Global and local molecular dynamics of a bacterial carboxylesterase provide insight into its catalytic mechanism

PubMed Central

Yu, Xiaozhen; Sigler, Sara C.; Hossain, Delwar; Wierdl, Monika; Gwaltney, Steven R.; Potter, Philip M.; Wadkins, Randy M.

2013-01-01

Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of xenobiotics. In humans, substrates for these enzymes are far-ranging, and include the street drug heroin and the anticancer agent irinotecan. Hence, their ability to bind and metabolize substrates is of broad interest to biomedical science. In this study, we focused our attention on dynamic motions of a CE from B. subtilis (pnbCE), with emphasis on the question of what individual domains of the enzyme might contribute to its catalytic activity. We used a 10 ns all-atom molecular dynamics simulation, normal mode calculations, and enzyme kinetics to understand catalytic consequences of structural changes within this enzyme. Our results shed light on how molecular motions are coupled with catalysis. During molecular dynamics, we observed a distinct C-C bond rotation between two conformations of Glu310. Such a bond rotation would alternately facilitate and impede protonation of the active site His399 and act as a mechanism by which the enzyme alternates between its active and inactive conformation. Our normal mode results demonstrate that the distinct low-frequency motions of two loops in pnbCE, coil_5 and coil_21, are important in substrate conversion and seal the active site. Mutant CEs lacking these external loops show significantly reduced rates of substrate conversion, suggesting this sealing motion prevents escape of substrate. Overall, the results of our studies give new insight into the structure-function relationship of CEs and have implications for the entire family of α/β fold family of hydrolases, of which this CE is a member. PMID:22127613

Geometrical criteria for characterizing open and closed states of WPD-loop in PTP1B

NASA Astrophysics Data System (ADS)

Shinde, Ranajit Nivrutti; Elizabeth Sobhia, M.

2012-06-01

Distinctive movement of WPD-loop occurs during the catalysis of phosphotyrosine by protein tyrosine phosphatase 1B (PTP1B). This loop is in the "open" state in apo-form whereas it is catalytically competent in the "closed" state. During the closure of this loop, unique hydrogen bond interactions are formed between different residues of the PTP1B. Present study examines such interactions from the available 118 crystal structures of PTP1B. It gives insights into the five novel hydrogen bonds essentially formed in the "closed" loop structures. Additionally, the study provides distance ranges between the atoms involved in the hydrogen bonds. This information can be used as a geometrical criterion in the characterization of conformational state of the WPD-loop especially in the molecular dynamics simulations.

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

PubMed Central

Miao, Li-Li; Hou, Yan-Jie; Fan, Hong-Xia; Qu, Jie; Qi, Chao; Liu, Ying

2016-01-01

Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures. PMID:26801571

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus.

PubMed

Miao, Li-Li; Hou, Yan-Jie; Fan, Hong-Xia; Qu, Jie; Qi, Chao; Liu, Ying; Li, De-Feng; Liu, Zhi-Pei

2016-01-22

Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Conservative Tryptophan Mutants of the Protein Tyrosine Phosphatase YopH Exhibit Impaired WPD-Loop Function and Crystallize with Divanadate Esters in Their Active Sites

PubMed Central

Moise, Gwendolyn; Gallup, Nathan M.; Alexandrova, Anastassia N.; Hengge, Alvan C.; Johnson, Sean J.

2016-01-01

Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution. PMID:26445170

Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics

PubMed Central

Smirnov, Ivan; Carletti, Eugénie; Kurkova, Inna; Nachon, Florian; Nicolet, Yvain; Mitkevich, Vladimir A.; Débat, Hélène; Avalle, Bérangère; Belogurov, Alexey A.; Kuznetsov, Nikita; Reshetnyak, Andrey; Masson, Patrick; Tonevitsky, Alexander G.; Ponomarenko, Natalia; Makarov, Alexander A.; Friboulet, Alain; Tramontano, Alfonso; Gabibov, Alexander

2011-01-01

Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (VL) and variable heavy (VH) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes. PMID:21896761

Loop-loop interactions govern multiple steps in indole-3-glycerol phosphate synthase catalysis

PubMed Central

Zaccardi, Margot J; O'Rourke, Kathleen F; Yezdimer, Eric M; Loggia, Laura J; Woldt, Svenja; Boehr, David D

2014-01-01

Substrate binding, product release, and likely chemical catalysis in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) are dependent on the structural dynamics of the β1α1 active-site loop. Statistical coupling analysis and molecular dynamic simulations had previously indicated that covarying residues in the β1α1 and β2α2 loops, corresponding to Arg54 and Asn90, respectively, in the Sulfolobus sulfataricus enzyme (ssIGPS), are likely important for coordinating functional motions of these loops. To test this hypothesis, we characterized site mutants at these positions for changes in catalytic function, protein stability and structural dynamics for the thermophilic ssIGPS enzyme. Although there were only modest changes in the overall steady-state kinetic parameters, solvent viscosity and solvent deuterium kinetic isotope effects indicated that these amino acid substitutions change the identity of the rate-determining step across multiple temperatures. Surprisingly, the N90A substitution had a dramatic effect on the general acid/base catalysis of the dehydration step, as indicated by the loss of the descending limb in the pH rate profile, which we had previously assigned to Lys53 on the β1α1 loop. These changes in enzyme function are accompanied with a quenching of ps-ns and µs-ms timescale motions in the β1α1 loop as measured by nuclear magnetic resonance studies. Altogether, our studies provide structural, dynamic and functional rationales for the coevolution of residues on the β1α1 and β2α2 loops, and highlight the multiple roles that the β1α1 loop plays in IGPS catalysis. Thus, substitution of covarying residues in the active-site β1α1 and β2α2 loops of indole-3-glycerol phosphate synthase results in functional, structural, and dynamic changes, highlighting the multiple roles that the β1α1 loop plays in enzyme catalysis and the importance of regulating the structural dynamics of this loop through noncovalent interactions with nearby structural elements. PMID:24403092

A mobile loop near the active site acts as a switch between the dual activities of a viral protease/deubiquitinase

PubMed Central

Ayach, Maya; Fieulaine, Sonia

2017-01-01

The positive-strand RNA virus Turnip yellow mosaic virus (TYMV) encodes an ovarian tumor (OTU)-like protease/deubiquitinase (PRO/DUB) protein domain involved both in proteolytic processing of the viral polyprotein through its PRO activity, and in removal of ubiquitin chains from ubiquitylated substrates through its DUB activity. Here, the crystal structures of TYMV PRO/DUB mutants and molecular dynamics simulations reveal that an idiosyncratic mobile loop participates in reversibly constricting its unusual catalytic site by adopting "open", "intermediate" or "closed" conformations. The two cis-prolines of the loop form a rigid flap that in the most closed conformation zips up against the other side of the catalytic cleft. The intermediate and closed conformations also correlate with a reordering of the TYMV PRO/DUB catalytic dyad, that then assumes a classical, yet still unusually mobile, OTU DUB alignment. Further structure-based mutants designed to interfere with the loop's mobility were assessed for enzymatic activity in vitro and in vivo, and were shown to display reduced DUB activity while retaining PRO activity. This indicates that control of the switching between the dual PRO/DUB activities resides prominently within this loop next to the active site. Introduction of mutations into the viral genome revealed that the DUB activity contributes to the extent of viral RNA accumulation both in single cells and in whole plants. In addition, the conformation of the mobile flap was also found to influence symptoms severity in planta. Such mutants now provide powerful tools with which to study the specific roles of reversible ubiquitylation in viral infection. PMID:29117247

Active site loop dynamics of a class IIa fructose 1,6-bisphosphate aldolase from M. tuberculosis

PubMed Central

Pegan, Scott D.; Rukseree, Kamolchanok; Capodagli, Glenn C.; Baker, Erica A; Krasnykh, Olga; Franzblau, Scott G; Mesecar, Andrew D

2014-01-01

Class II fructose 1,6-bisphosphate aldolases (FBA; E.C. 4.1.2.13) comprise one of two families of aldolases. Instead of forming a Schiff-base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs has been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies on class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation/deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI/DHAP bound form of the enzyme and determined the X-ray structure of MtFBA-PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information plus site-directed mutagenesis and kinetic studies conducted on a series of residues within the active-site loop revealed that E169 facilitates a water mediated deprotonation/protonation step of the MtFBA reaction mechanism. Also, secondary isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form. PMID:23298222

Active site loop dynamics of a class IIa fructose 1,6-bisphosphate aldolase from Mycobacterium tuberculosis.

PubMed

Pegan, Scott D; Rukseree, Kamolchanok; Capodagli, Glenn C; Baker, Erica A; Krasnykh, Olga; Franzblau, Scott G; Mesecar, Andrew D

2013-02-05

Class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprise one of two families of aldolases. Instead of forming a Schiff base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs have been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies of class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria, and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation-deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and determined the X-ray structure of the MtFBA-PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information and site-directed mutagenesis and kinetic studies conducted on a series of residues within the active site loop revealed that E169 facilitates a water-mediated deprotonation-protonation step of the MtFBA reaction mechanism. Also, solvent isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.

Active Site Loop Dynamics of a Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pegan, Scott D.; Rukseree, Kamolchanok; Capodagli, Glenn C.

The class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprises one of two families of aldolases. Instead of forming a Schiff base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs have been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies of class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria, and protozoa have been reported,more » the structure of the active site loop responsible for catalyzing the protonation–deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and determined the X-ray structure of the MtFBA–PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information and site-directed mutagenesis and kinetic studies conducted on a series of residues within the active site loop revealed that E169 facilitates a water-mediated deprotonation–protonation step of the MtFBA reaction mechanism. Furthermore, solvent isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.« less

An auto-inhibitory helix in CTP:phosphocholine cytidylyltransferase hijacks the catalytic residue and constrains a pliable, domain-bridging helix pair

PubMed Central

Ramezanpour, Mohsen; Lee, Jaeyong; Taneva, Svetla G.; Tieleman, D. Peter; Cornell, Rosemary B.

2018-01-01

The activity of CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme in phosphatidylcholine synthesis, is regulated by reversible interactions of a lipid-inducible amphipathic helix (domain M) with membrane phospholipids. When dissociated from membranes, a portion of the M domain functions as an auto-inhibitory (AI) element to suppress catalysis. The AI helix from each subunit binds to a pair of α helices (αE) that extend from the base of the catalytic dimer to create a four-helix bundle. The bound AI helices make intimate contact with loop L2, housing a key catalytic residue, Lys122. The impacts of the AI helix on active-site dynamics and positioning of Lys122 are unknown. Extensive MD simulations with and without the AI helix revealed that backbone carbonyl oxygens at the point of contact between the AI helix and loop L2 can entrap the Lys122 side chain, effectively competing with the substrate, CTP. In silico, removal of the AI helices dramatically increased αE dynamics at a predicted break in the middle of these helices, enabling them to splay apart and forge new contacts with loop L2. In vitro cross-linking confirmed the reorganization of the αE element upon membrane binding of the AI helix. Moreover, when αE bending was prevented by disulfide engineering, CCT activation by membrane binding was thwarted. These findings suggest a novel two-part auto-inhibitory mechanism for CCT involving capture of Lys122 and restraint of the pliable αE helices. We propose that membrane binding enables bending of the αE helices, bringing the active site closer to the membrane surface. PMID:29519816

Conformational motions regulate phosphoryl transfer in related protein tyrosine phosphatases

PubMed Central

Whittier, Sean K.; Hengge, Alvan C.; Loria, J. Patrick

2014-01-01

Many studies have implicated a role for conformational motions during the catalytic cycle, acting to optimize the binding pocket or facilitate product release, but a more intimate role in the chemical reaction has not been described. We address this by monitoring active-site loop motion in two protein tyrosine phosphatases (PTPs) using NMR spectroscopy. The PTPs, YopH and PTP1B, have very different catalytic rates, however we find in both that the active-site loop closes to its catalytically competent position at rates that mirror the phosphotyrosine cleavage kinetics. This loop contains the catalytic acid, suggesting that loop closure occurs concomitantly with the protonation of the leaving group tyrosine and explains the different kinetics of two otherwise chemically and mechanistically indistinguishable enzymes. PMID:23970698

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bodenheimer, Annette M.; Meilleur, Flora

Trichoderma reesei Cel7A efficiently hydrolyses cellulose. We report here the crystallographic structures of the wild-type TrCel7A catalytic domain (CD) in an open state and, for the first time, in a closed state. Molecular dynamics (MD) simulations indicate that the loops along the CD tunnel move in concerted motions. Together, the crystallographic and MD data suggest that the CD cycles between the tense and relaxed forms that are characteristic of work producing enzymes. Analysis of the interactions formed by R251 provides a structural rationale for the concurrent decrease in product inhibition and catalytic efficiency measured for product-binding site mutants.

Role of coupled dynamics in the catalytic activity of prokaryotic-like prolyl-tRNA synthetases.

PubMed

Sanford, Brianne; Cao, Bach; Johnson, James M; Zimmerman, Kurt; Strom, Alexander M; Mueller, Robyn M; Bhattacharyya, Sudeep; Musier-Forsyth, Karin; Hati, Sanchita

2012-03-13

Prolyl-tRNA synthetases (ProRSs) have been shown to activate both cognate and some noncognate amino acids and attach them to specific tRNA(Pro) substrates. For example, alanine, which is smaller than cognate proline, is misactivated by Escherichia coli ProRS. Mischarged Ala-tRNA(Pro) is hydrolyzed by an editing domain (INS) that is distinct from the activation domain. It was previously shown that deletion of the INS greatly reduced cognate proline activation efficiency. In this study, experimental and computational approaches were used to test the hypothesis that deletion of the INS alters the internal protein dynamics leading to reduced catalytic function. Kinetic studies with two ProRS variants, G217A and E218A, revealed decreased amino acid activation efficiency. Molecular dynamics studies showed motional coupling between the INS and protein segments containing the catalytically important proline-binding loop (PBL, residues 199-206). In particular, the complete deletion of INS, as well as mutation of G217 or E218 to alanine, exhibited significant effects on the motion of the PBL. The presence of coupled dynamics between neighboring protein segments was also observed through in silico mutations and essential dynamics analysis. Altogether, this study demonstrates that structural elements at the editing domain-activation domain interface participate in coupled motions that facilitate amino acid binding and catalysis by bacterial ProRSs, which may explain why truncated or defunct editing domains have been maintained in some systems, despite the lack of catalytic activity.

Role of Coupled-Dynamics in the Catalytic Activity of Prokaryotic-like Prolyl-tRNA Synthetases

PubMed Central

Sanford, Brianne; Cao, Bach; Johnson, James M.; Zimmerman, Kurt; Strom, Alexander M.; Mueller, Robyn M.; Bhattacharyya, Sudeep; Musier-Forsyth, Karin; Hati, Sanchita

2012-01-01

Prolyl-tRNA synthetases (ProRSs) have been shown to activate both cognate and some noncognate amino acids and attach them to specific tRNAPro substrates. For example, alanine, which is smaller than cognate proline, is misactivated by Escherichia coli ProRS. Mischarged Ala-tRNAPro is hydrolyzed by an editing domain (INS) that is distinct from the activation domain. It was previously shown that deletion of the INS greatly reduced cognate proline activation efficiency. In the present study, experimental and computational approaches were used to test the hypothesis that INS deletion alters the internal protein dynamics leading to reduce catalytic function. Kinetic studies with two ProRS variants, G217A and E218A, revealed decreased amino acid activation efficiency. Molecular dynamics studies showed motional coupling between the INS and protein segments containing the catalytically important proline-binding loop (PBL, residues 199–206). In particular, the complete deletion of INS, as well as mutation of G217 or E218 to alanine, exhibited significant effects on the motion of the PBL. The presence of coupled-dynamics between neighboring protein segments was also observed through in silico mutations and essential dynamics analysis. Taken together, the present study demonstrates that structural elements at the editing domain-activation domain interface participate in coupled motions that facilitate amino acid binding and catalysis by bacterial ProRSs, which may explain why truncated or defunct editing domains have been maintained in some systems, despite the lack of catalytic activity. PMID:22356126

Engineering a hyper-catalytic enzyme by photo-activated conformation modulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agarwal, Pratul K

2012-01-01

Enzyme engineering for improved catalysis has wide implications. We describe a novel chemical modification of Candida antarctica lipase B that allows modulation of the enzyme conformation to promote catalysis. Computational modeling was used to identify dynamical enzyme regions that impact the catalytic mechanism. Surface loop regions located distal to active site but showing dynamical coupling to the reaction were connected by a chemical bridge between Lys136 and Pro192, containing a derivative of azobenzene. The conformational modulation of the enzyme was achieved using two sources of light that alternated the azobenzene moiety in cis and trans conformations. Computational model predicted thatmore » mechanical energy from the conformational fluctuations facilitate the reaction in the active-site. The results were consistent with predictions as the activity of the engineered enzyme was found to be enhanced with photoactivation. Preliminary estimations indicate that the engineered enzyme achieved 8-52 fold better catalytic activity than the unmodulated enzyme.« less

Crystal structure analysis, covalent docking, and molecular dynamics calculations reveal a conformational switch in PhaZ7 PHB depolymerase.

PubMed

Kellici, Tahsin F; Mavromoustakos, Thomas; Jendrossek, Dieter; Papageorgiou, Anastassios C

2017-07-01

An open and a closed conformation of a surface loop in PhaZ7 extracellular poly(3-hydroxybutyrate) depolymerase were identified in two high-resolution crystal structures of a PhaZ7 Y105E mutant. Molecular dynamics (MD) simulations revealed high root mean square fluctuations (RMSF) of the 281-295 loop, in particular at residue Asp289 (RMSF 7.62 Å). Covalent docking between a 3-hydroxybutyric acid trimer and the catalytic residue Ser136 showed that the binding energy of the substrate is significantly more favorable in the open loop conformation compared to that in the closed loop conformation. MD simulations with the substrate covalently bound depicted 1 Å RMSF higher values for the residues 281-295 in comparison to the apo (substrate-free) form. In addition, the presence of the substrate in the active site enhanced the ability of the loop to adopt a closed form. Taken together, the analysis suggests that the flexible loop 281-295 of PhaZ7 depolymerase can act as a lid domain to control substrate access to the active site of the enzyme. Proteins 2017; 85:1351-1361. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A competent catalytic active site is necessary for substrate induced dimer assembly in triosephosphate isomerase.

PubMed

Jimenez-Sandoval, Pedro; Vique-Sanchez, Jose Luis; Hidalgo, Marisol López; Velazquez-Juarez, Gilberto; Diaz-Quezada, Corina; Arroyo-Navarro, Luis Fernando; Moran, Gabriela Montero; Fattori, Juliana; Jessica Diaz-Salazar, A; Rudiño-Pinera, Enrique; Sotelo-Mundo, Rogerio; Figueira, Ana Carolina Migliorini; Lara-Gonzalez, Samuel; Benítez-Cardoza, Claudia G; Brieba, Luis G

2017-11-01

The protozoan parasite Trichomonas vaginalis contains two nearly identical triosephosphate isomerases (TvTIMs) that dissociate into stable monomers and dimerize upon substrate binding. Herein, we compare the role of the "ball and socket" and loop 3 interactions in substrate assisted dimer assembly in both TvTIMs. We found that point mutants at the "ball" are only 39 and 29-fold less catalytically active than their corresponding wild-type counterparts, whereas Δloop 3 deletions are 1502 and 9400-fold less active. Point and deletion mutants dissociate into stable monomers. However, point mutants assemble as catalytic competent dimers upon binding of the transition state substrate analog PGH, whereas loop 3 deletions remain monomeric. A comparison between crystal structures of point and loop 3 deletion monomeric mutants illustrates that the catalytic residues in point mutants and wild-type TvTIMs are maintained in the same orientation, whereas the catalytic residues in deletion mutants show an increase in thermal mobility and present structural disorder that may hamper their catalytic role. The high enzymatic activity present in monomeric point mutants correlates with the formation of dimeric TvTIMs upon substrate binding. In contrast, the low activity and lack of dimer assembly in deletion mutants suggests a role of loop 3 in promoting the formation of the active site as well as dimer assembly. Our results suggest that in TvTIMs the active site is assembled during dimerization and that the integrity of loop 3 and ball and socket residues is crucial to stabilize the dimer. Copyright © 2017 Elsevier B.V. All rights reserved.

