spectroscopy also revealed that the sorbed mother CIP molecule decayed to other surface species over a period of at least 65 h. This was supported by the detection of three daughter products in the aqueous phase by LC/MS. The appearance of NH3(+) groups during the course of these experiments, revealed by cryogenic XPS, provides further evidence that CIP oxidation proceeds through an opening of piperazine ring via N-dealkylation. Additional in vacuo FTIR experiments under temperature-programmed desorption also showed that oxidation of sorbed byproducts were effectively degraded beyond 450 °C, a result denoting considerably strong (inter)molecular bonds of both mother and daughter products. This work also showed that rougher, possibly multidomainic particles (H80) generated slower rates of CIP decomposition but occurring through more complex schemes than at smoother particle surfaces (H10). This work thus uncovered key aspects of the binding of an important antibiotic at iron oxide surfaces, and therefore provided additional constraints to our growing understanding of the fate of emerging contaminants in the environment.

  3. Substrate specificity and kinetic mechanism of Escherichia coli ribulokinase.

    PubMed

    Lee, L V; Gerratana, B; Cleland, W W

    2001-12-15

    L-ribulokinase is unusual among kinases since it phosphorylates all four 2-ketopentoses with almost the same k(cat) values. The K(m)'s differ, however, being 0.14 mM for L- and 0.39 mM for d-ribulose and 3.4 mM for l- and 16 mM for d-xylulose. In addition, L-arabitol is phosphorylated at C-5 (K(m) 4 mM) and ribitol (adonitol) is phosphorylated to D-ribitol-5-phosphate (K(m) 5.5 mM), but D-arabitol, xylitol, and aldopentoses are not substrates. The K(m)'s for MgATP depend on the substrates, being 0.02 mM with L-ribulose, 0.027 mM with D-ribulose and L-xylulose, and 0.3-0.5 mM with the other substrates. In the absence of a sugar substrate there is an ATPase with K(m) of 7 mM and k(cat) 1% of that with sugar substrates. The initial velocity pattern is intersecting, and MgAMPPNP is competitive vs MgATP and uncompetitive vs L-ribulose. L-Erythrulose is competitive vs L-ribulose and when MgATP concentration is varied induces substrate inhibition which is partial. These data show that the mechanism is random, but there is a high level of synergism in the binding of sugar and MgATP, and the path in which the sugar adds first is strongly preferred. (c)2001 Elsevier Science.

  4. Detailed Chemical Kinetic Reaction Mechanism for Biodiesel Components Methyl Stearate and Methyl Oleate

    SciTech Connect

    Naik, C; Westbrook, C K; Herbinet, O; Pitz, W J; Mehl, M

    2010-01-22

    New chemical kinetic reaction mechanisms are developed for two of the five major components of biodiesel fuel, methyl stearate and methyl oleate. The mechanisms are produced using existing reaction classes and rules for reaction rates, with additional reaction classes to describe other reactions unique to methyl ester species. Mechanism capabilities were examined by computing fuel/air autoignition delay times and comparing the results with more conventional hydrocarbon fuels for which experimental results are available. Additional comparisons were carried out with measured results taken from jet-stirred reactor experiments for rapeseed methyl ester fuels. In both sets of computational tests, methyl oleate was found to be slightly less reactive than methyl stearate, and an explanation of this observation is made showing that the double bond in methyl oleate inhibits certain low temperature chain branching reaction pathways important in methyl stearate. The resulting detailed chemical kinetic reaction mechanism includes more approximately 3500 chemical species and more than 17,000 chemical reactions.

  5. Kinetics and Mechanisms of NO(x) - Char Reduction

    SciTech Connect

    Suuberg, E.M.; Lilly, W.D.; Aarna, I.

    1997-09-01

    The emission of nitrogen oxides from combustion of coal remains a problem of considerable interest, whether the concern is with acid rain, stratospheric ozone chemistry, or greenhouse gases. Whereas earlier the concern was focused mainly on NO (as a primary combustion product) and to a lesser extent N0{sub 2} (since it is mainly a secondary product of combustion, e.g. see ref. 1), in recent years the emissions of N{sub 2}0 have also captured considerable attention, particularly in the context of fluidized bed combustion, in which the problem appears to be most acute. The research community has only recently begun to take solid hold on the N{sub 2}0 problem. This is in part because earlier estimates of the importance of N{sub 2}0 in combustion processes were clouded by artifacts in sampling which have now been resolved. This project is concerned with the mechanism of reduction of both NO and N{sub 2}0 by carbons. It was recognized some years ago that NO formed during fluidized bed coal combustion can be heterogeneously reduced in-situ by the carbonaceous solid intermediates of combustion. This has been recently supplemented by the knowledge that heterogeneous reaction with carbon can also play an important role in reducing emissions of N{sub 2}0{sub 2}, but that the NO-carbon reactions might also contribute to formation of N{sub 2}0{sub 2}. The precise role of carbon in N{sub 2}0 reduction and formation has yet to be established, since in one case the authors of a recent study were compelled to comment that the basic knowledge of N{sub 2}0 formation and reduction still has to be improved. The same can be said of the NO-carbon system. Interest in the NO- and N{sub 2}0-char reactions has been significant in connection with both combustor modeling, as well as in design of post-combustion NO{sub x} control strategies.