The SH2 Domain Regulates c-Abl Kinase Activation by a Cyclin-Like Mechanism and Remodulation of the Hinge Motion

PubMed Central

Dölker, Nicole; Górna, Maria W.; Sutto, Ludovico; Torralba, Antonio S.; Superti-Furga, Giulio; Gervasio, Francesco L.

2014-01-01

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors. PMID:25299346

The SH2 domain regulates c-Abl kinase activation by a cyclin-like mechanism and remodulation of the hinge motion.

PubMed

Dölker, Nicole; Górna, Maria W; Sutto, Ludovico; Torralba, Antonio S; Superti-Furga, Giulio; Gervasio, Francesco L

2014-10-01

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors.

Computational simulations assessment of mutations impact on streptokinase (SK) from a group G streptococci with enhanced activity - insights into the functional roles of structural dynamics flexibility of SK and stabilization of SK-μplasmin catalytic complex.

PubMed

Kazemi, Faegheh; Arab, Seyed Shahriar; Mohajel, Nasir; Keramati, Malihe; Niknam, Niloofar; Aslani, Mohammad Mehdi; Roohvand, Farzin

2018-05-28

Streptokinase (SK), a plasminogen activator (PA) that converts inactive plasminogen (Pg) to plasmin (Pm), is a protein secreted by groups A, C, and G streptococci (GAS, GCS, and GGS, respectively), with high sequence divergence and functional heterogeneity. While roles of some residual changes in altered SK functionality are shown, the underlying structural mechanisms are less known. Herein, using computational approaches, we analyzed the conformational basis for the increased activity of SK from a GGS (SKG132) isolate with four natural residual substitutions (Ile33Phe, Arg45Gln, Asn228Lys, Phe287Ile) compared to the standard GCS (SKC). Using the crystal structure of SK.Pm catalytic complex as main template SKC.μPm catalytic complex was modeled through homology modeling process and validated by several online validation servers. Subsequently, SKG132.μPm structure was constructed by altering the corresponding residual substitutions. Results of three independent MD simulations showed increased RMSF values for SKG132.μPm, indicating the enhanced structural flexibility compared to SKC.μPm, specially in 170 and 250 loops and three regions: R1 (149-161), R2 (182-215) and R3 (224-229). In parallel, the average number of Hydrogen bonds in 170 loop, R2 and R3 (especially for Asn228Lys) of SKG132 compared to that of the SKC was decreased. Accordingly, residue interaction networks (RINs) analyses indicated that Asn228Lys might induce more level of structural flexibility by generation of free Lys256, while Phe287Ile and Ile33Phe enhanced the stabilization of the SKG132.μPm catalytic complex. These results denoted the potential role of the optimal dynamic state and stabilized catalytic complex for increased PA potencies of SK as a thrombolytic drug.

Computational Investigation of the pH Dependence of Loop Flexibility and Catalytic Function in Glycoside Hydrolases*

PubMed Central

Bu, Lintao; Crowley, Michael F.; Himmel, Michael E.; Beckham, Gregg T.

2013-01-01

Cellulase enzymes cleave glycosidic bonds in cellulose to produce cellobiose via either retaining or inverting hydrolysis mechanisms, which are significantly pH-dependent. Many fungal cellulases function optimally at pH ∼5, and their activities decrease dramatically at higher or lower pH. To understand the molecular-level implications of pH in cellulase structure, we use a hybrid, solvent-based, constant pH molecular dynamics method combined with pH-based replica exchange to determine the pKa values of titratable residues of a glycoside hydrolase (GH) family 6 cellobiohydrolase (Cel6A) and a GH family 7 cellobiohydrolase (Cel7A) from the fungus Hypocrea jecorina. For both enzymes, we demonstrate that a bound substrate significantly affects the pKa values of the acid residues at the catalytic center. The calculated pKa values of catalytic residues confirm their proposed roles from structural studies and are consistent with the experimentally measured apparent pKa values. Additionally, GHs are known to impart a strained pucker conformation in carbohydrate substrates in active sites for catalysis, and results from free energy calculations combined with constant pH molecular dynamics suggest that the correct ring pucker is stable near the optimal pH for both Cel6A and Cel7A. Much longer molecular dynamics simulations of Cel6A and Cel7A with fixed protonation states based on the calculated pKa values suggest that pH affects the flexibility of tunnel loops, which likely affects processivity and substrate complexation. Taken together, this work demonstrates several molecular-level effects of pH on GH enzymes important for cellulose turnover in the biosphere and relevant to biomass conversion processes. PMID:23504310

Stem-Loop V of Varkud Satellite RNA Exhibits Characteristics of the Mg2+ Bound Structure in the Presence of Monovalent Ions

PubMed Central

2015-01-01

The Varkud Satellite RNA contains a self-cleaving ribozyme that has been shown to function independently of its surroundings. This 160 nucleotide ribozyme adopts a catalytically active tertiary structure that includes a kissing hairpin complex formed by stem-loop I and stem-loop V (SLV). The five-nucleotide 5′-rUGACU loop of the isolated SLV has been shown to adopt a Mg2+-dependent U-turn structure by solution NMR. This U-turn hairpin is examined here by molecular dynamics simulations in the presence of monovalent and divalent ions. Simulations confirm on an all-atom level the hypotheses for the role of the Mg2+ ions in stabilizing the loop, as well as the role of the solvent exposed U700 base. Additionally, these simulations suggest the Mg2+-free stem-loop adopts a wide range of structures, including energetically favorable structures similar to the Mg2+-bound loop structure. We propose this structure is a “gatekeeper” or precursor to Mg2+ binding when those ions are present. PMID:26328924

Proteins with Novel Structure, Function and Dynamics

NASA Technical Reports Server (NTRS)

Pohorille, Andrew

2014-01-01

Recently, a small enzyme that ligates two RNA fragments with the rate of 10(exp 6) above background was evolved in vitro (Seelig and Szostak, Nature 448:828-831, 2007). This enzyme does not resemble any contemporary protein (Chao et al., Nature Chem. Biol. 9:81-83, 2013). It consists of a dynamic, catalytic loop, a small, rigid core containing two zinc ions coordinated by neighboring amino acids, and two highly flexible tails that might be unimportant for protein function. In contrast to other proteins, this enzyme does not contain ordered secondary structure elements, such as alpha-helix or beta-sheet. The loop is kept together by just two interactions of a charged residue and a histidine with a zinc ion, which they coordinate on the opposite side of the loop. Such structure appears to be very fragile. Surprisingly, computer simulations indicate otherwise. As the coordinating, charged residue is mutated to alanine, another, nearby charged residue takes its place, thus keeping the structure nearly intact. If this residue is also substituted by alanine a salt bridge involving two other, charged residues on the opposite sides of the loop keeps the loop in place. These adjustments are facilitated by high flexibility of the protein. Computational predictions have been confirmed experimentally, as both mutants retain full activity and overall structure. These results challenge our notions about what is required for protein activity and about the relationship between protein dynamics, stability and robustness. We hypothesize that small, highly dynamic proteins could be both active and fault tolerant in ways that many other proteins are not, i.e. they can adjust to retain their structure and activity even if subjected to mutations in structurally critical regions. This opens the doors for designing proteins with novel functions, structures and dynamics that have not been yet considered.

The Local Dinucleotide Preference of APOBEC3G Can Be Altered from 5′-CC to 5′-TC by a Single Amino Acid Substitution

PubMed Central

Rathore, Anurag; Carpenter, Michael A; Demir, Özlem; Ikeda, Terumasa; Li, Ming; Shaban, Nadine; Law, Emily K.; Anokhin, Dmitry; Brown, William L.; Amaro, Rommie E.; Harris, Reuben S.

2013-01-01

APOBEC3A and APOBEC3G are DNA cytosine deaminases with biological functions in foreign DNA and retrovirus restriction, respectively. APOBEC3A has an intrinsic preference for cytosine preceded by thymine (5′-TC) in single-stranded DNA substrates, whereas APOBEC3G prefers the target cytosine to be preceded by another cytosine (5′-CC). To determine the amino acids responsible for these strong dinucleotide preferences, we analyzed a series of chimeras in which putative DNA binding loop regions of APOBEC3G were replaced with the corresponding regions from APOBEC3A. Loop 3 replacement enhanced APOBEC3G catalytic activity but did not alter its intrinsic 5′-CC dinucleotide substrate preference. Loop 7 replacement caused APOBEC3G to become APOBEC3A-like and strongly prefer 5′-TC substrates. Simultaneous loop 3/7 replacement resulted in a hyperactive APOBEC3G variant that also preferred 5′-TC dinucleotides. Single amino acid exchanges revealed D317 as a critical determinant of dinucleotide substrate specificity. Multi-copy explicitly solvated all-atom molecular dynamics simulations suggested a model in which D317 acts as a helix-capping residue by constraining the mobility of loop 7, forming a novel binding pocket that favorably accommodates cytosine. All catalytically active APOBEC3G variants, regardless of dinucleotide preference, retained HIV-1 restriction activity. These data support a model in which the loop 7 region governs the selection of local dinucleotide substrates for deamination but is unlikely to be part of the higher level targeting mechanisms that direct these enzymes to biological substrates such as HIV-1 cDNA. PMID:23938202

Conformational Flexibility of a Short Loop near the Active Site of the SARS-3CLpro is Essential to Maintain Catalytic Activity

NASA Astrophysics Data System (ADS)

Li, Chunmei; Teng, Xin; Qi, Yifei; Tang, Bo; Shi, Hailing; Ma, Xiaomin; Lai, Luhua

2016-02-01

The SARS 3C-like proteinase (SARS-3CLpro), which is the main proteinase of the SARS coronavirus, is essential to the virus life cycle. This enzyme has been shown to be active as a dimer in which only one protomer is active. However, it remains unknown how the dimer structure maintains an active monomer conformation. It has been observed that the Ser139-Leu141 loop forms a short 310-helix that disrupts the catalytic machinery in the inactive monomer structure. We have tried to disrupt this helical conformation by mutating L141 to T in the stable inactive monomer G11A/R298A/Q299A. The resulting tetra-mutant G11A/L141T/R298A/Q299A is indeed enzymatically active as a monomer. Molecular dynamics simulations revealed that the L141T mutation disrupts the 310-helix and helps to stabilize the active conformation. The coil-310-helix conformational transition of the Ser139-Leu141 loop serves as an enzyme activity switch. Our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations.

Multiple roles of mobile active center loops in the E1 component of the Escherichia coli pyruvate dehydrogenase complex - Linkage of protein dynamics to catalysis

PubMed Central

Jordan, Frank; Arjunan, Palaniappa; Kale, Sachin; Nemeria, Natalia S.; Furey, William

2009-01-01

The region encompassing residues 401–413 on the E1 component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli comprises a loop (the inner loop) which was not seen in the X-ray structure in the presence of thiamin diphosphate, the required cofactor for the enzyme. This loop is seen in the presence of a stable analogue of the pre-decarboxylation intermediate, the covalent adduct between the substrate analogue methyl acetylphosphonate and thiamin diphosphate, C2α-phosphonolactylthiamin diphosphate. It has been shown that the residue H407 and several other residues on this loop are required to reduce the mobility of the loop so electron density corresponding to it can be seen once the pre-decarboxylation intermediate is formed. Concomitantly, the loop encompassing residues 541–557 (the outer loop) appears to work in tandem with the inner loop and there is a hydrogen bond between the two loops ensuring their correlated motion. The inner loop was shown to: a) sequester the active center from carboligase side reactions; b) assist the interaction between the E1 and the E2 components, thereby affecting the overall reaction rate of the entire multienzyme complex; c) control substrate access to the active center. Using viscosity effects on kinetics it was shown that formation of the pre-decarboxylation intermediate is specifically affected by loop movement. A cysteine-less variant was created for the E1 component, onto which cysteines were substituted at selected loop positions. Introducing an electron spin resonance spin label and an 19F NMR label onto these engineered cysteines, the loop mobility was examined: a) both methods suggested that in the absence of ligand, the loop exists in two conformations; b) line-shape analysis of the NMR signal at different temperatures, enabled estimation of the rate constant for loop movement, and this rate constant was found to be of the same order of magnitude as the turnover number for the enzyme under the same conditions. Furthermore, this analysis gave important insights into rate-limiting thermal loop dynamics. Overall, the results suggest that the dynamic properties correlate with catalytic events on the E1 component of the pyruvate dehydrogenase complex. PMID:20160956

Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase

PubMed Central

Helmstaedt, Kerstin; Heinrich, Gabriele; Lipscomb, William N.; Braus, Gerhard H.

2002-01-01

The yeast chorismate mutase is regulated by tyrosine as feedback inhibitor and tryptophan as crosspathway activator. The monomer consists of a catalytic and a regulatory domain covalently linked by the loop L220s (212–226), which functions as a molecular hinge. Two monomers form the active dimeric enzyme stabilized by hydrophobic interactions in the vicinity of loop L220s. The role of loop L220s and its environment for enzyme regulation, dimerization, and stability was analyzed. Substitution of yeast loop L220s in place of the homologous loop from the corresponding and similarly regulated Aspergillus enzyme (and the reverse substitution) changed tyrosine inhibition to activation. Yeast loop L220s substituted into the Aspergillus enzyme resulted in a tryptophan-inhibitable enzyme. Monomeric yeast chorismate mutases could be generated by substituting two hydrophobic residues in and near the hinge region. The resulting Thr-212→Asp–Phe-28→Asp enzyme was as stable as wild type, but lost allosteric regulation and showed reduced catalytic activity. These results underline the crucial role of this molecular hinge for inhibition, activation, quaternary structure, and stability of yeast chorismate mutase. PMID:11997452

Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding

DOE PAGES

Chen, Qi; Luan, Zheng -Jiao; Yu, Hui -Lei; ...

2015-10-31

A new carboxylic esterase RhEst1 which catalyzes the hydrolysis of (S)-(+)-2,2-dimethylcyclopropanecarboxylate (S-DmCpCe), the key chiral building block of cilastatin, was identified and subsequently crystallized in our previous work. Mutant RhEst 1A147I/V148F/G254A was found to show a 5-fold increase in the catalytic activity. In this work, molecular dynamic simulations were performed to elucidate the molecular determinant of the enzyme activity. Our simulations show that the substrate binds much more strongly in the A147I/V148F/G254A mutant than in wild type, with more hydrogen bonds formed between the substrate and the catalytic triad and the oxyanion hole. The OH group of the catalytic residuemore » Ser101 in the mutant is better positioned to initiate the nucleophilic attack on S-DmCpCe. Interestingly, the "170-179" loop which is involved in shaping the catalytic sites and facilitating the product release shows remarkable dynamic differences in the two systems. Based on the simulation results, six residues were identified as potential "hot-spots" for further experimental testing. Consequently, the G126S and R133L mutants show higher catalytic efficiency as compared with the wild type. In conclusion, this work provides molecular-level insights into the substrate binding mechanism of carboxylic esterase RhEst1, facilitating future experimental efforts toward developing more efficient RhEst1 variants for industrial applications.« less

Importance of Loop L1 Dynamics for Substrate Capture and Catalysis in Pseudomonas aeruginosa d-Arginine Dehydrogenase.

PubMed

Ouedraogo, Daniel; Souffrant, Michael; Vasquez, Sheena; Hamelberg, Donald; Gadda, Giovanni

2017-05-16

Mobile loops located at the active site entrance in enzymes often participate in conformational changes required to shield the reaction from bulk solvent, to control the access of the substrate to the active site, and to position residues for substrate binding and catalysis. In d-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH), previous crystallographic data suggested that residues 45-47 in the FAD-binding domain and residues 50-56 in the substrate-binding domain in loop L1 could adopt two distinct conformations. In this study, we have used molecular dynamics, kinetics, and fluorescence spectroscopy on the S45A and A46G enzyme variants of PaDADH to investigate the impact of mutations in loop L1 on the catalytic function of the enzyme. Molecular dynamics showed that the mutant enzymes have probabilities of being in open conformations that are higher than that of wild-type PaDADH of loop L1, yielding an increased level of solvent exposure of the active site. In agreement, the flavin fluorescence intensity was ∼2-fold higher in the S45A and A46G enzymes than in wild-type PaDADH, with a 9 nm bathochromic shift of the emission band. In the variant enzymes, the k cat /K m values with d-arginine were ∼13-fold lower than in wild-type PaDADH. Moreover, the pH profiles for the k cat value with d-arginine showed a hollow, consistent with restricted proton movements in catalysis, and no saturation was achieved with the alternate substrate d-leucine in the reductive half-reaction of the variant enzymes. Taken together, the computational and experimental data are consistent with the dynamics of loop L1 being important for substrate capture and catalysis in PaDADH.

Activation Loop Dynamics Determine the Different Catalytic Efficiencies of B Cell- and T Cell-Specific Tec Kinases

PubMed Central

Joseph, Raji E.; Kleino, Iivari; Wales, Thomas E.; Xie, Qian; Fulton, D. Bruce; Engen, John R.; Berg, Leslie J.; Andreotti, Amy H.

2014-01-01

Itk and Btk are nonreceptor tyrosine kinases of the Tec family that signal downstream of the T cell receptor (TCR) and B cell receptor (BCR), respectively. Despite their high sequence similarity and related signaling roles, Btk is a substantially more active kinase than Itk. We showed that substitution of six of the 619 amino acid residues of Itk with those of Btk was sufficient to completely switch the activities of Itk and Btk. The substitutions responsible for the swap in activity are all localized to the activation segment of the kinase domain. Nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses revealed that Itk and Btk had distinct protein dynamics in this region, which could explain the observed differences in catalytic efficiency between these kinases. Introducing Itk with enhanced activity into T cells led to enhanced and prolonged TCR signaling compared to that in cells with wild-type Itk. These findings imply that evolutionary pressures have led to Tec kinases having distinct enzymatic properties depending on the cellular context. We suggest that the weaker catalytic activities observed for T cell–specific kinases is one mechanism to regulate cellular activation and prevent aberrant immune responses. PMID:23982207

[Deletion of a dynamic surface loop improves thermostability of (R)-selective amine transaminase from Aspergillus terreus].