  6. Kinetics and mechanism of the catalytic reaction between alcohols and dimethyl carbonate

    NASA Astrophysics Data System (ADS)

    Koledina, K. F.; Koledin, S. N.; Shchadneva, N. A.; Gubaidullin, I. M.

    2017-03-01

    The mechanism of the reaction between alcohols and dimethyl carbonate, catalyzed by dicobalt octacarbonyl Co2(CO)8, is studied by means of mathematical modeling. Kinetic models for possible schemes of chemical transformations are constructed at different initial concentrations of the catalyst. Based on a comparative analysis of activation energies of possible stages of chemical transformations, possible reaction pathways are determined and an appropriate mechanism is selected.

  7. Coupling between chemical kinetics and mechanics that is both nonlinear and compatible with thermodynamics.

    PubMed

    Klika, Václav; Grmela, Miroslav

    2013-01-01

    Motivated by biological applications (e.g., bone tissue development and regeneration) we investigate coupling between mesoscopic mechanics and chemical kinetics. Governing equations of both dynamical systems are first written in a form expressing manifestly their compatibility with microscopic mechanics and thermodynamics. The same form is then required from governing equations of the coupled dynamics. The main result of the paper is an admissible form of the coupled dynamics.

  8. Evolving Stress State and Deformation Mechanism in the Himalayan Foreland Fold-and-Thrust Belt, Northern Pakistan

    NASA Astrophysics Data System (ADS)

    Ahmad, I.; Dasti, N.

    2010-12-01

    Crustal deformation along with shortening due to northward under-thrusting of the Indian plate beneath the Eurasian plate continues to create active tectonic features on the northern fringes of the Indian craton since major collision began in the Eocene. Here the study provides insights on the evolving stress state and deformation mechanism of the Salt Range and Potwar area of Northern Pakistan. This part of Himalayan foreland fold-and-thrust-belt has severe history of deformation during 5.1 Ma and 2 Ma. This foreland area lies between Main Boundary Thrust (MBT) in the north, Himalayan Frontal Thrust (HFT) in the south and Jhelum fault of sinistral nature in the east & Kalabagh fault of dextral nature in the west. An integrated data from seismic reflection profiles and drilling logs reveal that the subsurface deformation encompasses pop-ups, imbricates, duplexes with in-sequence and out-of-sequence thrusting. It also depicts that intensity of deformation increases from the northern margin of Soan geosyncline towards north due to lacking of evaporites while in the south it decreases due to gradual increase in salt thickness. Surface geologic mapping glimpses a series of thrust sheets and anticlines trending ENE-SWS in the eastern and central part of the study area; whereas in the western part, the trend is almost E-W. This variation in the trend of structures is the result of counter clock rotational behaviour (~10°deviation from north to the west) of north-western part of the Indian lithospheric plate. Current outcrop-scale natural fracture data collected from selected anticlinal structures of the study area is presented to manifest the stress evolution and deformation styles under the established tectonic framework. Collected data is analysed for the evaluation of tectonic stress direction and deformation mechanism. The genetic arrangement and types of fractures observed in the study area indicate that the whole area is under compression. The data also testify

  9. Bacteriorhodopsin folds through a poorly organized transition state.

    PubMed

    Schlebach, Jonathan P; Woodall, Nicholas B; Bowie, James U; Park, Chiwook

    2014-11-26

    The folding mechanisms of helical membrane proteins remain largely uncharted. Here we characterize the kinetics of bacteriorhodopsin folding and employ φ-value analysis to explore the folding transition state. First, we developed and confirmed a kinetic model that allowed us to assess the rate of folding from SDS-denatured bacteriorhodopsin (bRU) and provides accurate thermodynamic information even under influence of retinal hydrolysis. Next, we obtained reliable φ-values for 16 mutants of bacteriorhodopsin with good coverage across the protein. Every φ-value was less than 0.4, indicating the transition state is not uniquely structured. We suggest that the transition state is a loosely organized ensemble of conformations.

  10. A Chemical Kinetic Mechanism for the Ignition of Silane/Hydrogen Mixtures

    NASA Technical Reports Server (NTRS)

    Jachimowski, C. J.; Mclain, A. G.