PubMed

Xie, Dongfang; Lv, Changjiang; Fang, Hui; Yang, Weikang; Hu, Sheng; Zhao, Weirui; Huang, Jun; Mei, Lehe

2017-12-25

Chiral amines are important building blocks for the synthesis of pharmaceutical products and fine chemicals. Highly stereoselective synthesis of chiral amines compounds through asymmetric amination has attracted more and more attention. ω-transaminases (ω-TAs) are a promising class of natural biocatalysts which provide an efficient and environment-friendly access to production of chiral amines with stringent enantioselectivity and excellent catalytic efficiency. Compared with (S)-ω-TA, the research focused on (R)-ω-TA was relatively less. However, increasing demand for chiral (R)-amines as pharmaceutical intermediates has rendered industrial applications of (R)-ω-TA more attractive. Improving the thermostability of (R)-ω-TA with potential biotechnological application will facilitate the preparation of chiral amines. In this study, the dynamic surface loop with higher B-factor from Aspergillus terreus (R)-ω-TA was predicted by two computer softwares (PyMOL and YASARA). Then mutant enzymes were obtained by deleting amino acid residues of a dynamic surface loop using site-directed mutagenesis. The results showed that the best two mutants R131del and P132-E133del improved thermostability by 2.6 ℃ and 0.9 ℃ in T₅₀¹⁰ (41.1 ℃ and 39.4 ℃, respectively), and 2.2-fold and 1.5-fold in half-life (t1/2) at 40 ℃ (15.0 min and 10.0 min, respectively), compared to that of wild type. Furtherly, the thermostability mechanism of the mutant enzymes was investigated by molecular dynamics (MD) simulation and intermolecular interaction analysis. R131del in the loop region has lower root mean square fluctuation (RMSF) than the wild type at 400 K for 10 ns, and mutant enzyme P132-E133del increases four hydrogen bonds in the loop region. In this study, we obtain two stability-increased mutants of (R)-ω-TA from A. terreus by deleting its dynamic surface loop and also provide methodological guidance for the use of rational design to enhance the thermal stability of other enzymes.

A comprehensive functional analysis of PTEN mutations: implications in tumor- and autism-related syndromes.

PubMed

Rodríguez-Escudero, Isabel; Oliver, María D; Andrés-Pons, Amparo; Molina, María; Cid, Víctor J; Pulido, Rafael

2011-11-01

The PTEN (phosphatase and tensin homolog) phosphatase is unique in mammals in terms of its tumor suppressor activity, exerted by dephosphorylation of the lipid second messenger PIP(3) (phosphatidylinositol 3,4,5-trisphosphate), which activates the phosphoinositide 3-kinase/Akt/mTOR (mammalian target of rapamycin) oncogenic pathway. Loss-of-function mutations in the PTEN gene are frequent in human cancer and in the germline of patients with PTEN hamartoma tumor-related syndromes (PHTSs). In addition, PTEN is mutated in patients with autism spectrum disorders (ASDs), although no functional information on these mutations is available. Here, we report a comprehensive in vivo functional analysis of human PTEN using a heterologous yeast reconstitution system. Ala-scanning mutagenesis at the catalytic loops of PTEN outlined the critical role of residues within the P-catalytic loop for PIP(3) phosphatase activity in vivo. PTEN mutations that mimic the P-catalytic loop of mammalian PTEN-like proteins (TPTE, TPIP, tensins and auxilins) affected PTEN function variably, whereas tumor- or PHTS-associated mutations targeting the PTEN P-loop produced complete loss of function. Conversely, Ala-substitutions, as well as tumor-related mutations at the WPD- and TI-catalytic loops, displayed partial activity in many cases. Interestingly, a tumor-related D92N mutation was partially active, supporting the notion that the PTEN Asp92 residue might not function as the catalytic general acid. The analysis of a panel of ASD-associated hereditary PTEN mutations revealed that most of them did not substantially abrogate PTEN activity in vivo, whereas most of PHTS-associated mutations did. Our findings reveal distinctive functional patterns among PTEN mutations found in tumors and in the germline of PHTS and ASD patients, which could be relevant for therapy.

Homology models of main proteinase from coronavirus associated with SARS

NASA Astrophysics Data System (ADS)

Liu, Hsuan-Liang; Lin, Jin-Chung; Ho, Yih; Chen, Chin-Wen

2005-01-01

In this study, two homology models of the main proteinase (M pro) from the novel coronavirus associated with severe acute respiratory syndrome (SARS-CoV) were constructed. These models reveal three distinct functional domains, in which an intervening loop connecting domains II and III as well as a catalytic cleft containing the substrate binding subsites S1 and S2 between domains I and II are observed. S2 exhibits structural variations more significantly than S1 during the 200 ps molecular dynamics simulations because it is located at the open mouth of the catalytic cleft and the amino acid residues lining up this subsite are least conserved. In addition, the higher structural variation of S2 makes it flexible enough to accommodate a bulky hydrophobic residue from the substrate.

Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding.

PubMed

Chen, Qi; Luan, Zheng-Jiao; Yu, Hui-Lei; Cheng, Xiaolin; Xu, Jian-He

2015-11-01

A new carboxylic esterase RhEst1 which catalyzes the hydrolysis of (S)-(+)-2,2-dimethylcyclopropanecarboxylate (S-DmCpCe), the key chiral building block of cilastatin, was identified and subsequently crystallized in our previous work. Mutant RhEst1A147I/V148F/G254A was found to show a 5-fold increase in the catalytic activity. In this work, molecular dynamic simulations were performed to elucidate the molecular determinant of the enzyme activity. Our simulations show that the substrate binds much more strongly in the A147I/V148F/G254A mutant than in wild type, with more hydrogen bonds formed between the substrate and the catalytic triad and the oxyanion hole. The OH group of the catalytic residue Ser101 in the mutant is better positioned to initiate the nucleophilic attack on S-DmCpCe. Interestingly, the "170-179" loop which is involved in shaping the catalytic sites and facilitating the product release shows remarkable dynamic differences in the two systems. Based on the simulation results, six residues were identified as potential "hot-spots" for further experimental testing. Consequently, the G126S and R133L mutants show higher catalytic efficiency as compared with the wild type. This work provides molecular-level insights into the substrate binding mechanism of carboxylic esterase RhEst1, facilitating future experimental efforts toward developing more efficient RhEst1 variants for industrial applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of protonation state of Asp181 and position of active site water molecules on the conformation of PTP1B.

PubMed

Ozcan, Ahmet; Olmez, Elif Ozkirimli; Alakent, Burak

2013-05-01

In protein tyrosine phosphatase 1B (PTP1B), the flexible WPD loop adopts a closed conformation (WPDclosed ) in the active state of PTP1B, bringing the catalytic Asp181 close to the active site pocket, while WPD loop is in an open conformation (WPDopen ) in the inactive state. Previous studies showed that Asp181 may be protonated at physiological pH, and ordered water molecules exist in the active site. In the current study, molecular dynamics simulations are employed at different Asp181 protonation states and initial positions of active site water molecules, and compared with the existing crystallographic data of PTP1B. In WPDclosed conformation, the active site is found to maintain its conformation only in the protonated state of Asp181 in both free and liganded states, while Asp181 is likely to be deprotonated in WPDopen conformation. When the active site water molecule network that is a part of the free WPDclosed crystal structure is disrupted, intermediate WPD loop conformations, similar to that in the PTPRR crystal structure, are sampled in the MD simulations. In liganded PTP1B, one active site water molecule is found to be important for facilitating the orientation of Cys215 and the phosphate ion, thus may play a role in the reaction. In conclusion, conformational stability of WPD loop, and possibly catalytic activity of PTP1B, is significantly affected by the protonation state of Asp181 and position of active site water molecules, showing that these aspects should be taken into consideration both in MD simulations and inhibitor design. Copyright © 2013 Wiley Periodicals, Inc.

Optimal management of nutrient reserves in microorganisms under time-varying environmental conditions.

PubMed

Nev, Olga A; Nev, Oleg A; van den Berg, Hugo A

2017-09-21

Intracellular reserves are a conspicuous feature of many bacteria; such internal stores are often present in the form of inclusions in which polymeric storage compounds are accumulated. Such reserves tend to increase in times of plenty and be used up in times of scarcity. Mathematical models that describe the dynamical nature of reserve build-up and use are known as "cell quota," "dynamic energy/nutrient budget," or "variable-internal-stores" models. Here we present a stoichiometrically consistent macro-chemical model that accounts for variable stores as well as adaptive allocation of building blocks to various types of catalytic machinery. The model posits feedback loops linking expression of assimilatory machinery to reserve density. The precise form of the "regulatory law" at the heart of such a loop expresses how the cell manages internal stores. We demonstrate how this "regulatory law" can be recovered from experimental data using several empirical data sets. We find that stores should be expected to be negligibly small in stable growth-sustaining environments, but prominent in environments characterised by marked fluctuations on time scales commensurate with the inherent dynamic time scale of the organismal system. Copyright © 2017 Elsevier Ltd. All rights reserved.

Epoxide Hydrolase Conformational Heterogeneity for the Resolution of Bulky Pharmacologically Relevant Epoxide Substrates.

PubMed

Serrano-Hervás, Eila; Casadevall, Guillem; Garcia-Borràs, Marc; Feixas, Ferran; Osuna, Sílvia

2018-04-06

The conformational landscape of Bacillus megaterium epoxide hydrolase (BmEH) and how it is altered by mutations that confer the enzyme the ability to accept bulky epoxide substrates has been investigated. Extensive molecular dynamics (MD) simulations coupled to active site volume calculations have unveiled relevant features of the enzyme conformational dynamics and function. Our long-timescale MD simulations identify key conformational states not previously observed by means of X-ray crystallography and short MD simulations that present the loop containing one of the catalytic residues, Asp239, in a wide-open conformation, which is likely involved in the binding of the epoxide substrate. Introduction of mutations M145S and F128A dramatically alters the conformational landscape of the enzyme. These singly mutated variants can accept bulky epoxide substrates due to the disorder induced by mutation in the α-helix containing the catalytic Tyr144 and some parts of the lid domain. These changes impact the enzyme active site, which is substantially wider and more complementary to the bulky pharmacologically relevant epoxide substrates. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Connecting Active-Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue 96 in the Plasmodial Enzyme.

PubMed

Pareek, Vidhi; Samanta, Moumita; Joshi, Niranjan V; Balaram, Hemalatha; Murthy, Mathur R N; Balaram, Padmanabhan

2016-04-01

Despite extensive research into triosephosphate isomerases (TIMs), there exists a gap in understanding of the remarkable conjunction between catalytic loop-6 (residues 166-176) movement and the conformational flip of Glu165 (catalytic base) upon substrate binding that primes the active site for efficient catalysis. The overwhelming occurrence of serine at position 96 (98% of the 6277 unique TIM sequences), spatially proximal to E165 and the loop-6 residues, raises questions about its role in catalysis. Notably, Plasmodium falciparum TIM has an extremely rare residue--phenylalanine--at this position whereas, curiously, the mutant F96S was catalytically defective. We have obtained insights into the influence of residue 96 on the loop-6 conformational flip and E165 positioning by combining kinetic and structural studies on the PfTIM F96 mutants F96Y, F96A, F96S/S73A, and F96S/L167V with sequence conservation analysis and comparative analysis of the available apo and holo structures of the enzyme from diverse organisms. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Redox 2-Cys Mechanism Regulates the Catalytic Activity of Divergent Cyclophilins1[W

PubMed Central

Campos, Bruna Medéia; Sforça, Mauricio Luis; Ambrosio, Andre Luis Berteli; Domingues, Mariane Noronha; Brasil de Souza, Tatiana de Arruda Campos; Barbosa, João Alexandre Ribeiro Gonçalvez; Leme, Adriana Franco Paes; Perez, Carlos Alberto; Whittaker, Sara Britt-Marie; Murakami, Mario Tyago; Zeri, Ana Carolina de Matos; Benedetti, Celso Eduardo

2013-01-01

The citrus (Citrus sinensis) cyclophilin CsCyp is a target of the Xanthomonas citri transcription activator-like effector PthA, required to elicit cankers on citrus. CsCyp binds the citrus thioredoxin CsTdx and the carboxyl-terminal domain of RNA polymerase II and is a divergent cyclophilin that carries the additional loop KSGKPLH, invariable cysteine (Cys) residues Cys-40 and Cys-168, and the conserved glutamate (Glu) Glu-83. Despite the suggested roles in ATP and metal binding, the functions of these unique structural elements remain unknown. Here, we show that the conserved Cys residues form a disulfide bond that inactivates the enzyme, whereas Glu-83, which belongs to the catalytic loop and is also critical for enzyme activity, is anchored to the divergent loop to maintain the active site open. In addition, we demonstrate that Cys-40 and Cys-168 are required for the interaction with CsTdx and that CsCyp binds the citrus carboxyl-terminal domain of RNA polymerase II YSPSAP repeat. Our data support a model where formation of the Cys-40-Cys-168 disulfide bond induces a conformational change that disrupts the interaction of the divergent and catalytic loops, via Glu-83, causing the active site to close. This suggests a new type of allosteric regulation in divergent cyclophilins, involving disulfide bond formation and a loop-displacement mechanism. PMID:23709667

Functional roles of H98 and W99 and β2α2 loop dynamics in the α-l-arabinofuranosidase from Thermobacillus xylanilyticus.

PubMed

Arab-Jaziri, Faten; Bissaro, Bastien; Barbe, Sophie; Saurel, Olivier; Débat, Hélène; Dumon, Claire; Gervais, Virginie; Milon, Alain; André, Isabelle; Fauré, Régis; O'Donohue, Michael J

2012-10-01

This study is focused on the elucidation of the functional role of the mobile β2α2 loop in the α-L-arabinofuranosidase from Thermobacillus xylanilyticus, and particularly on the roles of loop residues H98 and W99. Using site-directed mutagenesis, coupled to characterization methods including isothermal titration calorimetry (ITC) and saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy, and molecular dynamics simulations, it has been possible to provide a molecular level view of interactions and the consequences of mutations. Binding of para-nitrophenyl α-L-arabinofuranoside (pNP-α-l-Araf) to the wild-type arabinofuranosidase was characterized by K(d) values (0.32 and 0.16 mm, from ITC and STD-NMR respectively) that highly resembled that of the arabinoxylo-oligosaccharide XA(3)XX (0.21 mm), and determination of the thermodynamic parameters of enzyme : pNP-α-L-Araf binding revealed that this process is driven by favourable entropy, which is linked to the movement of the β2α2 loop. Loop closure relocates the solvent-exposed W99 into a buried location, allowing its involvement in substrate binding and in the formation of a functional active site. Similarly, the data underline the role of H98 in the ‘dynamic’ formation and definition of a catalytically operational active site, which may be a specific feature of a subset of GH51 arabinofuranosidases. Substitution of H98 and W99 by alanine or phenylalanine revealed that mutations affected K(M) and/or k(cat). Molecular dynamics performed on W99A implied that this mutation causes the loss of a hydrogen bond and leads to an alternative binding mode that is detrimental for catalysis. STD-NMR experiments revealed altered binding of the aglycon motif in the active site, combined with reduced STD intensities of the α-L-arabinofuranosyl moiety for W99 substitutions. © 2012 The Authors Journal compilation © 2012 FEBS.

A double-headed cathepsin B inhibitor devoid of warhead

PubMed Central

Schenker, Patricia; Alfarano, Pietro; Kolb, Peter; Caflisch, Amedeo; Baici, Antonio

2008-01-01

Most synthetic inhibitors of peptidases have been targeted to the active site for inhibiting catalysis through reversible competition with the substrate or by covalent modification of catalytic groups. Cathepsin B is unique among the cysteine peptidase for the presence of a flexible segment, known as the occluding loop, which can block the primed subsites of the substrate binding cleft. With the occluding loop in the open conformation cathepsin B acts as an endopeptidase, and it acts as an exopeptidase when the loop is closed. We have targeted the occluding loop of human cathepsin B at its surface, outside the catalytic center, using a high-throughput docking procedure. The aim was to identify inhibitors that would interact with the occluding loop thereby modulating enzyme activity without the help of chemical warheads against catalytic residues. From a large library of compounds, the in silico approach identified [2-[2-(2,4-dioxo-1,3-thiazolidin-3-yl)ethylamino]-2-oxoethyl] 2-(furan-2-carbonylamino) acetate, which fulfills the working hypothesis. This molecule possesses two distinct binding moieties and behaves as a reversible, double-headed competitive inhibitor of cathepsin B by excluding synthetic and protein substrates from the active center. The kinetic mechanism of inhibition suggests that the occluding loop is stabilized in its closed conformation, mainly by hydrogen bonds with the inhibitor, thus decreasing endoproteolytic activity of the enzyme. Furthermore, the dioxothiazolidine head of the compound sterically hinders binding of the C-terminal residue of substrates resulting in inhibition of the exopeptidase activity of cathepsin B in a physiopathologically relevant pH range. PMID:18796695

Accurate placement of substrate RNA by Gar1 in H/ACA RNA-guided pseudouridylation.

PubMed

Wang, Peng; Yang, Lijiang; Gao, Yi Qin; Zhao, Xin Sheng

2015-09-03

H/ACA RNA-guided ribonucleoprotein particle (RNP), the most complicated RNA pseudouridylase so far known, uses H/ACA guide RNA for substrate capture and four proteins (Cbf5, Nop10, L7Ae and Gar1) for pseudouridylation. Although it was shown that Gar1 not only facilitates the product release, but also enhances the catalytic activity, the chemical role that Gar1 plays in this complicated machinery is largely unknown. Kinetics measurement on Pyrococcus furiosus RNPs at different temperatures making use of fluorescence anisotropy showed that Gar1 reduces the catalytic barrier through affecting the activation entropy instead of enthalpy. Site-directed mutagenesis combined with molecular dynamics simulations demonstrated that V149 in the thumb loop of Cbf5 is critical in placing the target uridine to the right position toward catalytic D85 of Cbf5. The enzyme elegantly aligns the position of uridine in the catalytic site with the help of Gar1. In addition, conversion of uridine to pseudouridine results in a rigid syn configuration of the target nucleotide in the active site and causes Gar1 to pull out the thumb. Both factors guarantee the efficient release of the product. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis

PubMed Central

O’Brien, Darragh P.; Durand, Dominique; Voegele, Alexis; Hourdel, Véronique; Davi, Marilyne; Chamot-Rooke, Julia; Vachette, Patrice; Brier, Sébastien; Ladant, Daniel

2017-01-01

Once translocated into the cytosol of target cells, the catalytic domain (AC) of the adenylate cyclase toxin (CyaA), a major virulence factor of Bordetella pertussis, is potently activated by binding calmodulin (CaM) to produce supraphysiological levels of cAMP, inducing cell death. Using a combination of small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), and synchrotron radiation circular dichroism (SR-CD), we show that, in the absence of CaM, AC exhibits significant structural disorder, and a 75-residue-long stretch within AC undergoes a disorder-to-order transition upon CaM binding. Beyond this local folding, CaM binding induces long-range allosteric effects that stabilize the distant catalytic site, whilst preserving catalytic loop flexibility. We propose that the high enzymatic activity of AC is due to a tight balance between the CaM-induced decrease of structural flexibility around the catalytic site and the preservation of catalytic loop flexibility, allowing for fast substrate binding and product release. The CaM-induced dampening of AC conformational disorder is likely relevant to other CaM-activated enzymes. PMID:29287065

Tryptophan as a Molecular Shovel in the Glycosyl Transfer Activity of Trypanosoma cruzi Trans-sialidase

PubMed Central

Mitchell, Felicity L.; Miles, Steven M.; Neres, João; Bichenkova, Elena V.; Bryce, Richard A.

2010-01-01

Abstract Molecular dynamics investigations into active site plasticity of Trypanosoma cruzi trans-sialidase, a protein implicated in Chagas disease, suggest that movement of the Trp312 loop plays an important role in the enzyme's sialic acid transfer mechanism. The observed Trp312 flexibility equates to a molecular shovel action, which leads to the expulsion of the donor aglycone leaving group from the catalytic site. These computational simulations provide detailed structural insights into sialyl transfer by the trans-sialidase and may aid the design of inhibitors effective against this neglected tropical disease. PMID:20441732

Conformational Rigidity and Protein Dynamics at Distinct Timescales Regulate PTP1B Activity and Allostery.