    1983-01-01

    A chemical kinetic reaction mechanism for the oxidation of silane/hydrogen mixtures is presented and discussed. Shock-tube ignition delay time data were used to evaluate and refine the mechanism. Good agreement between experimental results and the results predicted by the mechanism was obtained by adjusting the rate coefficient for the reaction SiH3 + O2 yields SiH2O + OH. The reaction mechanism was used to theoretically investigate the ignition characteristics of silane/hydrogen mixtures. The results revealed that over the entire range of temperature examined (800 K to 1200 K), substantial reduction in ignition delay times is obtained when silane is added to hydrogen.

  11. Development of Non-Equilibrium Plasma-Flame Kinetic Mechanism and its Validation Using Gliding Arc Integrated with Counterflow Burner

    DTIC Science & Technology

    2010-02-21

    FINAL REPORT: FA9550-07-1-0136, Dec. 2006 – Nov. 2009 Development of Non-Equilibrium Plasma-Flame Kinetic Mechanism and its...Non-Equilibrium Plasma-Flame Kinetic 5a. CONTRACT NUMBER Mechanism and its Validation Using Gliding Arc Integrated with FA9550-07-1...DISTRIBUTION / AVAILABILITY STATEMENT 13. SUPPLEMENTARY NOTES Kinetic enhancements of NOx, O3, and O2(a1Δg) on ignition and flame propagation

  12. Identification of multiple folding pathways of monellin using pulsed thiol labeling and mass spectrometry.

    PubMed

    Jha, Santosh Kumar; Dasgupta, Amrita; Malhotra, Pooja; Udgaonkar, Jayant B

    2011-04-19

    Protein folding reactions often display multiexponential kinetics of changes in intrinsic optical signals, as a manifestation of heterogeneity, either on one folding pathway or on multiple folding pathways. Delineating the origin of this heterogeneity is difficult because different coexisting structural forms of a protein cannot be easily distinguished by optical probes. In this study, the complex folding reaction of single-chain monellin has been investigated using a pulsed thiol labeling (SX) methodology in conjunction with mass spectrometry, which measures the kinetics of burial of a cysteine side chain thiol during folding. Because it can directly distinguish between unfolded and folded molecules and can measure the disappearance of the former during folding, the pulsed SX methodology is an ideal method for investigating whether multiple pathways are operative during folding. The kinetics of burial of the C42 thiol of monellin was observed to follow biexponential kinetics. To determine whether this was because the fast phase leads to the partial protection of the thiol group in all the molecules or to complete protection in only a fraction of the molecules, the duration and intensity of the labeling pulse were varied. The observation that the extent of labeling did not vary with the duration of the pulse cannot be explained by a simple sequential folding mechanism. Two parallel folding pathways are shown to be operative, with one leading to the formation of thiol-protective structure more rapidly than the other.

  13. Evaluating endoglucanase Cel7B-lignin interaction mechanisms and kinetics using quartz crystal microgravimetry.

    PubMed

    Pfeiffer, Katherine A; Sorek, Hagit; Roche, Christine M; Strobel, Kathryn L; Blanch, Harvey W; Clark, Douglas S

    2015-11-01

    The kinetics and mechanisms of protein interactions with solid surfaces are important to fields as diverse as industrial biocatalysis, biomedical engineering, food science, and cell biology. The nonproductive adsorption of cellulase enzymes to lignin, a plant cell wall polymer, reduces their effectiveness in saccharifying biomass. Cellulase has been shown to interact with lignin, but the heterogeneity of lignin surfaces, challenges in measuring irreversible components of these interactions, and fast adsorption rates make quantifying the reaction kinetics difficult. This work employs quartz crystal microgravimetry with dissipation monitoring (QCM-D) for real-time measurement of adsorbed mass on a flat lignin surface. We have developed a method for casting homogeneous lignin films that are chemically similar to lignin found in pretreated biomass, and used QCM-D to compare three models of reversible-irreversible binding behavior: a single-site transition model, a transition model with changing adsorbate footprint, and a two-site transition model. Of the three models tested, the two-site transition model provides the only kinetic mechanism able to describe the behavior of Cel7B binding to lignin. While the direct implications of lignin-cellulase interactions may be limited to biomass deconstruction for renewable energy and green chemistry, the analytical and experimental methods demonstrated in this work are relevant to any system in which the kinetics and reaction mechanism of reversible and irreversible protein adsorption at a solid-liquid interface are important.