PubMed

Choy, Meng S; Li, Yang; Machado, Luciana E S F; Kunze, Micha B A; Connors, Christopher R; Wei, Xingyu; Lindorff-Larsen, Kresten; Page, Rebecca; Peti, Wolfgang

2017-02-16

Protein function originates from a cooperation of structural rigidity, dynamics at different timescales, and allostery. However, how these three pillars of protein function are integrated is still only poorly understood. Here we show how these pillars are connected in Protein Tyrosine Phosphatase 1B (PTP1B), a drug target for diabetes and cancer that catalyzes the dephosphorylation of numerous substrates in essential signaling pathways. By combining new experimental and computational data on WT-PTP1B and ≥10 PTP1B variants in multiple states, we discovered a fundamental and evolutionarily conserved CH/π switch that is critical for positioning the catalytically important WPD loop. Furthermore, our data show that PTP1B uses conformational and dynamic allostery to regulate its activity. This shows that both conformational rigidity and dynamics are essential for controlling protein activity. This connection between rigidity and dynamics at different timescales is likely a hallmark of all enzyme function. Copyright © 2017 Elsevier Inc. All rights reserved.

Atomistic insights into regulatory mechanisms of the HER2 tyrosine kinase domain: a molecular dynamics study.

PubMed

Telesco, Shannon E; Radhakrishnan, Ravi

2009-03-18

HER2 (ErbB2/Neu) is a receptor tyrosine kinase belonging to the epidermal growth factor receptor (EGFR)/ErbB family and is overexpressed in 20-30% of human breast cancers. Although several crystal structures of ErbB kinases have been solved, the precise mechanism of HER2 activation remains unknown, and it has been suggested that HER2 is unique in its requirement for phosphorylation of Y877, a key tyrosine residue located in the activation loop. To elucidate mechanistic details of kinase domain regulation, we performed molecular dynamics simulations of a homology-modeled HER2 kinase structure in active and inactive conformations. Principal component analysis of the atomistic fluctuations reveals a tight coupling between the activation loop and catalytic loop that may contribute to alignment of residues required for catalysis in the active kinase. The free energy perturbation method is also employed to predict a role for phosphorylated Y877 in stabilizing the kinase conformations. Finally, simulation results are presented for a HER2/EGFR heterodimer and reveal that the dimeric interface induces a rearrangement of the alphaC helix toward the active conformation. Elucidation of the molecular regulatory mechanisms in HER2 will help establish structure-function relationships in the wild-type kinase, as well as predict mutations with a propensity for constitutive activation in HER2-mediated cancers.

A comparative molecular dynamics study of thermophilic and mesophilic β-fructosidase enzymes.

PubMed

Mazola, Yuliet; Guirola, Osmany; Palomares, Sucel; Chinea, Glay; Menéndez, Carmen; Hernández, Lázaro; Musacchio, Alexis

2015-09-01

Arabidopsis thaliana cell wall invertase 1 (AtcwINV1) and Thermotoga maritima β-fructosidase (BfrA) are among the best structurally studied members of the glycoside hydrolase family 32. Both enzymes hydrolyze sucrose as the main substrate but differ strongly in their thermal stability. Mesophilic AtcwINV1 and thermophilic BfrA have divergent sequence similarities in the N-terminal five bladed β-propeller catalytic domain (31 %) and the C-terminal β-sandwich domain (15 %) of unknown function. The two enzymes were subjected to 200 ns molecular dynamics simulations at 300 K (27 °C) and 353 K (80 °C). Regular secondary structure regions, but not loops, in AtcwINV1 and BfrA showed no significant fluctuation differences at both temperatures. BfrA was more rigid than AtcwINV1 at 300 K. The simulation at 353 K did not alter the structural stability of BfrA, but did increase the overall flexibility of AtcwINV1 exhibiting the most fluctuating regions in the β-propeller domain. The simulated heat treatment also increased the gyration radius and hydrophobic solvent accessible surface area of the plant enzyme, consistent with the initial steps of an unfolding process. The preservation of the conformational rigidity of BfrA at 353 K is linked to the shorter size of the protein loops. Shortening of BfrA loops appears to be a key mechanism for thermostability.

Primordial evolvability: Impasses and challenges.

PubMed

Vasas, Vera; Fernando, Chrisantha; Szilágyi, András; Zachár, István; Santos, Mauro; Szathmáry, Eörs

2015-09-21

While it is generally agreed that some kind of replicating non-living compounds were the precursors of life, there is much debate over their possible chemical nature. Metabolism-first approaches propose that mutually catalytic sets of simple organic molecules could be capable of self-replication and rudimentary chemical evolution. In particular, the graded autocatalysis replication domain (GARD) model, depicting assemblies of amphiphilic molecules, has received considerable interest. The system propagates compositional information across generations and is suggested to be a target of natural selection. However, evolutionary simulations indicate that the system lacks selectability (i.e. selection has negligible effect on the equilibrium concentrations). We elaborate on the lessons learnt from the example of the GARD model and, more widely, on the issue of evolvability, and discuss the implications for similar metabolism-first scenarios. We found that simple incorporation-type chemistry based on non-covalent bonds, as assumed in GARD, is unlikely to result in alternative autocatalytic cycles when catalytic interactions are randomly distributed. An even more serious problem stems from the lognormal distribution of catalytic factors, causing inherent kinetic instability of such loops, due to the dominance of efficiently catalyzed components that fail to return catalytic aid. Accordingly, the dynamics of the GARD model is dominated by strongly catalytic, but not auto-catalytic, molecules. Without effective autocatalysis, stable hereditary propagation is not possible. Many repetitions and different scaling of the model come to no rescue. Despite all attempts to show the contrary, the GARD model is not evolvable, in contrast to reflexively autocatalytic networks, complemented by rare uncatalyzed reactions and compartmentation. The latter networks, resting on the creation and breakage of chemical bonds, can generate novel ('mutant') autocatalytic loops from a given set of environmentally available compounds. Real chemical reactions that make or break covalent bonds, rather than mere incorporation of components, are necessary for open-ended evolvability. The issue of whether or not several concrete chemical systems (rather than singular curiosities) could realize reflexively autocatalytic macromolecular networks will ultimately determine the relevance of metabolism-first approaches to the origin of life, as stepping stones towards true open-endedness that requires the combination of rich combinatorial chemistry controlled by information stored in template replicators. Copyright © 2015 Elsevier Ltd. All rights reserved.

Entropic stabilization of a deubiquitinase provides conformational plasticity and slow unfolding kinetics beneficial for functioning on the proteasome

PubMed Central

Lee, Yun-Tzai Cloud; Chang, Chia-Yun; Chen, Szu-Yu; Pan, Yun-Ru; Ho, Meng-Ru; Hsu, Shang-Te Danny

2017-01-01

Human ubiquitin C-terminal hydrolyase UCH-L5 is a topologically knotted deubiquitinase that is activated upon binding to the proteasome subunit Rpn13. The length of its intrinsically disordered cross-over loop is essential for substrate recognition. Here, we showed that the catalytic domain of UCH-L5 exhibits higher equilibrium folding stability with an unfolding rate on the scale of 10−8 s−1, over four orders of magnitudes slower than its paralogs, namely UCH-L1 and -L3, which have shorter cross-over loops. NMR relaxation dynamics analysis confirmed the intrinsic disorder of the cross-over loop. Hydrogen deuterium exchange analysis further revealed a positive correlation between the length of the cross-over loop and the degree of local fluctuations, despite UCH-L5 being thermodynamically and kinetically more stable than the shorter UCHs. Considering the role of UCH-L5 in removing K48-linked ubiquitin to prevent proteasomal degradation of ubiquitinated substrates, our findings offered mechanistic insights into the evolution of UCH-L5. Compared to its paralogs, it is entropically stabilized to withstand mechanical unfolding by the proteasome while maintaining structural plasticity. It can therefore accommodate a broad range of substrate geometries at the cost of unfavourable entropic loss. PMID:28338014

MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding.

PubMed

Asamitsu, Kaori; Hirokawa, Takatsugu; Okamoto, Takashi

2017-01-01

In this study, we applied molecular dynamics (MD) simulation to analyze the dynamic behavior of the Tat/CycT1/CDK9 tri-molecular complex and revealed the structural changes of P-TEFb upon Tat binding. We found that Tat could deliberately change the local flexibility of CycT1. Although the structural coordinates of the H1 and H2 helices did not substantially change, H1', H2', and H3' exhibited significant changes en masse. Consequently, the CycT1 residues involved in Tat binding, namely Tat-recognition residues (TRRs), lost their flexibility with the addition of Tat to P-TEFb. In addition, we clarified the structural variation of CDK9 in complex with CycT1 in the presence or absence of Tat. Interestingly, Tat addition significantly reduced the structural variability of the T-loop, thus consolidating the structural integrity of P-TEFb. Finally, we deciphered the formation of the hidden catalytic cavity of CDK9 upon Tat binding. MD simulation revealed that the PITALRE signature sequence of CDK9 flips the inactive kinase cavity of CDK9 into the active form by connecting with Thr186, which is crucial for its activity, thus presumably recruiting the substrate peptide such as the C-terminal domain of RNA pol II. These findings provide vital information for the development of effective novel anti-HIV drugs with CDK9 catalytic activity as the target.

Structural prediction of a novel chitinase from the psychrophilic Glaciozyma antarctica PI12 and an analysis of its structural properties and function

NASA Astrophysics Data System (ADS)

Ramli, Aizi Nor Mazila; Mahadi, Nor Muhammad; Shamsir, Mohd Shahir; Rabu, Amir; Joyce-Tan, Kwee Hong; Murad, Abdul Munir Abdul; Illias, Rosli Md.

2012-08-01

The structure of psychrophilic chitinase (CHI II) from Glaciozyma antarctica PI12 has yet to be studied in detail. Due to its low sequence identity (<30 %), the structural prediction of CHI II is a challenge. A 3D model of CHI II was built by first using a threading approach to search for a suitable template and to generate an optimum target-template alignment, followed by model building using MODELLER9v7. Analysis of the catalytic insertion domain structure in CHI II revealed an increase in the number of aromatic residues and longer loops compared to mesophilic and thermophilic chitinases. A molecular dynamics simulation was used to examine the stability of the CHI II structure at 273, 288 and 300 K. Structural analysis of the substrate-binding cleft revealed a few exposed aromatic residues. Substitutions of certain amino acids in the surface and loop regions of CHI II conferred an increased flexibility to the enzyme, allowing for an adaptation to cold temperatures. A substrate binding comparison of CHI II with the mesophilic chitinase from Coccidioides immitis, 1D2K, suggested that the psychrophilic adaptation and catalytic activity at low temperatures were achieved through a reduction in the number of salt bridges, fewer hydrogen bonds and an increase in the exposure of the hydrophobic side chains to the solvent.

Backbone resonance assignment of an insect arylalkylamine N-acetyltransferase from Bombyx mori reveals conformational heterogeneity.

PubMed

Aboalroub, Adam A; Zhang, Ziming; Keramisanou, Dimitra; Gelis, Ioannis

2017-04-01

Arylalkylamine N-acetyltransferases (AANATs) catalyze the transfer of an acetyl group from the acetyl-group donor, acetyl-CoA, to an arylalkylamine acceptor. Although a single AANAT has been identified in mammals, insects utilize multiple AANATs in a diverse array of biological processes. AANATs belong to the GCN5-related acetyltransferase (GNAT) superfamily of enzymes, which despite their overall very low sequence homology, are characterized by a well conserved catalytic core domain. The structural properties of many GNATs have been extensively studied by X-ray crystallography that revealed common features during the catalytic cycle. Here we report the 1 H, 13 C and 15 N backbone NMR resonance assignment of the 24 kDa AANAT3 from Bombyx mori (bmAANAT3) as a first step towards understanding the role of protein dynamics in the catalytic properties of AANATs. Our preliminary solution NMR studies reveal that bmAANAT3 is well-folded in solution. The P-loop, which is responsible for cofactor binding, is flexible in the free-state, while a large region of the enzyme interconverts between two distinct conformations in the slow exchange regime.

Acetyl group coordinated progression through the catalytic cycle of an arylalkylamine N-acetyltransferase.

PubMed

Aboalroub, Adam A; Bachman, Ashleigh B; Zhang, Ziming; Keramisanou, Dimitra; Merkler, David J; Gelis, Ioannis

2017-01-01

The transfer of an acetyl group from acetyl-CoA to an acceptor amine is a ubiquitous biochemical transformation catalyzed by Gcn5-related N-acetyltransferases (GNATs). Although it is established that the reaction proceeds through a sequential ordered mechanism, the role of the acetyl group in driving the ordered formation of binary and ternary complexes remains elusive. Herein, we show that CoA and acetyl-CoA alter the conformation of the substrate binding site of an arylalkylamine N-acetyltransferase (AANAT) to facilitate interaction with acceptor substrates. However, it is the presence of the acetyl group within the catalytic funnel that triggers high affinity binding. Acetyl group occupancy is relayed through a conserved salt bridge between the P-loop and the acceptor binding site, and is manifested as differential dynamics in the CoA and acetyl-CoA-bound states. The capacity of the acetyl group carried by an acceptor to promote its tight binding even in the absence of CoA, but also its mutually exclusive position to the acetyl group of acetyl-CoA underscore its importance in coordinating the progression of the catalytic cycle.

Acetyl group coordinated progression through the catalytic cycle of an arylalkylamine N-acetyltransferase

PubMed Central

Aboalroub, Adam A.; Bachman, Ashleigh B.; Zhang, Ziming; Keramisanou, Dimitra; Merkler, David J.

2017-01-01

The transfer of an acetyl group from acetyl-CoA to an acceptor amine is a ubiquitous biochemical transformation catalyzed by Gcn5-related N-acetyltransferases (GNATs). Although it is established that the reaction proceeds through a sequential ordered mechanism, the role of the acetyl group in driving the ordered formation of binary and ternary complexes remains elusive. Herein, we show that CoA and acetyl-CoA alter the conformation of the substrate binding site of an arylalkylamine N-acetyltransferase (AANAT) to facilitate interaction with acceptor substrates. However, it is the presence of the acetyl group within the catalytic funnel that triggers high affinity binding. Acetyl group occupancy is relayed through a conserved salt bridge between the P-loop and the acceptor binding site, and is manifested as differential dynamics in the CoA and acetyl-CoA-bound states. The capacity of the acetyl group carried by an acceptor to promote its tight binding even in the absence of CoA, but also its mutually exclusive position to the acetyl group of acetyl-CoA underscore its importance in coordinating the progression of the catalytic cycle. PMID:28486510

Tryptophan as a molecular shovel in the glycosyl transfer activity of Trypanosoma cruzi trans-sialidase.

PubMed

Mitchell, Felicity L; Miles, Steven M; Neres, João; Bichenkova, Elena V; Bryce, Richard A

2010-05-19

Molecular dynamics investigations into active site plasticity of Trypanosoma cruzi trans-sialidase, a protein implicated in Chagas disease, suggest that movement of the Trp(312) loop plays an important role in the enzyme's sialic acid transfer mechanism. The observed Trp(312) flexibility equates to a molecular shovel action, which leads to the expulsion of the donor aglycone leaving group from the catalytic site. These computational simulations provide detailed structural insights into sialyl transfer by the trans-sialidase and may aid the design of inhibitors effective against this neglected tropical disease. Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Deletion of loop fragment adjacent to active site diminishes the stability and activity of exo-inulinase.

PubMed

Arjomand, Maryam Rezaei; Habibi-Rezaei, Mehran; Ahmadian, Gholamreza; Hassanzadeh, Malihe; Karkhane, Ali Asghar; Asadifar, Mandana; Amanlou, Massoud

2016-11-01

Inulinases are classified as hydrolases and widely used in the food and medical industries. Here, we report the deletion of a six-membered adjacent active site loop fragment ( 74 YGSDVT 79 sequence) from third Ω-loop of the exo-inulinase containing aspartate residue from Aspergillus niger to study its structural and functional importance. Site-directed mutagenesis was used to create the mutant of the exo-inulinase (Δ6SL). To investigate the stability of the region spanning this loop, MD simulations were performed 80ns for 20-85 residues. Molecular docking was performed to compare the interactions in the active sites of enzymes with fructose as a ligand. Accordingly, the functional thermostability of the exo-inulinase was significantly decreased upon loop fragment deletion. Evaluation of the kinetics parameters (V max , K m , k cat and, k cat /K m ) and activation energy (E a ) of the catalysis of enzymes indicated the importance of the deleted sequence on the catalytic performance of the enzyme. In conclusion, six-membered adjacent active site loop fragment not only plays a crucial role in the stability of the enzyme, but also it involves in the enzyme catalysis through lowering the activation energy of the catalysis and effective improving the catalytic performance. Copyright © 2016. Published by Elsevier B.V.

Efficient Ligation of the Schistosoma Hammerhead Ribozyme †

PubMed Central

Canny, Marella D.; Jucker, Fiona M.; Pardi, Arthur

2011-01-01

The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by a ~2,000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2–3 at physiological Mg2+ ion concentrations (0.1 –1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules, and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme. PMID:17319693

Structure of an E. coli integral membrane sulfurtransferase and its structural transition upon SCN- binding defined by EPR-based hybrid method

NASA Astrophysics Data System (ADS)

Ling, Shenglong; Wang, Wei; Yu, Lu; Peng, Junhui; Cai, Xiaoying; Xiong, Ying; Hayati, Zahra; Zhang, Longhua; Zhang, Zhiyong; Song, Likai; Tian, Changlin

2016-01-01

Electron paramagnetic resonance (EPR)-based hybrid experimental and computational approaches were applied to determine the structure of a full-length E. coli integral membrane sulfurtransferase, dimeric YgaP, and its structural and dynamic changes upon ligand binding. The solution NMR structures of the YgaP transmembrane domain (TMD) and cytosolic catalytic rhodanese domain were reported recently, but the tertiary fold of full-length YgaP was not yet available. Here, systematic site-specific EPR analysis defined a helix-loop-helix secondary structure of the YagP-TMD monomers using mobility, accessibility and membrane immersion measurements. The tertiary folds of dimeric YgaP-TMD and full-length YgaP in detergent micelles were determined through inter- and intra-monomer distance mapping and rigid-body computation. Further EPR analysis demonstrated the tight packing of the two YgaP second transmembrane helices upon binding of the catalytic product SCN-, which provides insight into the thiocyanate exportation mechanism of YgaP in the E. coli membrane.

Stopped-in-loop flow analysis of trace vanadium in water.

PubMed

Teshima, Norio; Ohno, Shinsuke; Sakai, Tadao

2007-01-01

The new concept of stopped-in-loop flow analysis (SIL-FA) is proposed, and an SIL-FA method for the catalytic determination of vanadium is demonstrated. In an SIL format, a sample solution merges with reagent(s), and the well-mixed solution is loaded into a loop. The solution in the loop is separated by a six-way switching valve from the main stream. While the reaction proceeds in the stationary loop, the SIL-FA system does not need to establish a baseline continuously. This leads to a reduction in reagent consumption and waste generation compared with traditional flow injection analysis.