  14. Roles of the Kinetic and Dynamic Mechanisms in the Lp -Ep Relation

    NASA Astrophysics Data System (ADS)

    Qin, Yi-Ping; Chen, Zhi-Fu

    2013-04-01

    The Lp -Ep relation is a well-known relation in gamma-ray bursts (GRBs). Its implication remains unclear. We propose to investigate the underlying mechanisms of this relation by considering the corresponding kinetic and dynamic mechanisms separately. In this way, one can tell how much the kinetic or dynamic mechanism contributes to the index of the relationship. Our analysis gives rise to several conclusions. (1) The index of the kinetic effect in the Lp -Ep relation can simply be derived from the theory of special relativity, which is generally larger than 2, depending on the situation concerned. (2) The index of the dynamic effect in the relation can be deduced from observation once a model of jets is adopted. According to current GRB data, we find that the dynamic effect alone tends to give rise to an anti-correlation between Lp and Ep ; in terms of statistics, the dynamic effect is obviously smaller than the kinetic effect; in the situation of jets with moving discrete radio clouds that move directly toward the observer, the index of the dynamic effect is currently constrained within (- 1.6, -1), while in other situations of jets, the constraints are different; both internal and external shocks can account for the current data.

  15. Site-specific experiments on folding/unfolding of Jun coiled coils: thermodynamic and kinetic parameters from spin inversion transfer nuclear magnetic resonance at leucine-18.

    PubMed

    d'Avignon, D André; Bretthorst, G Larry; Holtzer, Marilyn Emerson; Schwarz, Kathleen A; Angeletti, Ruth Hogue; Mints, Lisa; Holtzer, Alfred

    2006-10-15

    The 32-residue leucine zipper subsequence, called here Jun-lz, associates in benign media to form a parallel two-stranded coiled coil. Studies are reported of its thermal unfolding/folding transition by circular dichroism (CD) on samples of natural isotopic abundance and by both equilibrium and spin inversion transfer (SIT) nuclear magnetic resonance (NMR) on samples labeled at the leucine-18 alpha-carbon with 99% 13C. The data cover a wide range of temperature and concentration, and show that Jun-lz unfolds below room temperature, being far less stable than some other leucine zippers such as GCN4. 13C-NMR shows two well-separated resonances. We ascribe the upfield one to 13C spins on unfolded single chains and the downfield one to 13C spins on coiled-coil dimers. Their relative intensities provide a measure of the unfolding equilibrium constant. In SIT NMR, the recovery of the equilibrium magnetization after one resonance is inverted is modulated in part by the unfolding and folding rate constants, which are accessible from the data. Global Bayesian analysis of the equilibrium and SIT NMR data provide values for the standard enthalpy, entropy, and heat capacity of unfolding, and show the latter to be unusually large. The CD results are compatible with the NMR findings. Global Bayesian analysis of the SIT NMR data yields the corresponding activation parameters for unfolding and folding. The results show that both reaction directions are activated processes. Activation for unfolding is entropy driven, enthalpy opposed. Activation for folding is strongly enthalpy opposed and somewhat entropy opposed, falsifying the idea that the barrier for folding is solely due to a purely entropic search for properly registered partners. The activation heat capacity is much larger for folding, so almost the entire overall change is due to the folding direction. This latter finding, if it applies to GCN4 leucine zippers, clears up an extant apparent disagreement between folding rate

  16. Controlling Self-Assembly Kinetics of DNA-Functionalized Liposomes Using Toehold Exchange Mechanism.

    PubMed

    Parolini, Lucia; Kotar, Jurij; Di Michele, Lorenzo; Mognetti, Bortolo M

    2016-02-23

    The selectivity of Watson-Crick base pairing has allowed the design of DNA-based functional materials bearing an unprecedented level of accuracy. Examples include DNA origami, made of tiles assembling into arbitrarily complex shapes, and DNA coated particles featuring rich phase behaviors. Frequently, the realization of conceptual DNA-nanotechnology designs has been hampered by the lack of strategies for effectively controlling relaxations. In this article, we address the problem of kinetic control on DNA-mediated interactions between Brownian objects. We design a kinetic pathway based on toehold-exchange mechanisms that enables rearrangement of DNA bonds without the need for thermal denaturation, and test it on suspensions of DNA-functionalized liposomes, demonstrating tunability of aggregation rates over more than 1 order of magnitude. While the possibility to design complex phase behaviors using DNA as a glue is already well recognized, our results demonstrate control also over the kinetics of such systems.

  17. New mechanism of kinetic exchange interaction induced by strong magnetic anisotropy

    PubMed Central

    Iwahara, Naoya; Chibotaru, Liviu F.

    2016-01-01

    It is well known that the kinetic exchange interaction between single-occupied magnetic orbitals (s-s) is always antiferromagnetic, while between single- and double-occupied orbitals (s-d) is always ferromagnetic and much weaker. Here we show that the exchange interaction between strongly anisotropic doublets of lanthanides, actinides and transition metal ions with unquenched orbital momentum contains a new s-d kinetic contribution equal in strength with the s-s one. In non-collinear magnetic systems, this s-d kinetic mechanism can cause an overall ferromagnetic exchange interaction which can become very strong for transition metal ions. These findings are fully confirmed by DFT based analysis of exchange interaction in several Ln3+ complexes. PMID:27098292

  18. Mechanisms and Kinetics of Tellurium Precipitation in CdTe-based Materials

    NASA Astrophysics Data System (ADS)

    Lordi, Vincenzo

    2012-02-01

    CdTe and related alloys are important materials for solar photovoltaic application as well as for high-resolution room-temperature gamma radiation detectors. However, the performance of devices, particularly in high-energy applications, is limited by various material defects. Among the most important defects are Te precipitates of various sizes caused by non-stoichiometric growth conditions. In this work, we study the kinetics of Te aggregation and precipitation at the atomic scale. Density functional theory is used to compute the energetics, migration rates, and binding energies of point defects involved in Te aggregation, which include various interstitials, vacancies, and anti-site defects. Kinetic Monte Carlo is then used to simulate the aggregation process leading to precipitation nuclei. The mechanisms and kinetics of formation of these Te-rich regions are analyzed for various conditions. Prepared by LLNL under Contract DE-AC52-07NA27344.

  19. Regulatory motifs on ISWI chromatin remodelers: molecular mechanisms and kinetic proofreading

    NASA Astrophysics Data System (ADS)

    Brysbaert, Guillaume; Lensink, Marc F.; Blossey, Ralf

    2015-02-01

    Recently, kinetic proofreading scenarios have been proposed for the regulation of chromatin remodeling, first on purely theoretical grounds (Blossey and Schiessel 2008 HFSP J. 2 167-70) and deduced from experiments on the ISWI/ACF system (Narlikar 2010 Curr. Opin. Chem. Biol. 14 660). In the kinetic proofreading scenario of chromatin remodeling, the combination of the recognition of a histone tail state and ATP-hydrolysis in the remodeler motor act together to select (i.e. proofread) a nucleosomal substrate. ISWI remodelers have recently been shown to have an additional level of regulation as they contain auto-inhibitory motifs which need to be inactivated through an interaction with the nucleosome. In this paper we show that the auto-regulatory effect enhances substrate recognition in kinetic proofreading. We further report some suggestive additional insights into the molecular mechanism underlying ISWI-autoregulation.

  20. Chemical Kinetic Reaction Mechanisms for Combustion of Hydrocarbon and Other Types of Chemical Fuels

    DOE Data Explorer

    The central feature of the Combustion Chemistry project at LLNL is the development, validation, and application of detailed chemical kinetic reaction mechanisms for the combustion of hydrocarbon and other types of chemical fuels. For the past 30 years, LLNL's Chemical Sciences Division has built hydrocarbon mechanisms for fuels from hydrogen and methane through much larger fuels including heptanes and octanes. Other classes of fuels for which models have been developed include flame suppressants such as halons and organophosphates, and air pollutants such as soot and oxides of nitrogen and sulfur. Reaction mechanisms have been tested and validated extensively through comparisons between computed results and measured data from laboratory experiments (e.g., shock tubes, laminar flames, rapid compression machines, flow reactors, stirred reactors) and from practical systems (e.g., diesel engines, spark-ignition engines, homogeneous charge, compression ignition (HCCI) engines). These kinetic models are used to examine a wide range of combustion systems.

  1. The Role of Comprehensive Detailed Chemical Kinetic Reaction Mechanisms in Combustion Research

    SciTech Connect

    Westbrook, C K; Pitz, W J; Curran, H J; Mehl, M

    2008-07-16

    Recent developments by the authors in the field of comprehensive detailed chemical kinetic reaction mechanisms for hydrocarbon fuels are reviewed. Examples are given of how these mechanisms provide fundamental chemical insights into a range of combustion applications. Practical combustion consists primarily of chemical heat release from reactions between a fuel and an oxidizer, and computer simulations of practical combustion systems have become an essential tool of combustion research (Westbrook et al., 2005). At the heart of most combustion simulations, the chemical kinetic submodel frequently is the most detailed, complex and computationally costly part of a system model. Historically, the chemical submodel equations are solved using time-implicit numerical algorithms, due to the extreme stiffness of the coupled rate equations, with a computational cost that varies roughly with the cube of the number of chemical species in the model. While early mechanisms (c. 1980) for apparently simple fuels such as methane (Warnatz, 1980) or methanol (Westbrook and Dryer, 1979) included perhaps 25 species, current detailed mechanisms for much larger, more complex fuels such as hexadecane (Fournet et al., 2001; Ristori et al., 2001; Westbrook et al., 2008) or methyl ester methyl decanoate (Herbinet et al., 2008) have as many as 2000 or even 3000 species. Rapid growth in capabilities of modern computers has been an essential feature in this rapid growth in the size and complexity of chemical kinetic reaction mechanisms.