Rice Cellulose SynthaseA8 Plant-Conserved Region Is a Coiled-Coil at the Catalytic Core Entrance1[OPEN

PubMed Central

Rushton, Phillip S.; Olek, Anna T.; Makowski, Lee; Badger, John

2017-01-01

The crystallographic structure of a rice (Oryza sativa) cellulose synthase, OsCesA8, plant-conserved region (P-CR), one of two unique domains in the catalytic domain of plant CesAs, was solved to 2.4 Å resolution. Two antiparallel α-helices form a coiled-coil domain linked by a large extended connector loop containing a conserved trio of aromatic residues. The P-CR structure was fit into a molecular envelope for the P-CR domain derived from small-angle X-ray scattering data. The P-CR structure and molecular envelope, combined with a homology-based chain trace of the CesA8 catalytic core, were modeled into a previously determined CesA8 small-angle X-ray scattering molecular envelope to produce a detailed topological model of the CesA8 catalytic domain. The predicted position for the P-CR domain from the molecular docking models places the P-CR connector loop into a hydrophobic pocket of the catalytic core, with the coiled-coil aligned near the entrance of the substrate UDP-glucose into the active site. In this configuration, the P-CR coiled-coil alone is unlikely to regulate substrate access to the active site, but it could interact with other domains of CesA, accessory proteins, or other CesA catalytic domains to control substrate delivery. PMID:27879387

Crystal structure analysis of a bacterial aryl acylamidase belonging to the amidase signature enzyme family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Saeyoung; Park, Eun-Hye; Ko, Hyeok-Jin

2015-11-13

The atomic structure of a bacterial aryl acylamidase (EC 3.5.1.13; AAA) is reported and structural features are investigated to better understand the catalytic profile of this enzyme. Structures of AAA were determined in its native form and in complex with the analgesic acetanilide, p-acetaminophenol, at 1.70 Å and 1.73 Å resolutions, respectively. The overall structural fold of AAA was identified as an α/β fold class, exhibiting an open twisted β-sheet core surrounded by α-helices. The asymmetric unit contains one AAA molecule and the monomeric form is functionally active. The core structure enclosing the signature sequence region, including the canonical Ser-cisSer-Lys catalytic triad,more » is conserved in all members of the Amidase Signature enzyme family. The structure of AAA in a complex with its ligand reveals a unique organization in the substrate-binding pocket. The binding pocket consists of two loops (loop1 and loop2) in the amidase signature sequence and one helix (α10) in the non-amidase signature sequence. We identified two residues (Tyr{sup 136} and Thr{sup 330}) that interact with the ligand via water molecules, and a hydrogen-bonding network that explains the catalytic affinity over various aryl acyl compounds. The optimum activity of AAA at pH > 10 suggests that the reaction mechanism employs Lys{sup 84} as the catalytic base to polarize the Ser{sup 187} nucleophile in the catalytic triad. - Highlights: • We determined the first structure of a bacterial aryl acylamidase (EC 3.5.1.13). • Structure revealed spatially distinct architecture of the substrate-binding pocket. • Hydrogen-bonding with Tyr{sup 136} and Thr{sup 330} mediates ligand-binding and substrate.« less

Structural dynamics of a methionine γ-lyase for calicheamicin biosynthesis: Rotation of the conserved tyrosine stacking with pyridoxal phosphate

DOE PAGES

Cao, Hongnan; Tan, Kemin; Wang, Fengbin; ...

2016-04-29

CalE6 from Micromonospora echinospora is a (pyridoxal 50 phosphate) PLP-dependent methionine γ-lyase involved in the biosynthesis of calicheamicins. Here, we report the crystal structure of a CalE6 2-(N-morpholino)ethanesulfonic acid complex showing ligand-induced rotation of Tyr100, which stacks with PLP, resembling the corresponding tyrosine rotation of true catalytic intermediates of CalE6 homologs. Elastic network modeling and crystallographic ensemble refinement reveal mobility of the N-terminal loop, which involves both tetrameric assembly and PLP binding. Modeling and comparative structural analysis of PLP-dependent enzymes involved in Cys/Met metabolism shine light on the functional implications of the intrinsic dynamic properties of CalE6 in catalysis andmore » holoenzyme maturation.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rushton, Phillip S.; Olek, Anna T.; Makowski, Lee

The crystallographic structure of a rice (Oryza sativa) cellulose synthase, OsCesA8, plant-conserved region (P-CR), one of two unique domains in the catalytic domain of plant CesAs, was solved to 2.4 Å resolution. Two antiparallel α-helices form a coiled-coil domain linked by a large extended connector loop containing a conserved trio of aromatic residues. The P-CR structure was fit into a molecular envelope for the P-CR domain derived from small-angle X-ray scattering data. The P-CR structure and molecular envelope, combined with a homology-based chain trace of the CesA8 catalytic core, were modeled into a previously determined CesA8 small-angle X-ray scattering molecularmore » envelope to produce a detailed topological model of the CesA8 catalytic domain. The predicted position for the P-CR domain from the molecular docking models places the P-CR connector loop into a hydrophobic pocket of the catalytic core, with the coiled-coil aligned near the entrance of the substrate UDP-glucose into the active site. In this configuration, the P-CR coiled-coil alone is unlikely to regulate substrate access to the active site, but it could interact with other domains of CesA, accessory proteins, or other CesA catalytic domains to control substrate delivery.« less

Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase).

PubMed

Rice, Amy J; Lei, Hao; Santarsiero, Bernard D; Lee, Hyun; Johnson, Michael E

2016-10-01

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine synthesis pathway and is responsible for the reversible cyclization of carbamyl-aspartate (Ca-asp) to dihydroorotate (DHO). DHOase is further divided into two classes based on several structural characteristics, one of which is the length of the flexible catalytic loop that interacts with the substrate, Ca-asp, regulating the enzyme activity. Here, we present the crystal structure of Class I Bacillus anthracis DHOase with Ca-asp in the active site, which shows the peptide backbone of glycine in the shorter loop forming the necessary hydrogen bonds with the substrate, in place of the two threonines found in Class II DHOases. Despite the differences in the catalytic loop, the structure confirms that the key interactions between the substrate and active site residues are similar between Class I and Class II DHOase enzymes, which we further validated by mutagenesis studies. B. anthracis DHOase is also a potential antibacterial drug target. In order to identify prospective inhibitors, we performed high-throughput screening against several libraries using a colorimetric enzymatic assay and an orthogonal fluorescence thermal binding assay. Surface plasmon resonance was used for determining binding affinity (KD) and competition analysis with Ca-asp. Our results highlight that the primary difference between Class I and Class II DHOase is the catalytic loop. We also identify several compounds that can potentially be further optimized as potential B. anthracis inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bae, Brian; Nayak, Dhananjaya; Ray, Ananya

RNA polymerase inhibitors like the CBR class that target the enzyme’s complex catalytic center are attractive leads for new antimicrobials. The catalysis by RNA polymerase involves multiple rearrangements of bridge helix, trigger loop, and active-center side chains that isomerize the triphosphate of bound NTP and two Mg 2+ ions from a preinsertion state to a reactive configuration. CBR inhibitors target a crevice between the N-terminal portion of the bridge helix and a surrounding cap region within which the bridge helix is thought to rearrange during the nucleotide addition cycle. Here, we report crystal structures of CBR inhibitor/Escherichia coli RNA polymerasemore » complexes as well as biochemical tests that establish two distinct effects of the inhibitors on the RNA polymerase catalytic site. One effect involves inhibition of trigger-loop folding via the F loop in the cap, which affects both nucleotide addition and hydrolysis of 3'-terminal dinucleotides in certain backtracked complexes. The second effect is trigger-loop independent, affects only nucleotide addition and pyrophosphorolysis, and may involve inhibition of bridge-helix movements that facilitate reactive triphosphate alignment.« less

The Role of the β5-α11 Loop in the Active-Site Dynamics of Acylated Penicillin-Binding Protein A from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fedarovich, Alena; Nicholas, Robert A.; Davies, Christopher

Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the second-order acylation rate constantsmore » for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the β5–α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5–α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states.« less

Identification and characterization of the sodium-binding site of activated protein C.

PubMed

He, X; Rezaie, A R

1999-02-19

Activated protein C (APC) requires both Ca2+ and Na+ for its optimal catalytic function. In contrast to the Ca2+-binding sites, the Na+-binding site(s) of APC has not been identified. Based on a recent study with thrombin, the 221-225 loop is predicted to be a potential Na+-binding site in APC. The sequence of this loop is not conserved in trypsin. We engineered a Gla domainless form of protein C (GDPC) in which the 221-225 loop was replaced with the corresponding loop of trypsin. We found that activated GDPC (aGDPC) required Na+ (or other alkali cations) for its amidolytic activity with dissociation constant (Kd(app)) = 44.1 +/- 8.6 mM. In the presence of Ca2+, however, the requirement for Na+ by aGDPC was eliminated, and Na+ stimulated the cleavage rate 5-6-fold with Kd(app) = 2.3 +/- 0.3 mM. Both cations were required for efficient factor Va inactivation by aGDPC. In the presence of Ca2+, the catalytic function of the mutant was independent of Na+. Unlike aGDPC, the mutant did not discriminate among monovalent cations. We conclude that the 221-225 loop is a Na+-binding site in APC and that an allosteric link between the Na+ and Ca2+ binding loops modulates the structure and function of this anticoagulant enzyme.

Structural dynamics of Casein Kinase I (CKI) from malarial parasite Plasmodium falciparum (Isolate 3D7): Insights from theoretical modelling and molecular simulations.

PubMed

Dehury, Budheswar; Behera, Santosh Kumar; Mahapatra, Namita

2017-01-01

The protein kinases (PKs), belonging to serine/threonine kinase (STKs), are important drug targets for a wide spectrum of diseases in human. Among protein kinases, the Casein Kinases (CKs) are vastly expanded in various organisms, where, the malarial parasite Plasmodium falciparum possesses a single member i.e., PfCKI, which can phosphorylate various proteins in parasite extracts in vitro condition. But, the structure-function relationship of PfCKI and dynamics of ATP binding is yet to be understood. Henceforth, an attempt was made to study the dynamics, stability, and ATP binding mechanisms of PfCKI through computational modelling, docking, molecular dynamics (MD) simulations, and MM/PBSA binding free energy estimation. Bi-lobed catalytic domain of PfCKI shares a high degree of secondary structure topology with CKI domains of rice, human, and mouse indicating co-evolution of these kinases. Molecular docking study revealed that ATP binds to the active site where the glycine-rich ATP-binding motif (G16-X-G18-X-X-G21) along with few conserved residues plays a crucial role maintaining stability of the complex. Structural superposition of PfCKI with close structural homologs depicted that the location and length of important loops are different, indicating the dynamic properties of these loops among CKIs, which is consistent with principal component analysis (PCA). PCA displayed that the overall global motion of ATP-bound form is comparatively higher than that of apo form. The present study provides insights into the structural features of PfCKI, which could contribute towards further understanding of related protein structures, dynamics of catalysis and phosphorylation mechanism in these important STKs from malarial parasite in near future. Copyright © 2016 Elsevier Inc. All rights reserved.

The catalytic mechanism of hairpin ribozyme studied by hydrostatic pressure

PubMed Central

Tobé, Sylvia; Heams, Thomas; Vergne, Jacques; Hervé, Guy; Maurel, Marie-Christine

2005-01-01

The discovery of ribozymes strengthened the RNA world hypothesis, which assumes that these precursors of modern life both stored information and acted as catalysts. For the first time among extensive studies on ribozymes, we have investigated the influence of hydrostatic pressure on the hairpin ribozyme catalytic activity. High pressures are of interest when studying life under extreme conditions and may help to understand the behavior of macromolecules at the origins of life. Kinetic studies of the hairpin ribozyme self-cleavage were performed under high hydrostatic pressure. The activation volume of the reaction (34 ± 5 ml/mol) calculated from these experiments is of the same order of magnitude as those of common protein enzymes, and reflects an important compaction of the RNA molecule during catalysis, associated to a water release. Kinetic studies were also carried out under osmotic pressure and confirmed this interpretation and the involvement of water movements (78 ± 4 water molecules per RNA molecule). Taken together, these results are consistent with structural studies indicating that loops A and B of the ribozyme come into close contact during the formation of the transition state. While validating baro-biochemistry as an efficient tool for investigating dynamics at work during RNA catalysis, these results provide a complementary view of ribozyme catalytic mechanisms. PMID:15870387

Structural Basis for the ATP-dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase*

PubMed Central

Chen, Yaozong; Sun, Yueru; Song, Haigang; Guo, Zhihong

2015-01-01

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of novel antibiotics in the menaquinone biosynthesis. Using its crystal structures in a ligand-free form or in complex with nucleotides, a conserved pattern is identified in the interaction between ATP and adenylating enzymes, including acyl/aryl-CoA synthetases, adenylation domains of nonribosomal peptide synthetases, and luciferases. It involves tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction creates a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and in-line alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling. In addition, the ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction has also clarified the positioning and catalytic role of a conserved lysine residue in stabilization of the transition state. These findings provide new insights into the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylate-forming enzymes. PMID:26276389

Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of cAMP-dependent protein kinase complexed with the peptide inhibitor PKI(5-24) and adenosine.

PubMed

Narayana, N; Cox, S; Shaltiel, S; Taylor, S S; Xuong, N

1997-04-15

The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H6-rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heat-stable protein kinase inhibitor PKI(5-24) and adenosine, was determined at 2.2 A resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final crystallographic R-factor of 18.2% with good stereochemical parameters. The "active" enzyme adopts a "closed" conformation as found in rC:PKI(5-24) [Knighton et al. (1991a,b) Science 253, 407-414, 414-420] and packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nucleotide binding site found in the protein kinase family. The adenosine occupies a mostly hydrophobic pocket at the base of the cleft between the two lobes and is completely buried. The missing triphosphate moiety of ATP is filled with a water molecule (Wtr 415) which replaces the gamma-phosphate of ATP. The glycine-rich loop between beta1 and beta2 helps to anchor the phosphates while the ribose ring is buried beneath beta-strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in beta-strand 1, Glu 127 in the linker strand between the two lobes, Tyr 330, and a third water molecule, Wtr 359. The conserved nucleotide fold can be defined as a lid comprised of beta-strand 1, the glycine-rich loop, and beta-strand 2. The adenine ring is buried beneath beta-strand 1 and the linker strand (120-127) that joins the small and large lobes. The C-terminal tail containing Tyr 330, a segment that lies outside the conserved core, covers this fold and anchors it in a closed conformation. The main-chain atoms of the flexible glycine-rich loop (residues 50-55) in the ATP binding domain have a mean B-factor of 41.4 A2. This loop is quite mobile, in striking contrast to the other conserved loops that converge at the active site cleft. The catalytic loop (residues 166-171) and the Mg2+ positioning loop (residues 184-186) are a stable part of the large lobe and have low B-factors in all structures solved to date. The stability of the glycine-rich loop is highly dependent on the ligands that occupy the active site cleft with maximum stability achieved in the ternary complex containing Mg x ATP and the peptide inhibitor. In this ternary complex the gamma-phosphate is secured between both lobes by hydrogen bonds to the backbone amide of Ser 53 in the glycine-rich loop and the amino group of Lys 168 in the catalytic loop. In the adenosine ternary complex the water molecule replacing the gamma-phosphate hydrogen bonds between Lys 168 and Asp 166 and makes no contact with the small lobe. This glycine-rich loop is thus the most mobile component of the active site cleft, with the tip of the loop being highly sensitive to what occupies the gamma-subsite.

Improving the reversibility of thermal denaturation and catalytic efficiency of Bacillus licheniformis α-amylase through stabilizing a long loop in domain B.

PubMed

Li, Zhu; Duan, Xuguo; Chen, Sheng; Wu, Jing

2017-01-01

The reversibility of thermal denaturation and catalytic efficiency of Bacillus licheniformis α-amylase were improved through site-directed mutagenesis. By using multiple sequence alignment and PoPMuSiC algorithm, Ser187 and Asn188, which located within a long loop in Domain B of Bacillus licheniformis α-amylase, were selected for mutation. In addition, Ala269, which is adjacent to Ser187 and Asn188, was also investigated. Seven mutants carrying the mutations S187D, N188T, N188S, A269K, A269K/S187D, S187D/N188T, and A269K/S187D/N188T were generated and characterized. The most thermostable mutant, A269K/S187D/N188T, exhibited a 9-fold improvement in half-life at 95°C and pH 5.5, compared with that of the wild-type enzyme. Mutant A269K/S187D/N188T also exhibited improved catalytic efficiency. The catalytic efficiency of mutant A269K/S187D/N188T reached 5.87×103±0.17 g·L-1·s-1 at pH 5.5, which is 1.84-fold larger than the corresponding value determined for the wild-type enzyme. Furthermore, the structure analysis showed that immobilization of the loop containing Ser187 and Asn188 plays a significant role in developing the properties of Bacillus licheniformis α-amylase.

Improving the reversibility of thermal denaturation and catalytic efficiency of Bacillus licheniformis α-amylase through stabilizing a long loop in domain B

PubMed Central

Li, Zhu; Duan, Xuguo; Chen, Sheng; Wu, Jing

2017-01-01

The reversibility of thermal denaturation and catalytic efficiency of Bacillus licheniformis α-amylase were improved through site-directed mutagenesis. By using multiple sequence alignment and PoPMuSiC algorithm, Ser187 and Asn188, which located within a long loop in Domain B of Bacillus licheniformis α-amylase, were selected for mutation. In addition, Ala269, which is adjacent to Ser187 and Asn188, was also investigated. Seven mutants carrying the mutations S187D, N188T, N188S, A269K, A269K/S187D, S187D/N188T, and A269K/S187D/N188T were generated and characterized. The most thermostable mutant, A269K/S187D/N188T, exhibited a 9-fold improvement in half-life at 95°C and pH 5.5, compared with that of the wild-type enzyme. Mutant A269K/S187D/N188T also exhibited improved catalytic efficiency. The catalytic efficiency of mutant A269K/S187D/N188T reached 5.87×103±0.17 g·L-1·s-1 at pH 5.5, which is 1.84-fold larger than the corresponding value determined for the wild-type enzyme. Furthermore, the structure analysis showed that immobilization of the loop containing Ser187 and Asn188 plays a significant role in developing the properties of Bacillus licheniformis α-amylase. PMID:28253342

Regenerable Incinerator Exhaust Purification and Trace Contaminant Control System

NASA Technical Reports Server (NTRS)

Finn, John E.; Cho, Shelia Y.; LeVan, M. Douglas

2003-01-01

In this novel approach to air purification, contaminants removed from a process air stream by a high-capacity adsorbent are displaced periodically by a warm, high-humidity, reverse-flow air stream. Displaced contaminants flow into a closed regeneration loop, in which organic compounds are oxidized catalytically and acid gases are removed by a gas- water contactor (which also serves as the source of the water vapor). These features are expected to result in a design that has few expendables and lower energy consumption than alternative regenerable techniques. The joint project between NASA Ames Research Center and Vanderbilt University has completed its third year. Breadboard development continues at NASA Ames, while Vanderbilt has completed most of its adsorption equilibria development. Vanderbilt has completed its fixed-bed apparatus for investigation of dynamic adsorption and desorption processes for trace organic compounds and water vapor, and is continuing its development of the mathematical model describing the column dynamics.

Interdomain Hydrophobic Interactions Modulate the Thermostability of Microbial Esterases from the Hormone-Sensitive Lipase Family*

PubMed Central

Li, Ping-Yi; Chen, Xiu-Lan; Ji, Peng; Li, Chun-Yang; Wang, Peng; Zhang, Yi; Xie, Bin-Bin; Qin, Qi-Long; Su, Hai-Nan; Zhou, Bai-Cheng; Zhang, Yu-Zhong; Zhang, Xi-Ying

2015-01-01

Microbial hormone-sensitive lipases (HSLs) contain a CAP domain and a catalytic domain. However, it remains unclear how the CAP domain interacts with the catalytic domain to maintain the stability of microbial HSLs. Here, we isolated an HSL esterase, E40, from a marine sedimental metagenomic library. E40 exhibited the maximal activity at 45 °C and was quite thermolabile, with a half-life of only 2 min at 40 °C, which may be an adaptation of E40 to the permanently cold sediment environment. The structure of E40 was solved to study its thermolability. Structural analysis showed that E40 lacks the interdomain hydrophobic interactions between loop 1 of the CAP domain and α7 of the catalytic domain compared with its thermostable homologs. Mutational analysis showed that the introduction of hydrophobic residues Trp202 and Phe203 in α7 significantly improved E40 stability and that a further introduction of hydrophobic residues in loop 1 made E40 more thermostable because of the formation of interdomain hydrophobic interactions. Altogether, the results indicate that the absence of interdomain hydrophobic interactions between loop 1 and α7 leads to the thermolability of E40. In addition, a comparative analysis of the structures of E40 and other thermolabile and thermostable HSLs suggests that the interdomain hydrophobic interactions between loop 1 and α7 are a key element for the thermostability of microbial HSLs. Therefore, this study not only illustrates the structural element leading to the thermolability of E40 but also reveals a structural determinant for HSL thermostability. PMID:25771540

Low and medium heating value coal gas catalytic combustor characterization

NASA Technical Reports Server (NTRS)

Schwab, J. A.