  2. Mixed Reversible Covalent Crosslink Kinetics Enable Precise, Hierarchical Mechanical Tuning of Hydrogel Networks.

    PubMed

    Yesilyurt, Volkan; Ayoob, Andrew M; Appel, Eric A; Borenstein, Jeffrey T; Langer, Robert; Anderson, Daniel G

    2017-05-01

    Hydrogels play a central role in a number of medical applications and new research aims to engineer their mechanical properties to improve their capacity to mimic the functional dynamics of native tissues. This study shows hierarchical mechanical tuning of hydrogel networks by utilizing mixtures of kinetically distinct reversible covalent crosslinks. A methodology is described to precisely tune stress relaxation in PEG networks formed from mixtures of two different phenylboronic acid derivatives with unique diol complexation rates, 4-carboxyphenylboronic acid, and o-aminomethylphenylboronic acid. Gel relaxation time and the mechanical response to dynamic shear are exquisitely controlled by the relative concentrations of the phenylboronic acid derivatives. The differences observed in the crossover frequencies corresponding to pKa differences in the phenylboronic acid derivatives directly connect the molecular kinetics of the reversible crosslinks to the macroscopic dynamic mechanical behavior. Mechanical tuning by mixing reversible covalent crosslinking kinetics is found to be independent of other attributes of network architecture, such as molecular weight between crosslinks. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Distinct folding pathways of two homologous disulfide proteins: bovine pancreatic trypsin inhibitor and tick anticoagulant peptide.

    PubMed

    Chang, Jui-Yoa

    2011-01-01

    The folding pathways of disulfide proteins vary substantially (Arolas et al., Trends Biochem Sci 31: 292-301, 2006). The diversity is mainly manifested by (a) the extent of heterogeneity of folding intermediates, (b) the extent of presence of native-like intermediates, and (c) the variation of folding kinetics. Even among structurally similar proteins, the difference can be enormous. This is demonstrated in this concise review with two structurally homologous kunitz-type protease inhibitors, bovine pancreatic trypsin inhibitor and tick anticoagulant peptide, as well as a group of cystine knot proteins. The diversity of their folding mechanisms is illustrated with two different folding techniques: (a) the conventional method of disulfide oxidation (oxidative folding), and (b) the novel method of disulfide scrambling (Chang, J Biol Chem 277: 120-126, 2002). This review also highlights the convergence of folding models concluded form the conventional conformational folding and those obtained by oxidative folding.

  4. Identifying Kinetic Barriers to Mechanical Unfolding of the T. thermophila Ribozyme

    NASA Astrophysics Data System (ADS)

    Onoa, Bibiana; Dumont, Sophie; Liphardt, Jan; Smith, Steven B.; Tinoco, Ignacio; Bustamante, Carlos

    2003-03-01

    Mechanical unfolding trajectories for single molecules of the Tetrahymena thermophila ribozyme display eight intermediates corresponding to discrete kinetic barriers that oppose mechanical unfolding with lifetimes of seconds and rupture forces between 10 and 30 piconewtons. Barriers are magnesium dependent and correspond to known intra- and interdomain interactions. Several barrier structures are ``brittle,'' breakage requiring high forces but small (1 to 3 nanometers) deformations. Barrier crossing is stochastic, leading to variable unfolding paths. The response of complex RNA structures to locally applied mechanical forces may be analogous to the responses of RNA during translation, messenger RNA export from the nucleus, and viral replication.

  5. Identifying Kinetic Barriers to Mechanical Unfolding of the T. thermophila Ribozyme

    PubMed Central

    Onoa, Bibiana; Dumont, Sophie; Liphardt, Jan; Smith, Steven B.; Tinoco, Ignacio; Bustamante, Carlos

    2006-01-01

    Mechanical unfolding trajectories for single molecules of the Tetrahymena thermophila ribozyme display eight intermediates corresponding to discrete kinetic barriers that oppose mechanical unfolding with lifetimes of seconds and rupture forces between 10 and 30 piconewtons. Barriers are magnesium dependent and correspond to known intra- and interdomain interactions. Several barrier structures are “brittle,” breakage requiring high forces but small (1 to 3 nanometers) deformations. Barrier crossing is stochastic, leading to variable unfolding paths. The response of complex RNA structures to locally applied mechanical forces may be analogous to the responses of RNA during translation, messenger RNA export from the nucleus, and viral replication. PMID:12649482

  6. Relaxation kinetics and mechanical stability of metallic glasses and supercooled melts

    NASA Astrophysics Data System (ADS)

    Mayr, S. G.

    2009-02-01

    Metallic glasses are characterized by a rather complex viscoelastic response and the occurrence of the glass transition, while the atomistic origins are still poorly understood. Using a realistic CuTi model glass we employ global and local elasticity tensors for a thorough analysis of relaxation kinetics and mechanical stability. We obtain strong indication that (i) α relaxation is governed by an underlying process (identified as slow β relaxation) which resembles diffusion in its temperature dependence, (ii) glasses reveal intrinsic mechanical instabilities, which are closely linked to collective shear events within shear transformation zones, and (iii) glass transition can be understood as a percolation transition of mechanically unstable regions.