1982-01-01

Catalytic combustion with both low and medium heating value coal gases obtained from an operating gasifier was demonstrated. A practical operating range for efficient operation was determined, and also to identify potential problem areas were identified for consideration during stationary gas turbine engine design. The test rig consists of fuel injectors, a fuel-air premixing section, a catalytic reactor with thermocouple instrumentation and a single point, water cooled sample probe. The test rig included inlet and outlet transition pieces and was designed for installation into an existing test loop.

Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases.

PubMed

Val-Cid, Cristina; Biarnés, Xevi; Faijes, Magda; Planas, Antoni

2015-01-01

Hexosaminidases are involved in important biological processes catalyzing the hydrolysis of N-acetyl-hexosaminyl residues in glycosaminoglycans and glycoconjugates. The GH20 enzymes present diverse domain organizations for which we propose two minimal model architectures: Model A containing at least a non-catalytic GH20b domain and the catalytic one (GH20) always accompanied with an extra α-helix (GH20b-GH20-α), and Model B with only the catalytic GH20 domain. The large Bifidobacterium bifidum lacto-N-biosidase was used as a model protein to evaluate the minimal functional unit due to its interest and structural complexity. By expressing different truncated forms of this enzyme, we show that Model A architectures cannot be reduced to Model B. In particular, there are two structural requirements general to GH20 enzymes with Model A architecture. First, the non-catalytic domain GH20b at the N-terminus of the catalytic GH20 domain is required for expression and seems to stabilize it. Second, the substrate-binding cavity at the GH20 domain always involves a remote element provided by a long loop from the catalytic domain itself or, when this loop is short, by an element from another domain of the multidomain structure or from the dimeric partner. Particularly, the lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. We demonstrate restoration of activity of the inactive GH20b-GH20-α construct (model A architecture) by a complementation assay with the lectin-like domain. The engineering of minimal functional units of multidomain GH20 enzymes must consider these structural requirements.

Comparison of dynamics of wildtype and V94M human UDP-galactose 4-epimerase-A computational perspective on severe epimerase-deficiency galactosemia.

PubMed

Timson, David J; Lindert, Steffen

2013-09-10

UDP-galactose 4'-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr157) and the enzyme-bound NAD+ cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (kcat), with less substantial effects on Km. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia. Copyright © 2013 Elsevier B.V. All rights reserved.

Structure of an E. coli integral membrane sulfurtransferase and its structural transition upon SCN− binding defined by EPR-based hybrid method

PubMed Central

Ling, Shenglong; Wang, Wei; Yu, Lu; Peng, Junhui; Cai, Xiaoying; Xiong, Ying; Hayati, Zahra; Zhang, Longhua; Zhang, Zhiyong; Song, Likai; Tian, Changlin

2016-01-01

Electron paramagnetic resonance (EPR)-based hybrid experimental and computational approaches were applied to determine the structure of a full-length E. coli integral membrane sulfurtransferase, dimeric YgaP, and its structural and dynamic changes upon ligand binding. The solution NMR structures of the YgaP transmembrane domain (TMD) and cytosolic catalytic rhodanese domain were reported recently, but the tertiary fold of full-length YgaP was not yet available. Here, systematic site-specific EPR analysis defined a helix-loop-helix secondary structure of the YagP-TMD monomers using mobility, accessibility and membrane immersion measurements. The tertiary folds of dimeric YgaP-TMD and full-length YgaP in detergent micelles were determined through inter- and intra-monomer distance mapping and rigid-body computation. Further EPR analysis demonstrated the tight packing of the two YgaP second transmembrane helices upon binding of the catalytic product SCN−, which provides insight into the thiocyanate exportation mechanism of YgaP in the E. coli membrane. PMID:26817826

Structural assembly of the signaling competent ERK2–RSK1 heterodimeric protein kinase complex

PubMed Central

Alexa, Anita; Gógl, Gergő; Glatz, Gábor; Garai, Ágnes; Zeke, András; Varga, János; Dudás, Erika; Jeszenői, Norbert; Bodor, Andrea; Hetényi, Csaba; Reményi, Attila

2015-01-01

Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase–kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 “docking” groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they “readjust,” whereas generic kinase domain surface contacts bring them into a catalytically competent state. PMID:25730857

Catalytic Exhaust Gas Recirculation-Loop Reforming for High Efficiency in a Stoichiometric Spark-Ignited Engine through Thermochemical Recuperation and Dilution Limit Extension, Part 2: Engine Performance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Yan; Szybist, James P.; Pihl, Josh A.

This is the second part of a two-part investigation of on-board catalytic fuel reforming to increase the brake efficiency of a multicylinder, stoichiometric spark-ignited (SI) engine. In Part 1 of the investigation, we analytically and experimentally characterized the energetics and kinetics of a candidate reforming catalyst over a range of reforming equivalence ratios and oxygen concentration conditions to identify the best conditions for efficient reforming. In the present part of our investigation, we studied an engine strategy that combined exhaust gas recirculation (EGR)–loop reforming with dilution limit extension of the combustion. In our experiments, we found that, under an enginemore » operating condition of 2000 rpm and brake mean effective pressure (4 bar), catalytic EGR reforming made it possible to sustain stable combustion with a volumetric equivalent of 45%–55% EGR. Under this same operating condition with stoichiometric engine exhaust (and no reforming), we were only able to sustain stable combustion with EGR under 25%. In conclusion, these results indicate that multicylinder gasoline engine efficiency can be increased substantially with catalytic reforming combined with and higher EGR operation, resulting in a decrease of more than 8% in fuel consumption, compared to baseline operation.« less

Catalytic Exhaust Gas Recirculation-Loop Reforming for High Efficiency in a Stoichiometric Spark-Ignited Engine through Thermochemical Recuperation and Dilution Limit Extension, Part 2: Engine Performance

DOE PAGES

Chang, Yan; Szybist, James P.; Pihl, Josh A.; ...

2018-01-17

This is the second part of a two-part investigation of on-board catalytic fuel reforming to increase the brake efficiency of a multicylinder, stoichiometric spark-ignited (SI) engine. In Part 1 of the investigation, we analytically and experimentally characterized the energetics and kinetics of a candidate reforming catalyst over a range of reforming equivalence ratios and oxygen concentration conditions to identify the best conditions for efficient reforming. In the present part of our investigation, we studied an engine strategy that combined exhaust gas recirculation (EGR)–loop reforming with dilution limit extension of the combustion. In our experiments, we found that, under an enginemore » operating condition of 2000 rpm and brake mean effective pressure (4 bar), catalytic EGR reforming made it possible to sustain stable combustion with a volumetric equivalent of 45%–55% EGR. Under this same operating condition with stoichiometric engine exhaust (and no reforming), we were only able to sustain stable combustion with EGR under 25%. In conclusion, these results indicate that multicylinder gasoline engine efficiency can be increased substantially with catalytic reforming combined with and higher EGR operation, resulting in a decrease of more than 8% in fuel consumption, compared to baseline operation.« less

Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism.

PubMed

Fasching, Clare L; Cejka, Petr; Kowalczykowski, Stephen C; Heyer, Wolf-Dietrich

2015-02-19

The displacement loop (D loop) is a DNA strand invasion product formed during homologous recombination. Disruption of nascent D loops prevents recombination, and during synthesis-dependent strand annealing (SDSA), disruption of D loops extended by DNA polymerase ensures a non-crossover outcome. The proteins implicated in D loop disruption are DNA motor proteins/helicases that act by moving DNA junctions. Here we report that D loops can also be disrupted by DNA topoisomerase 3 (Top3), and this disruption depends on Top3's catalytic activity. Yeast Top3 specifically disrupts D loops mediated by yeast Rad51/Rad54; protein-free D loops or D loop mediated by bacterial RecA protein or human RAD51/RAD54 resist dissolution. Also, the human Topoisomerase IIIa-RMI1-RMI2 complex is capable of dissolving D loops. Consistent with genetic data, we suggest that the extreme growth defect and hyper-recombination phenotype of Top3-deficient yeast cells is partially a result of unprocessed D loops. Copyright © 2015 Elsevier Inc. All rights reserved.

Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

PubMed Central

Sakata, Souhei; Jinno, Yuka; Kawanabe, Akira; Okamura, Yasushi

2016-01-01

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane. PMID:27330112

Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid.

PubMed

Sakata, Souhei; Jinno, Yuka; Kawanabe, Akira; Okamura, Yasushi

2016-07-05

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane.

Enzymatic properties of a GH19 chitinase isolated from rice lacking a major loop structure involved in chitin binding.

PubMed

Tanaka, Jun; Fukamizo, Tamo; Ohnuma, Takayuki

2017-05-01

The catalytic domains of family GH19 chitinases have been found to consist of a conserved, α-helical core-region and different numbers (1-6) of loop structures, located at both ends of the substrate-binding groove and which extend over the glycon- and aglycon-binding sites. We expressed, purified and enzymatically characterized a GH19 chitinase from rice, Oryza sativa L. cv. Nipponbare (OsChia2a), lacking a major loop structure (loop III) connected to the functionally important β-stranded region. The new enzyme thus contained the five remaining loop structures (loops I, II, IV, V and C-term). The OsChia2a recombinant protein catalyzed hydrolysis of chitin oligosaccharides, (GlcNAc)n (n = 3-6), with inversion of anomeric configuration, indicating that OsChia2a correctly folded without loop III. From thermal unfolding experiments and calorimetric titrations using the inactive OsChia2a mutant (OsChia2a-E68Q), in which the catalytic residue Glu68 was mutated to glutamine, we found that the binding affinities towards (GlcNAc)n (n = 2-6) were almost proportional to the degree of polymerization of (GlcNAc)n, but were much lower than those obtained for a moss GH19 chitinase having only loop III [Ohnuma T, Sørlie M, Fukuda T, Kawamoto N, Taira T, Fukamizo T. 2011. Chitin oligosaccharide binding to a family GH19 chitinase from the moss, Bryum coronatum. FEBS J. 278:3991-4001]. Nevertheless, OsChia2a exhibited significant antifungal activity. It appears that loop III connected to the β-stranded region is important for (GlcNAc)n binding, but is not essential for antifungal activity. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Chimeric Proton-Pumping Rhodopsins Containing the Cytoplasmic Loop of Bovine Rhodopsin

PubMed Central

Sasaki, Kengo; Yamashita, Takahiro; Yoshida, Kazuho; Inoue, Keiichi; Shichida, Yoshinori; Kandori, Hideki

2014-01-01

G-protein-coupled receptors (GPCRs) transmit stimuli to intracellular signaling systems. Rhodopsin (Rh), which is a prototypical GPCR, possesses an 11-cis retinal. Photoisomerization of 11-cis to all-trans leads to structural changes in the protein of cytoplasmic loops, activating G-protein. Microbial rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. They possess an all-trans retinal, and photoisomerization to 13-cis triggers structural changes in protein. Despite these similarities, there is no sequence homology between visual and microbial rhodopsins, and microbial rhodopsins do not activate G-proteins. In this study, new chimeric proton-pumping rhodopsins, proteorhodopsin (PR) and Gloeobacter rhodopsin (GR) were designed by replacing cytoplasmic loops with bovine Rh loops. Although G-protein was not activated by the PR chimeras, all 12 GR chimeras activated G-protein. The GR chimera containing the second cytoplasmic loop of bovine Rh did not activate G-protein. However, the chimera with a second and third double-loop further enhanced G-protein activation. Introduction of an E132Q mutation slowed the photocycle 30-fold and enhanced activation. The highest catalytic activity of the GR chimera was still 3,200 times lower than bovine Rh but only 64 times lower than amphioxus Go-rhodopsin. This GR chimera showed a strong absorption change of the amide-I band on a light-minus-dark difference FTIR spectrum which could represent a larger helical opening, important for G-protein activation. The light-dependent catalytic activity of this GR chimera makes it a potential optogenetic tool for enzymatic activation by light. PMID:24621599

Asymmetric processing of a substrate protein in sequential allosteric cycles of AAA+ nanomachines

NASA Astrophysics Data System (ADS)

Kravats, Andrea N.; Tonddast-Navaei, Sam; Bucher, Ryan J.; Stan, George

2013-09-01

Essential protein quality control includes mechanisms of substrate protein (SP) unfolding and translocation performed by powerful ring-shaped AAA+ (ATPases associated with various cellular activities) nanomachines. These SP remodeling actions are effected by mechanical forces imparted by AAA+ loops that protrude into the central channel. Sequential intra-ring allosteric motions, which underlie repetitive SP-loop interactions, have been proposed to comprise clockwise (CW), counterclockwise (CCW), or random (R) conformational transitions of individual AAA+ subunits. To probe the effect of these allosteric mechanisms on unfoldase and translocase functions, we perform Langevin dynamics simulations of a coarse-grained model of an all-alpha SP processed by the single-ring ClpY ATPase or by the double-ring p97 ATPase. We find that, in all three allosteric mechanisms, the SP undergoes conformational transitions along a common set of pathways, which reveals that the active work provided by the ClpY machine involves single loop-SP interactions. Nevertheless, the rates and yields of SP unfolding and translocation are controlled by mechanism-dependent loop-SP binding events, as illustrated by faster timescales of SP processing in CW allostery compared with CCW and R allostery. The distinct efficacy of allosteric mechanisms is due to the asymmetric collaboration of adjacent subunits, which involves CW-biased structural motions of AAA+ loops and results in CW-compatible torque applied onto the SP. Additional simulations of mutant ClpY rings, which render a subset of subunits catalytically-defective or reduce their SP binding affinity, reveal that subunit-based conformational transitions play the major role in SP remodeling. Based on these results we predict that the minimally functional AAA+ ring includes three active subunits, only two of which are adjacent.

Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tong, Junsen; Yang, Huiseon; Eom, Soo Hyun

2014-09-12

Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domainmore » of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.« less

The Crystal Structure of the Ring-Hydroxylating Dioxygenase from Sphingomonas CHY-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jakoncic,J.; Jouanneau, Y.; Meyer, C.

The ring-hydroxylating dioxygenase (RHD) from Sphingomonas CHY-1 is remarkable due to its ability to initiate the oxidation of a wide range of polycyclic aromatic hydrocarbons (PAHs), including PAHs containing four- and five-fused rings, known pollutants for their toxic nature. Although the terminal oxygenase from CHY-1 exhibits limited sequence similarity with well characterized RHDs from the naphthalene dioxygenase family, the crystal structure determined to 1.85 {angstrom} by molecular replacement revealed the enzyme to share the same global {alpha}{sub 3}{beta}{sub 3} structural pattern. The catalytic domain distinguishes itself from other bacterial non-heme Rieske iron oxygenases by a substantially larger hydrophobic substrate bindingmore » pocket, the largest ever reported for this type of enzyme. While residues in the proximal region close to the mononuclear iron atom are conserved, the central region of the catalytic pocket is shaped mainly by the side chains of three amino acids, Phe350, Phe404 and Leu356, which contribute to the rather uniform trapezoidal shape of the pocket. Two flexible loops, LI and LII, exposed to the solvent seem to control the substrate access to the catalytic pocket and control the pocket length. Compared with other naphthalene dioxygenases residues Leu223 and Leu226, on loop LI, are moved towards the solvent, thus elongating the catalytic pocket by at least 2 {angstrom}. An 11 {angstrom} long water channel extends from the interface between the {alpha} and {beta} subunits to the catalytic site. The comparison of these structures with other known oxygenases suggests that the broad substrate specificity presented by the CHY-1 oxygenase is primarily due to the large size and particular topology of its catalytic pocket and provided the basis for the study of its reaction mechanism.« less

Rational engineering of the Neurospora VS ribozyme to allow substrate recognition via different kissing-loop interactions.

PubMed

Lacroix-Labonté, Julie; Girard, Nicolas; Dagenais, Pierre; Legault, Pascale

2016-08-19

The Neurospora VS ribozyme is a catalytic RNA that has the unique ability to specifically recognize and cleave a stem-loop substrate through formation of a highly stable kissing-loop interaction (KLI). In order to explore the engineering potential of the VS ribozyme to cleave alternate substrates, we substituted the wild-type KLI by other known KLIs using an innovative engineering method that combines rational and combinatorial approaches. A bioinformatic search of the protein data bank was initially performed to identify KLIs that are structurally similar to the one found in the VS ribozyme. Next, substrate/ribozyme (S/R) pairs that incorporate these alternative KLIs were kinetically and structurally characterized. Interestingly, several of the resulting S/R pairs allowed substrate cleavage with substantial catalytic efficiency, although with reduced activity compared to the reference S/R pair. Overall, this study describes an innovative approach for RNA engineering and establishes that the KLI of the trans VS ribozyme can be adapted to cleave other folded RNA substrates. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mapping the Structural and Dynamical Features of Multiple p53 DNA Binding Domains: Insights into Loop 1 Intrinsic Dynamics

PubMed Central

Lukman, Suryani; Lane, David P.; Verma, Chandra S.

2013-01-01

The transcription factor p53 regulates cellular integrity in response to stress. p53 is mutated in more than half of cancerous cells, with a majority of the mutations localized to the DNA binding domain (DBD). In order to map the structural and dynamical features of the DBD, we carried out multiple copy molecular dynamics simulations (totaling 0.8 μs). Simulations show the loop 1 to be the most dynamic element among the DNA-contacting loops (loops 1-3). Loop 1 occupies two major conformational states: extended and recessed; the former but not the latter displays correlations in atomic fluctuations with those of loop 2 (~24 Å apart). Since loop 1 binds to the major groove whereas loop 2 binds to the minor groove of DNA, our results begin to provide some insight into the possible mechanism underpinning the cooperative nature of DBD binding to DNA. We propose (1) a novel mechanism underlying the dynamics of loop 1 and the possible tread-milling of p53 on DNA and (2) possible mutations on loop 1 residues to restore the transcriptional activity of an oncogenic mutation at a distant site. PMID:24324553

Automated stopped-in-dual-loop flow analysis system for catalytic determination of vanadium in drinking water.

PubMed

Teshima, Norio; Kuno, Masami; Ueda, Minoru; Ueda, Hisashi; Ohno, Shinsuke; Sakai, Tadao

2009-07-15

An automated stopped-in-dual-loop flow analysis (SIDL-FA) system is proposed for the determination of vanadium in drinking water. The chemistry is based on the vanadium-catalyzed oxidation reaction of p-anisidine by bromate in the presence of Tiron as an activator to produce a dye (lambda(max)=510 nm). A SIDL-FA system basically consists of a selection valve, three pumps (one is for delivering of standard/sample, and others are for reagents), two six-way injection valves, a spectrophotometric detector and a data acquisition device. A 100-microL coiled loop around a heated device is fitted onto each six-way injection valve. A well-mixed solution containing reagents and standard/sample is loaded into the first loop on a six-way valve, and then the same solution is loaded into the second loop on another six-way valve. The solutions are isolated by switching these two six-way valves, so that the catalytic reaction can be promoted. The net waste can be zero in this stage, because all pumps are turned off. Then each resulting solution is dispensed to the detector with suitable time lag. A touchscreen controller is developed to automatically carry out the original SIDL-FA protocol. The proposed SIDL-FA method allows vanadium to be quantified in the range of 0.1-2 microg L(-1) and is applied to the determination of vanadium in drinking water samples.