  7. Interplay of Protein Binding Interactions, DNA Mechanics, and Entropy in DNA Looping Kinetics

    PubMed Central

    Mulligan, Peter J.; Chen, Yi-Ju; Phillips, Rob; Spakowitz, Andrew J.

    2015-01-01

    DNA looping plays a key role in many fundamental biological processes, including gene regulation, recombination, and chromosomal organization. The looping of DNA is often mediated by proteins whose structural features and physical interactions can alter the length scale at which the looping occurs. Looping and unlooping processes are controlled by thermodynamic contributions associated with mechanical deformation of the DNA strand and entropy arising from thermal fluctuations of the conformation. To determine how these confounding effects influence DNA looping and unlooping kinetics, we present a theoretical model that incorporates the role of the protein interactions, DNA mechanics, and conformational entropy. We show that for shorter DNA strands the interaction distance affects the transition state, resulting in a complex relationship between the looped and unlooped state lifetimes and the physical properties of the looped DNA. We explore the range of behaviors that arise with varying interaction distance and DNA length. These results demonstrate how DNA deformation and entropy dictate the scaling of the looping and unlooping kinetics versus the J-factor, establishing the connection between kinetic and equilibrium behaviors. Our results show how the twist-and-bend elasticity of the DNA chain modulates the kinetics and how the influence of the interaction distance fades away at intermediate to longer chain lengths, in agreement with previous scaling predictions. PMID:26244743

  8. Bi-stable vocal fold adduction: A mechanism of modal-falsetto register shifts and mixed registration

    PubMed Central

    Titze, Ingo R.

    2014-01-01

    The origin of vocal registers has generally been attributed to differential activation of cricothyroid and thyroarytenoid muscles in the larynx. Register shifts, however, have also been shown to be affected by glottal pressures exerted on vocal fold surfaces, which can change with loudness, pitch, and vowel. Here it is shown computationally and with empirical data that intraglottal pressures can change abruptly when glottal adductory geometry is changed relatively smoothly from convergent to divergent. An intermediate shape between large convergence and large divergence, namely, a nearly rectangular glottal shape with almost parallel vocal fold surfaces, is associated with mixed registration. It can be less stable than either of the highly angular shapes unless transglottal pressure is reduced and upper stiffness of vocal fold tissues is balanced with lower stiffness. This intermediate state of adduction is desirable because it leads to a low phonation threshold pressure with moderate vocal fold collision. Achieving mixed registration consistently across wide ranges of F0, lung pressure, and vocal tract shapes appears to be a balancing act of coordinating laryngeal muscle activation with vocal tract pressures. Surprisingly, a large transglottal pressure is not facilitative in this process, exacerbating the bi-stable condition and the associated register contrast. PMID:25235006

  9. Folding of beta-sandwich proteins: three-state transition of a fibronectin type III module.

    PubMed Central

    Cota, E.; Clarke, J.

    2000-01-01

    An analysis of the folding of the 94 residue tenth fibronectin type III (fnIII) domain of human fibronectin (FNfn10) is presented. Use of guanidine isothiocyanate as a denaturant allows us to obtain equilibrium and kinetic data across a broad range of denaturant concentrations that are unavailable in guanidine hydrochloride. Equilibrium unfolding experiments show that FNfn10 is significantly more stable than has been reported previously. Comparison of equilibrium and kinetic parameters reveals the presence of an intermediate that accumulates at low denaturant concentrations. This is the first demonstration of three-state folding kinetics for a fnIII domain. We have previously shown that a homologous domain from human tenascin (TNfn3) folds by a two-state mechanism, but this does not necessarily indicate that the two proteins fold by different folding pathways. PMID:10739253

  10. The Protein Folding Problem

    PubMed Central

    Dill, Ken A.; Ozkan, S. Banu; Shell, M. Scott; Weikl, Thomas R.

    2008-01-01

    The “protein folding problem” consists of three closely related puzzles: (a) What is the folding code? (b) What is the folding mechanism? (c) Can we predict the native structure of a protein from its amino acid sequence? Once regarded as a grand challenge, protein folding has seen great progress in recent years. Now, foldable proteins and nonbiological polymers are being designed routinely and moving toward successful applications. The structures of small proteins are now often well predicted by computer methods. And, there is now a testable explanation for how a protein can fold so quickly: A protein solves its large global optimization problem as a series of smaller local optimization problems, growing and assembling the native structure from peptide fragments, local structures first. PMID:18573083