Top3-Rmi1 dissolve Rad51-mediated D-loops by a topoisomerase-based mechanism

PubMed Central

Fasching, Clare L.; Cejka, Petr; Kowalczykowski, Stephen C.; Heyer, Wolf-Dietrich

2015-01-01

Summary The displacement loop (D-loop) is the DNA strand invasion product formed during homologous recombination. Disruption of nascent D-loops represents a mechanism of anti-recombination. During Synthesis-Dependent Strand Annealing D-loop disruption after extension of the invading strand is an integral step of the pathway and ensures a non-crossover outcome. The proteins implicated in D-loop disruption are DNA motor proteins/helicases acting by migrating DNA junctions. Here we report an unanticipated mechanism of D-loop dissolution mediated by DNA topoisomerase 3 (Top3) and dependent on its catalytic activity. D-loop dissolution catalyzed by yeast Top3 is highly specific for yeast Rad51/Rad54-mediated D-loops, whereas protein-free D-loops or D-loop mediated by bacterial RecA protein or human RAD51/RAD54 resist dissolution. Also the human Topoisomerase IIIα-RMI1–RMI2 complex is capable of dissolving D-loops. Consistent with genetic data, we suggest that the extreme growth defect and hyper-recombination phenotype of Top3-deficient yeast cells is in part a result of unprocessed D-loops. PMID:25699708

Computational study of β-N-acetylhexosaminidase from Talaromyces flavus, a glycosidase with high substrate flexibility.

PubMed

Kulik, Natallia; Slámová, Kristýna; Ettrich, Rüdiger; Křen, Vladimír

2015-01-28

β-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest. Computational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model. The main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.

The dynamic organization of fungal acetyl-CoA carboxylase

NASA Astrophysics Data System (ADS)

Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

2016-04-01

Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.

Catalytic site interactions in yeast OMP synthase.

PubMed

Hansen, Michael Riis; Barr, Eric W; Jensen, Kaj Frank; Willemoës, Martin; Grubmeyer, Charles; Winther, Jakob R

2014-01-15

The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5'-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding. Copyright © 2013. Published by Elsevier Inc.

A catalytic chiral gel microfluidic reactor assembled via dynamic covalent chemistry† †Electronic supplementary information (ESI) available: Experimental details, additional characterization and catalytic data. See DOI: 10.1039/c5sc00314h Click here for additional data file.

PubMed Central

Liu, Haoliang; Feng, Juan; Chen, Liuping

2015-01-01

A novel dynamic covalent gel strategy is reported to immobilize an asymmetric catalyst within the channels of a microfluidic flow reactor. A layer of a catalytically active Mn–salen dynamic covalent imine gel matrix was coated onto a functionalized capillary. Mn–salen active moiety was incorporated into dynamic covalent imine gel matrix via the reaction of a chiral Mn–salen dialdehyde unit with a tetraamine linker. The catalytic activity of the capillary reactor has been demonstrated in enantioselective kinetic resolution of secondary alcohols. PMID:28706652

Kinemage of action - Proposed reaction mechanism of glutamate-1-semialdehyde aminomutase at an atomic level

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sorensen, John L., E-mail: John_Sorensen@umanitoba.ca; Stetefeld, Joerg, E-mail: stetefel@cc.umanitoba.ca

2011-10-07

Highlights: {yields} Inhibitors of tetrapyrrole cofactor biosynthesis may be useful antibiotics. {yields} Mechanism of critical enzyme, glutamate-1-semialdehyde aminomutase, is presented. {yields} Unique vitamin B6-dependant enzyme traps intermediate in active site. {yields} Molecular dynamics show that a re-orientation of the substrate is required. -- Abstract: Glutamate-1-semialdehyde aminomutase (GSAM), a key enzyme in tetrapyrrole cofactor biosynthesis, performs a unique transamination on a single substrate. The substrate, glutamate-1-semialdehyde (GSA), undergoes a reaction that exchanges the position of an amine and a carbonyl group to produce 5-aminolevulinic acid (ALA). This transamination reaction is unique in the fact that is does not require an externalmore » cofactor to act as a nitrogen donor or acceptor in this transamination reaction. One of the other remarkable features of the catalytic mechanism is the release free in the enzyme active site of the intermediate 4,5-diaminovaleric acid (DAVA). The action of a gating loop prevents the escape of DAVA from the active site. In a MD simulation approach, using snapshots provided by X-ray crystallography and protein crystal absorption spectrometry data, the individual catalytic steps in this unique intramolecular transamination have been elucidated.« less

Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qian, Zhen; Horton, John R.; Cheng, Xiadong

2009-11-02

Circular permutation of Candida antarctica lipase B yields several enzyme variants with substantially increased catalytic activity. To better understand the structural and functional consequences of protein termini reorganization, we have applied protein engineering and x-ray crystallography to cp283, one of the most active hydrolase variants. Our initial investigation has focused on the role of an extended surface loop, created by linking the native N- and C-termini, on protein integrity. Incremental truncation of the loop partially compensates for observed losses in secondary structure and the permutants temperature of unfolding. Unexpectedly, the improvements are accompanied by quaternary-structure changes from monomer to dimer.more » The crystal structures of one truncated variant (cp283{Delta}7) in the apo-form determined at 1.49 {angstrom} resolution and with a bound phosphonate inhibitor at 1.69 {angstrom} resolution confirmed the formation of a homodimer by swapping of the enzyme's 35-residue N-terminal region. Separately, the new protein termini at amino acid positions 282/283 convert the narrow access tunnel to the catalytic triad into a broad crevice for accelerated substrate entry and product exit while preserving the native active-site topology for optimal catalytic turnover.« less

Crystal Structure of the Golgi-Associated Human Nα-Acetyltransferase 60 Reveals the Molecular Determinants for Substrate-Specific Acetylation.

PubMed

Støve, Svein Isungset; Magin, Robert S; Foyn, Håvard; Haug, Bengt Erik; Marmorstein, Ronen; Arnesen, Thomas

2016-07-06

N-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for the function of Naa60, we developed an Naa60 bisubstrate CoA-peptide conjugate inhibitor, determined its X-ray structure when bound to CoA and inhibitor, and carried out biochemical experiments. We show that Naa60 adapts an overall fold similar to that of the catalytic subunits of ribosome-associated NATs, but with the addition of two novel elongated loops that play important roles in substrate-specific binding. One of these loops mediates a dimer to monomer transition upon substrate-specific binding. Naa60 employs a catalytic mechanism most similar to Naa50. Collectively, these data reveal the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Architecture of a Diels-Alderase ribozyme with a preformed catalytic pocket.

PubMed

Keiper, Sonja; Bebenroth, Dirk; Seelig, Burckhard; Westhof, Eric; Jäschke, Andres

2004-09-01

Artificial ribozymes catalyze a variety of chemical reactions. Their structures and reaction mechanisms are largely unknown. We have analyzed a ribozyme catalyzing Diels-Alder cycloaddition reactions by comprehensive mutation analysis and a variety of probing techniques. New tertiary interactions involving base pairs between nucleotides of the 5' terminus and a large internal loop forming a pseudoknot fold were identified. The probing data indicate a preformed tertiary structure that shows no major changes on substrate or product binding. Based on these observations, a molecular architecture featuring a Y-shaped arrangement is proposed. The tertiary structure is formed in a rather unusual way; that is, the opposite sides of the asymmetric internal loop are clamped by the four 5'-terminal nucleotides, forming two adjacent two base-pair helices. It is proposed that the catalytic pocket is formed by a wedge within one of these helices.

Significance of the enzymatic properties of yeast S39A enolase to the catalytic mechanism.

PubMed

Brewer, J M; Glover, C V; Holland, M J; Lebioda, L

1998-04-02

The S39A mutant of yeast enolase (isozyme 1), prepared by site-directed mutagenesis, has a relative Vmax of 0.01% and an activation constant for Mg2+ ca. 10-fold higher, compared with native enzyme. It is correctly folded. There is little effect of solvent viscosity on activity. We think that the loop Ser36-His43 fails to move to the 'closed' position upon catalytic Mg2+ binding, weakening several electrostatic interactions involved in the mechanism.

Critical domain interactions for type A RNase P RNA catalysis with and without the specificity domain

PubMed Central

Mao, Guanzhong; Srivastava, Abhishek S.; Wu, Shiying; Kosek, David; Lindell, Magnus

2018-01-01

The natural trans-acting ribozyme RNase P RNA (RPR) is composed of two domains in which the catalytic (C-) domain mediates cleavage of various substrates. The C-domain alone, after removal of the second specificity (S-) domain, catalyzes this reaction as well, albeit with reduced efficiency. Here we provide experimental evidence indicating that efficient cleavage mediated by the Escherichia coli C-domain (Eco CP RPR) with and without the C5 protein likely depends on an interaction referred to as the "P6-mimic". Moreover, the P18 helix connects the C- and S-domains between its loop and the P8 helix in the S-domain (the P8/ P18-interaction). In contrast to the "P6-mimic", the presence of P18 does not contribute to the catalytic performance by the C-domain lacking the S-domain in cleavage of an all ribo model hairpin loop substrate while deletion or disruption of the P8/ P18-interaction in full-size RPR lowers the catalytic efficiency in cleavage of the same model hairpin loop substrate in keeping with previously reported data using precursor tRNAs. Consistent with that P18 is not required for cleavage mediated by the C-domain we show that the archaeal Pyrococcus furiosus RPR C-domain, which lacks the P18 helix, is catalytically active in trans without the S-domain and any protein. Our data also suggest that the S-domain has a larger impact on catalysis for E. coli RPR compared to P. furiosus RPR. Finally, we provide data indicating that the absence of the S-domain and P18, or the P8/ P18-interaction in full-length RPR influences the charge distribution near the cleavage site in the RPR-substrate complex to a small but reproducible extent. PMID:29509761

Molecular Dynamics Simulations of Family 7 Cellobiohydrolase Mutants Aimed at Reducing Product Inhibition.

PubMed

Silveira, Rodrigo L; Skaf, Munir S

2015-07-23

Enzymatic conversion of lignocellulosic biomass into biofuels and chemicals constitutes a potential route for sustainable development. Cellobiohydrolases are key enzymes used in industrial cocktails for depolymerization of crystalline cellulose, and their mechanism of action has been intensely studied in the past several years. Provided with a tunnel-like substrate-binding cavity, cellobiohydrolases possess the ability to processively hydrolyze glycosidic bonds of crystalline cellulose, yielding one molecule of cellobiose per catalytic cycle. As such, cellobiose expulsion from the product binding site is a necessary step in order to allow for the processive hydrolysis mechanism. However, the high-affinity binding of cellobiose to the enzyme impairs the process and causes activity inhibition due to reaction products. Here, we use molecular dynamics simulations to study the binding of cellobiose to the Trichoderma reesei Cel7A (TrCel7A) cellobiohydrolase and the effects of mutations that reduce cellobiose binding, without affecting the structural and dynamical integrities of the enzyme. We observe that the product binding site exhibits an intrinsic flexibility that can sterically hinder cellobiose release. Several point mutations in the product binding site reduce cellobiose-enzyme interactions, but not all modifications are able to maintain the structural integrity of the enzyme. In particular, mutation of charged residues in the TrCel7A product binding site causes perturbations that affect the structure of the loops that form the substrate-binding tunnel of the enzyme and, hence, may affect TrCel7A function in other steps of the hydrolysis mechanism. Our results suggest there is a trade-off between product inhibition and catalytic efficiency, and they provide directions for cellulases engineering.

NMR and Bioinformatics Discovery of Exosites That Tune Metalloelastase Specificity for Solubilized Elastin and Collagen Triple Helices*

PubMed Central

Palmier, Mark O.; Fulcher, Yan G.; Bhaskaran, Rajagopalan; Duong, Vinh Q.; Fields, Gregg B.; Van Doren, Steven R.

2010-01-01

The catalytic domain of metalloelastase (matrix metalloproteinase-12 or MMP-12) is unique among MMPs in exerting high proteolytic activity upon fibrils that resist hydrolysis, especially elastin from lungs afflicted with chronic obstructive pulmonary disease or arteries with aneurysms. How does the MMP-12 catalytic domain achieve this specificity? NMR interface mapping suggests that α-elastin species cover the primed subsites, a strip across the β-sheet from β-strand IV to the II–III loop, and a broad bowl from helix A to helix C. The many contacts may account for the comparatively high affinity, as well as embedding of MMP-12 in damaged elastin fibrils in vivo. We developed a strategy called BINDSIght, for bioinformatics and NMR discovery of specificity of interactions, to evaluate MMP-12 specificity without a structure of a complex. BINDSIght integration of the interface mapping with other ambiguous information from sequences guided choice mutations in binding regions nearer the active site. Single substitutions at each of ten locations impair specific activity toward solubilized elastin. Five of them impair release of peptides from intact elastin fibrils. Eight lesions also impair specific activity toward triple helices from collagen IV or V. Eight sites map to the “primed” side in the III–IV, V–B, and S1′ specificity loops. Two map to the “unprimed” side in the IV–V and B–C loops. The ten key residues circumscribe the catalytic cleft, form an exosite, and are distinctive features available for targeting by new diagnostics or therapeutics. PMID:20663866

STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF CIRCULAR PERMUTATION ON THE ACTIVE SITE OF OLD YELLOW ENZYME.

PubMed

Daugherty, Ashley B; Horton, John R; Cheng, Xiaodong; Lutz, Stefan

2015-02-06

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme's catalytic performance. Termini relocation into four regions of the protein (sectors I-IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I-III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location, but also provide a possible explanation for the catalytic gains in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290-310) of OYE1 which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such active site remodeling does not negatively impact the enzyme's activity and stereoselectivity, nor does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereo-selectivity for ( S )-carvone reduction. Our findings demonstrate the contribution of loop β6 toward determining the stereoselectivity of OYE1, an important insight for future OYE engineering efforts.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Daugherty, Ashley B.; Horton, John R.; Cheng, Xiaodong

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme’s catalytic performance. Termini relocation into four regions of the protein (sectors I–IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I–III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location but also provide a possible explanation for the catalytic gainsmore » in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290–310) of OYE1, which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such an active site remodeling does not negatively impact the enzyme’s activity and stereoselectivity; neither does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereoselectivity for (S)-carvone reduction. In conclusion, our findings demonstrate the contribution of loop β6 toward determining the stereoselectivity of OYE1, an important insight for future OYE engineering efforts.« less

Structural and Functional Consequences of Circular Permutation on the Active Site of Old Yellow Enzyme

DOE PAGES

Daugherty, Ashley B.; Horton, John R.; Cheng, Xiaodong; ...

2014-12-09

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme’s catalytic performance. Termini relocation into four regions of the protein (sectors I–IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I–III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location but also provide a possible explanation for the catalytic gainsmore » in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290–310) of OYE1, which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such an active site remodeling does not negatively impact the enzyme’s activity and stereoselectivity; neither does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereoselectivity for (S)-carvone reduction. In conclusion, our findings demonstrate the contribution of loop β6 toward determining the stereoselectivity of OYE1, an important insight for future OYE engineering efforts.« less

Initiator-catalyzed self-assembly of duplex-looped DNA hairpin motif based on strand displacement reaction for logic operations and amplified biosensing.

PubMed

Bi, Sai; Yue, Shuzhen; Wu, Qiang; Ye, Jiayan

2016-09-15

Here we program an initiator-catalyzed self-assembly of duplex-looped DNA hairpin motif based on strand displacement reaction. Due to the recycling of initiator and performance in a cascade manner, this system is versatilely extended to logic operations, including the construction of concatenated logic circuits with a feedback function and a biocomputing keypad-lock security system. Compared with previously reported molecular security systems, the prominent feature of our keypad lock is that it can be spontaneously reset and recycled with no need of any external stimulus and human intervention. Moreover, through integrating with an isothermal amplification technique of rolling circle amplification (RCA), this programming catalytic DNA self-assembly strategy readily achieves sensitive and selective biosensing of initiator. Importantly, a magnetic graphene oxide (MGO) is introduced to remarkably reduced background, which plays an important role in enhancing the signal-to-noise ratio and improving the detection sensitivity. Therefore, the proposed sophisticated DNA strand displacement-based methodology with engineering dynamic functions may find broad applications in the construction of programming DNA nanostructures, amplification biosensing platform, and large-scale DNA circuits. Copyright © 2016 Elsevier B.V. All rights reserved.

Accurate SHAPE-directed RNA secondary structure modeling, including pseudoknots.

PubMed

Hajdin, Christine E; Bellaousov, Stanislav; Huggins, Wayne; Leonard, Christopher W; Mathews, David H; Weeks, Kevin M

2013-04-02

A pseudoknot forms in an RNA when nucleotides in a loop pair with a region outside the helices that close the loop. Pseudoknots occur relatively rarely in RNA but are highly overrepresented in functionally critical motifs in large catalytic RNAs, in riboswitches, and in regulatory elements of viruses. Pseudoknots are usually excluded from RNA structure prediction algorithms. When included, these pairings are difficult to model accurately, especially in large RNAs, because allowing this structure dramatically increases the number of possible incorrect folds and because it is difficult to search the fold space for an optimal structure. We have developed a concise secondary structure modeling approach that combines SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) experimental chemical probing information and a simple, but robust, energy model for the entropic cost of single pseudoknot formation. Structures are predicted with iterative refinement, using a dynamic programming algorithm. This melded experimental and thermodynamic energy function predicted the secondary structures and the pseudoknots for a set of 21 challenging RNAs of known structure ranging in size from 34 to 530 nt. On average, 93% of known base pairs were predicted, and all pseudoknots in well-folded RNAs were identified.

Topological entropy of catalytic sets: Hypercycles revisited

NASA Astrophysics Data System (ADS)

Sardanyés, Josep; Duarte, Jorge; Januário, Cristina; Martins, Nuno

2012-02-01

The dynamics of catalytic networks have been widely studied over the last decades because of their implications in several fields like prebiotic evolution, virology, neural networks, immunology or ecology. One of the most studied mathematical bodies for catalytic networks was initially formulated in the context of prebiotic evolution, by means of the hypercycle theory. The hypercycle is a set of self-replicating species able to catalyze other replicator species within a cyclic architecture. Hypercyclic organization might arise from a quasispecies as a way to increase the informational containt surpassing the so-called error threshold. The catalytic coupling between replicators makes all the species to behave like a single and coherent evolutionary multimolecular unit. The inherent nonlinearities of catalytic interactions are responsible for the emergence of several types of dynamics, among them, chaos. In this article we begin with a brief review of the hypercycle theory focusing on its evolutionary implications as well as on different dynamics associated to different types of small catalytic networks. Then we study the properties of chaotic hypercycles with error-prone replication with symbolic dynamics theory, characterizing, by means of the theory of topological Markov chains, the topological entropy and the periods of the orbits of unimodal-like iterated maps obtained from the strange attractor. We will focus our study on some key parameters responsible for the structure of the catalytic network: mutation rates, autocatalytic and cross-catalytic interactions.

Probing the effect of the non-active-site mutation Y229W in New Delhi metallo-β-lactamase-1 by site-directed mutagenesis, kinetic studies, and molecular dynamics simulations.