  11. The Kinetic Reaction Mechanism of the Vibrio cholerae Sodium-dependent NADH Dehydrogenase*♦

    PubMed Central

    Tuz, Karina; Mezic, Katherine G.; Xu, Tianhao; Barquera, Blanca; Juárez, Oscar

    2015-01-01

    The sodium-dependent NADH dehydrogenase (Na+-NQR) is the main ion transporter in Vibrio cholerae. Its activity is linked to the operation of the respiratory chain and is essential for the development of the pathogenic phenotype. Previous studies have described different aspects of the enzyme, including the electron transfer pathways, sodium pumping structures, cofactor and subunit composition, among others. However, the mechanism of the enzyme remains to be completely elucidated. In this work, we have studied the kinetic mechanism of Na+-NQR with the use of steady state kinetics and stopped flow analysis. Na+-NQR follows a hexa-uni ping-pong mechanism, in which NADH acts as the first substrate, reacts with the enzyme, and the oxidized NAD leaves the catalytic site. In this conformation, the enzyme is able to capture two sodium ions and transport them to the external side of the membrane. In the last step, ubiquinone is bound and reduced, and ubiquinol is released. Our data also demonstrate that the catalytic cycle involves two redox states, the three- and five-electron reduced forms. A model that gathers all available information is proposed to explain the kinetic mechanism of Na+-NQR. This model provides a background to understand the current structural and functional information. PMID:26004776

  12. The Kinetic Reaction Mechanism of the Vibrio cholerae Sodium-dependent NADH Dehydrogenase.

    PubMed

    Tuz, Karina; Mezic, Katherine G; Xu, Tianhao; Barquera, Blanca; Juárez, Oscar

    2015-08-14

    The sodium-dependent NADH dehydrogenase (Na(+)-NQR) is the main ion transporter in Vibrio cholerae. Its activity is linked to the operation of the respiratory chain and is essential for the development of the pathogenic phenotype. Previous studies have described different aspects of the enzyme, including the electron transfer pathways, sodium pumping structures, cofactor and subunit composition, among others. However, the mechanism of the enzyme remains to be completely elucidated. In this work, we have studied the kinetic mechanism of Na(+)-NQR with the use of steady state kinetics and stopped flow analysis. Na(+)-NQR follows a hexa-uni ping-pong mechanism, in which NADH acts as the first substrate, reacts with the enzyme, and the oxidized NAD leaves the catalytic site. In this conformation, the enzyme is able to capture two sodium ions and transport them to the external side of the membrane. In the last step, ubiquinone is bound and reduced, and ubiquinol is released. Our data also demonstrate that the catalytic cycle involves two redox states, the three- and five-electron reduced forms. A model that gathers all available information is proposed to explain the kinetic mechanism of Na(+)-NQR. This model provides a background to understand the current structural and functional information.

  13. The Influence of Various Process Parameters on Dissolution Kinetics and Mechanism of Struvite Seed Crystals

    NASA Astrophysics Data System (ADS)

    Ariyanto, Eko; Ang, Ha Ming; Sen, Tushar Kanti

    2017-07-01

    The basic understanding of struvite dissolution chemistry is essential to designers and operators for anticipating struvite problem and remediating existing struvite damage in a wastewater treatment. The dissolution kinetic of struvite seed crystals is very important parameters to determine a solid substance entering in solvent to yield a solution. In this study the dissolution kinetics of struvite crystals (MgNH4PO4·6H2O) in deionized water was investigated in a batch crystallizer. The effects of stirrer speeds, temperature and seed crystals size on the dissolution rate were determined. The results showed that an increase of struvite dissolution rate with increasing stirring speed. Struvite dissolution occurred via a diffusion-controlled mechanism in the range of stirrer speeds 120-400 rpm but became interfacial-reaction-controlling at over 400 rpm. The influence of temperature on dissolution kinetic of struvite crystals was also investigated at stirrer speeds of 200 and 500 rpm. The dissolution rates increased with an increase in the temperature for both stirrer speeds. The change in activation energies at different stirrer speeds confirmed that the change of dissolution mechanism from a diffusion-controlled mechanism at low stirrer speeds to an interfacial-reaction-controlled mechanism at higher stirrer speeds. The dissolution rate of struvite crystals increased with smaller crystal sizes.

  14. Programmed folding of DNA origami structures through single-molecule force control

    NASA Astrophysics Data System (ADS)

    Bae, Wooli; Kim, Kipom; Min, Duyoung; Ryu, Je-Kyung; Hyeon, Changbong; Yoon, Tae-Young

    2014-12-01

    Despite the recent development in the design of DNA origami, its folding yet relies on thermal or chemical annealing methods. We here demonstrate mechanical folding of the DNA origami structure via a pathway that has not been accessible to thermal annealing. Using magnetic tweezers, we stretch a single scaffold DNA with mechanical tension to remove its secondary structures, followed by base pairing of the stretched DNA with staple strands. When the force is subsequently quenched, folding of the DNA nanostructure is completed through displacement between the bound staple strands. Each process in the mechanical folding is