PubMed

Chen, Jiao; Chen, Hui; Shi, Yun; Hu, Feng; Lao, Xingzhen; Gao, Xiangdong; Zheng, Heng; Yao, Wenbing

2013-01-01

New Delhi metallo-β-lactamase-1 (NDM-1) has attracted extensive attention for its high catalytic activities of hydrolyzing almost all β-lactam antibiotics. NDM-1 shows relatively higher similarity to subclass B1 metallo-β-lactamases (MβLs), but its residue at position 229 is identical to that of B2/B3 MβLs, which is a Tyr instead of a B1-MβL-conserved Trp. To elucidate the possible role of Y229 in the bioactivity of NDM-1, we performed mutagenesis study and molecular dynamics (MD) simulations. Although residue Y229 is spatially distant from the active site and not contacting directly with the substrate or zinc ions, the Y229W mutant was found to have higher kcat and Km values than those of wild-type NDM-1, resulting in 1 ∼ 7 fold increases in k(cat) /K(m) values against tested antibiotics. In addition, our MD simulations illustrated the enhanced flexibility of Loop 2 upon Y229W mutation, which could increase the kinetics of both substrate entrance (kon) and product egress (koff). The enhanced flexibility of Loop 2 might allow the enzyme to adjust the geometry of its active site to accommodate substrates with different structures, broadening its substrate spectrum. This study indicated the possible role of the residue at position 229 in the evolution of NDM-1.

Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

NASA Astrophysics Data System (ADS)

Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

2017-01-01

3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors.

System identification from closed-loop data with known output feedback dynamics

NASA Technical Reports Server (NTRS)

Phan, Minh; Juang, Jer-Nan; Horta, Lucas G.; Longman, Richard W.

1992-01-01

This paper presents a procedure to identify the open loop systems when it is operating under closed loop conditions. First, closed loop excitation data are used to compute the system open loop and closed loop Markov parameters. The Markov parameters, which are the pulse response samples, are then used to compute a state space representation of the open loop system. Two closed loop configurations are considered in this paper. The closed loop system can have either a linear output feedback controller or a dynamic output feedback controller. Numerical examples are provided to illustrate the proposed closed loop identification method.

Structure-function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A from Pseudomonas aeruginosa.

PubMed

Yates, Susan P; Taylor, Patricia L; Jørgensen, René; Ferraris, Dana; Zhang, Jie; Andersen, Gregers R; Merrill, A Rod

2005-02-01

The mono-ADPRT (mono-ADP-ribosyltransferase), Pseudomonas aeruginosa ETA (exotoxin A), catalyses the transfer of ADP-ribose from NAD+ to its protein substrate. A series of water-soluble compounds that structurally mimic the nicotinamide moiety of NAD+ was investigated for their inhibition of the catalytic domain of ETA. The importance of an amide locked into a hetero-ring structure and a core hetero-ring system that is planar was a trend evident by the IC50 values. Also, the weaker inhibitors have core ring structures that are less planar and thus more flexible. One of the most potent inhibitors, PJ34, was further characterized and shown to exhibit competitive inhibition with an inhibition constant K(i) of 140 nM. We also report the crystal structure of the catalytic domain of ETA in complex with PJ34, the first example of a mono-ADPRT in complex with an inhibitor. The 2.1 A (1 A=0.1 nm) resolution structure revealed that PJ34 is bound within the nicotinamide-binding pocket and forms stabilizing hydrogen bonds with the main chain of Gly-441 and to the side-chain oxygen of Gln-485, a member of a proposed catalytic loop. Structural comparison of this inhibitor complex with diphtheria toxin (a mono-ADPRT) and with PARPs [poly(ADP-ribose) polymerases] shows similarity of the catalytic residues; however, a loop similar to that found in ETA is present in diphtheria toxin but not in PARP. The present study provides insight into the important features required for inhibitors that mimic NAD+ and their binding to the mono-ADPRT family of toxins.

Dynamic formation of single-atom catalytic active sites on ceria-supported gold nanoparticles

PubMed Central

Wang, Yang-Gang; Mei, Donghai; Glezakou, Vassiliki-Alexandra; Li, Jun; Rousseau, Roger

2015-01-01

Catalysis by gold supported on reducible oxides has been extensively studied, yet issues such as the nature of the catalytic site and the role of the reducible support remain fiercely debated topics. Here we present ab initio molecular dynamics simulations of an unprecedented dynamic single-atom catalytic mechanism for the oxidation of carbon monoxide by ceria-supported gold clusters. The reported dynamic single-atom catalytic mechanism results from the ability of the gold cation to strongly couple with the redox properties of the ceria in a synergistic manner, thereby lowering the energy of redox reactions. The gold cation can break away from the gold nanoparticle to catalyse carbon monoxide oxidation, adjacent to the metal/oxide interface and subsequently reintegrate back into the nanoparticle after the reaction is completed. Our study highlights the importance of the dynamic creation of active sites under reaction conditions and their essential role in catalysis. PMID:25735407

Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions.

PubMed

Hoque, Md Anarul; Zhang, Yong; Chen, Liuqing; Yang, Guangyu; Khatun, Mst Afroza; Chen, Haifeng; Hao, Liu; Feng, Yan

2017-05-19

The remodeling of active sites to generate novel biocatalysts is an attractive and challenging task. We developed a stepwise loop insertion strategy (StLois), in which randomized residue pairs are inserted into active site loops. The phosphotriesterase-like lactonase from Geobacillus kaustophilus (GkaP-PLL) was used to investigate StLois's potential for changing enzyme function. By inserting six residues into active site loop 7, the best variant ML7-B6 demonstrated a 16-fold further increase in catalytic efficiency toward ethyl-paraoxon compared with its initial template, that is a 609-fold higher, >10 7 fold substrate specificity shift relative to that of wild-type lactonase. The remodeled variants displayed 760-fold greater organophosphate hydrolysis activity toward the organophosphates parathion, diazinon, and chlorpyrifos. Structure and docking computations support the source of notably inverted enzyme specificity. Considering the fundamental importance of active site loops, the strategy has potential for the rapid generation of novel enzyme functions by loop remodeling.

Indirect Identification of Linear Stochastic Systems with Known Feedback Dynamics

NASA Technical Reports Server (NTRS)

Huang, Jen-Kuang; Hsiao, Min-Hung; Cox, David E.

1996-01-01

An algorithm is presented for identifying a state-space model of linear stochastic systems operating under known feedback controller. In this algorithm, only the reference input and output of closed-loop data are required. No feedback signal needs to be recorded. The overall closed-loop system dynamics is first identified. Then a recursive formulation is derived to compute the open-loop plant dynamics from the identified closed-loop system dynamics and known feedback controller dynamics. The controller can be a dynamic or constant-gain full-state feedback controller. Numerical simulations and test data of a highly unstable large-gap magnetic suspension system are presented to demonstrate the feasibility of this indirect identification method.

Correlation of structure, function and protein dynamics in GH7 cellobiohydrolases from Trichoderma atroviride, T. reesei and T. harzianum.

PubMed

Borisova, Anna S; Eneyskaya, Elena V; Jana, Suvamay; Badino, Silke F; Kari, Jeppe; Amore, Antonella; Karlsson, Magnus; Hansson, Henrik; Sandgren, Mats; Himmel, Michael E; Westh, Peter; Payne, Christina M; Kulminskaya, Anna A; Ståhlberg, Jerry

2018-01-01

The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) Tre Cel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride ( Tat Cel7A) and Trichoderma harzianum ( Tha Cel7A) show high sequence identity with Tre Cel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate. The Tat Cel7A, Tha Cel7A, and Tre Cel7A enzymes were characterized for comparison of function. The catalytic domain of Tat Cel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with Tat Cel7A than either Tha Cel7A or Tre Cel7A. In synergistic saccharification of pretreated corn stover, both Tat Cel7A and Tha Cel7A were more efficient than Tre Cel7A, although Tat Cel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs. We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from Tat Cel7A with the thermostability features of Tre Cel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.

Correlation of structure, function and protein dynamics in GH7 cellobiohydrolases from Trichoderma atroviride, T. reesei and T. harzianum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borisova, Anna S.; Eneyskaya, Elena V.; Jana, Suvamay

The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate. The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. Themore » catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs. We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.« less

Correlation of structure, function and protein dynamics in GH7 cellobiohydrolases from Trichoderma atroviride, T. reesei and T. harzianum

DOE PAGES

Borisova, Anna S.; Eneyskaya, Elena V.; Jana, Suvamay; ...

2018-01-13

The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate. The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. Themore » catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs. We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.« less

The DUSP–Ubl domain of USP4 enhances its catalytic efficiency by promoting ubiquitin exchange

PubMed Central

Clerici, Marcello; Luna-Vargas, Mark P. A.; Faesen, Alex C.; Sixma, Titia K.

2014-01-01

Ubiquitin-specific protease USP4 is emerging as an important regulator of cellular pathways, including the TGF-β response, NF-κB signalling and splicing, with possible roles in cancer. Here we show that USP4 has its catalytic triad arranged in a productive conformation. Nevertheless, it requires its N-terminal DUSP–Ubl domain to achieve full catalytic turnover. Pre-steady-state kinetics measurements reveal that USP4 catalytic domain activity is strongly inhibited by slow dissociation of ubiquitin after substrate hydrolysis. The DUSP–Ubl domain is able to enhance ubiquitin dissociation, hence promoting efficient turnover. In a mechanism that requires all USP4 domains, binding of the DUSP–Ubl domain promotes a change of a switching loop near the active site. This ‘allosteric regulation of product discharge’ provides a novel way of regulating deubiquitinating enzymes that may have relevance for other enzyme classes. PMID:25404403

Negative Factor from SIV Binds to the Catalytic Subunit of the V-ATPase to Internalize CD4 and to Increase Viral Infectivity

PubMed Central

Mandic, Robert; Fackler, Oliver T.; Geyer, Matthias; Linnemann, Thomas; Zheng, Yong-Hui; Peterlin, B. Matija

2001-01-01

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIVmac239 for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity. PMID:11179428

All-digital signal-processing open-loop fiber-optic gyroscope with enlarged dynamic range.

PubMed

Wang, Qin; Yang, Chuanchuan; Wang, Xinyue; Wang, Ziyu

2013-12-15

We propose and realize a new open-loop fiber-optic gyroscope (FOG) with an all-digital signal-processing (DSP) system where an all-digital phase-locked loop is employed for digital demodulation to eliminate the variation of the source intensity and suppress the bias drift. A Sagnac phase-shift tracking method is proposed to enlarge the dynamic range, and, with its aid, a new open-loop FOG, which can achieve a large dynamic range and high sensitivity at the same time, is realized. The experimental results show that compared with the conventional open-loop FOG with the same fiber coil and optical devices, the proposed FOG reduces the bias instability from 0.259 to 0.018 deg/h, and the angle random walk from 0.031 to 0.006 deg/h(1/2), moreover, enlarges the dynamic range to ±360 deg/s, exceeding the maximum dynamic range ±63 deg/s of the conventional open-loop FOG.

Similarity in Shape Dictates Signature Intrinsic Dynamics Despite No Functional Conservation in TIM Barrel Enzymes

PubMed Central

Tiwari, Sandhya P.; Reuter, Nathalie

2016-01-01

The conservation of the intrinsic dynamics of proteins emerges as we attempt to understand the relationship between sequence, structure and functional conservation. We characterise the conservation of such dynamics in a case where the structure is conserved but function differs greatly. The triosephosphate isomerase barrel fold (TBF), renowned for its 8 β-strand-α-helix repeats that close to form a barrel, is one of the most diverse and abundant folds found in known protein structures. Proteins with this fold have diverse enzymatic functions spanning five of six Enzyme Commission classes, and we have picked five different superfamily candidates for our analysis using elastic network models. We find that the overall shape is a large determinant in the similarity of the intrinsic dynamics, regardless of function. In particular, the β-barrel core is highly rigid, while the α-helices that flank the β-strands have greater relative mobility, allowing for the many possibilities for placement of catalytic residues. We find that these elements correlate with each other via the loops that link them, as opposed to being directly correlated. We are also able to analyse the types of motions encoded by the normal mode vectors of the α-helices. We suggest that the global conservation of the intrinsic dynamics in the TBF contributes greatly to its success as an enzymatic scaffold both through evolution and enzyme design. PMID:27015412

CASPASE-9 CARD:CORE DOMAIN INTERACTIONS REQUIRE A PROPERLY-FORMED ACTIVE SITE

PubMed Central

Huber, Kristen L.; Serrano, Banyuhay P.; Hardy, Jeanne A.

2018-01-01

Caspase-9 is a critical factor in the initiation of apoptosis, and as a result is tightly regulated by a number of mechanisms. Caspase-9 contains a Caspase Activation and Recruitment Domain (CARD), which enables caspase-9 to form a tight interaction with the apoptosome, a heptameric activating platform. The caspase-9 CARD has been thought to be principally involved in recruitment to the apoptosome, but its roles outside this interaction have yet to be uncovered. In this work we show that the CARD is involved in physical interactions with the catalytic core of caspase-9 in the absence of the apoptosome; this interaction requires a properly formed caspase-9 active site. The active sites of caspases are composed of four extremely mobile loops. When the active-site loops are not properly ordered, the CARD and core domains of caspase-9 do not interact and behave independently, like loosely tethered beads. When the active-site loop bundle is properly ordered, the CARD domain interacts with the catalytic core, forming a single folding unit. Together these findings provide mechanistic insight into a new level of caspase-9 regulation, prompting speculation that the CARD may also play a role in the recruitment or recognition of substrate. PMID:29500231

Molecular dynamics simulation reveals insights into the mechanism of unfolding by the A130T/V mutations within the MID1 zinc-binding Bbox1 domain.

PubMed

Zhao, Yunjie; Zeng, Chen; Massiah, Michael A

2015-01-01

The zinc-binding Bbox1 domain in protein MID1, a member of the TRIM family of proteins, facilitates the ubiquitination of the catalytic subunit of protein phosphatase 2A and alpha4, a protein regulator of PP2A. The natural mutation of residue A130 to a valine or threonine disrupts substrate recognition and catalysis. While NMR data revealed the A130T mutant Bbox1 domain failed to coordinate both structurally essential zinc ions and resulted in an unfolded structure, the unfolding mechanism is unknown. Principle component analysis revealed that residue A130 served as a hinge point between the structured β-strand-turn-β-strand (β-turn-β) and the lasso-like loop sub-structures that constitute loop1 of the ββα-RING fold that the Bbox1 domain adopts. Backbone RMSD data indicate significant flexibility and departure from the native structure within the first 5 ns of the molecular dynamics (MD) simulation for the A130V mutant (>6 Å) and after 30 ns for A130T mutant (>6 Å). Overall RMSF values were higher for the mutant structures and showed increased flexibility around residues 125 and 155, regions with zinc-coordinating residues. Simulated pKa values of the sulfhydryl group of C142 located near A130 suggested an increased in value to ~9.0, paralleling the increase in the apparent dielectric constants for the small cavity near residue A130. Protonation of the sulfhydryl group would disrupt zinc-coordination, directly contributing to unfolding of the Bbox1. Together, the increased motion of residues of loop 1, which contains four of the six zinc-binding cysteine residues, and the increased pKa of C142 could destabilize the structure of the zinc-coordinating residues and contribute to the unfolding.

Plasticity of 150-loop in influenza neuraminidase explored by Hamiltonian replica exchange molecular dynamics simulations.

PubMed

Han, Nanyu; Mu, Yuguang

2013-01-01

Neuraminidase (NA) of influenza is a key target for antiviral inhibitors, and the 150-cavity in group-1 NA provides new insight in treating this disease. However, NA of 2009 pandemic influenza (09N1) was found lacking this cavity in a crystal structure. To address the issue of flexibility of the 150-loop, Hamiltonian replica exchange molecular dynamics simulations were performed on different groups of NAs. Free energy landscape calculated based on the volume of 150-cavity indicates that 09N1 prefers open forms of 150-loop. The turn A (residues 147-150) of the 150-loop is discovered as the most dynamical motif which induces the inter-conversion of this loop among different conformations. In the turn A, the backbone dynamic of residue 149 is highly related with the shape of 150-loop, thus can function as a marker for the conformation of 150-loop. As a contrast, the closed conformation of 150-loop is more energetically favorable in N2, one of group-2 NAs. The D147-H150 salt bridge is found having no correlation with the conformation of 150-loop. Instead the intimate salt bridge interaction between the 150 and 430 loops in N2 variant contributes the stabilizing factor for the closed form of 150-loop. The clustering analysis elaborates the structural plasticity of the loop. This enhanced sampling simulation provides more information in further structural-based drug discovery on influenza virus.

Plasticity of 150-Loop in Influenza Neuraminidase Explored by Hamiltonian Replica Exchange Molecular Dynamics Simulations

PubMed Central

Han, Nanyu; Mu, Yuguang

2013-01-01

Neuraminidase (NA) of influenza is a key target for antiviral inhibitors, and the 150-cavity in group-1 NA provides new insight in treating this disease. However, NA of 2009 pandemic influenza (09N1) was found lacking this cavity in a crystal structure. To address the issue of flexibility of the 150-loop, Hamiltonian replica exchange molecular dynamics simulations were performed on different groups of NAs. Free energy landscape calculated based on the volume of 150-cavity indicates that 09N1 prefers open forms of 150-loop. The turn A (residues 147–150) of the 150-loop is discovered as the most dynamical motif which induces the inter-conversion of this loop among different conformations. In the turn A, the backbone dynamic of residue 149 is highly related with the shape of 150-loop, thus can function as a marker for the conformation of 150-loop. As a contrast, the closed conformation of 150-loop is more energetically favorable in N2, one of group-2 NAs. The D147-H150 salt bridge is found having no correlation with the conformation of 150-loop. Instead the intimate salt bridge interaction between the 150 and 430 loops in N2 variant contributes the stabilizing factor for the closed form of 150-loop. The clustering analysis elaborates the structural plasticity of the loop. This enhanced sampling simulation provides more information in further structural-based drug discovery on influenza virus. PMID:23593372

A curved RNA helix incorporating an internal loop with G·A and A·A non-Watson–Crick base pairing

PubMed Central

Baeyens, Katrien J.; De Bondt, Hendrik L.; Pardi, Arthur; Holbrook, Stephen R.

1996-01-01

The crystal structure of the RNA dodecamer 5′-GGCC(GAAA)GGCC-3′ has been determined from x-ray diffraction data to 2.3-Å resolution. In the crystal, these oligomers form double helices around twofold symmetry axes. Four consecutive non-Watson–Crick base pairs make up an internal loop in the middle of the duplex, including sheared G·A pairs and novel asymmetric A·A pairs. This internal loop sequence produces a significant curvature and narrowing of the double helix. The helix is curved by 34° from end to end and the diameter is narrowed by 24% in the internal loop. A Mn2+ ion is bound directly to the N7 of the first guanine in the Watson–Crick region following the internal loop and the phosphate of the preceding residue. This Mn2+ location corresponds to a metal binding site observed in the hammerhead catalytic RNA. PMID:8917508

Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

PubMed Central

Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

2017-01-01

3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors. PMID:28079168

Functional Loop Dynamics of the Streptavidin-Biotin Complex

PubMed Central

Song, Jianing; Li, Yongle; Ji, Changge; Zhang, John Z. H.

2015-01-01

Accelerated molecular dynamics (aMD) simulation is employed to study the functional dynamics of the flexible loop3-4 in the strong-binding streptavidin-biotin complex system. Conventional molecular (cMD) simulation is also performed for comparison. The present study reveals the following important properties of the loop dynamics: (1) The transition of loop3-4 from open to closed state is observed in 200 ns aMD simulation. (2) In the absence of biotin binding, the open-state streptavidin is more stable, which is consistent with experimental evidences. The free energy (ΔG) difference is about 5 kcal/mol between two states. But with biotin binding, the closed state is more stable due to electrostatic and hydrophobic interactions between the loop3-4 and biotin. (3) The closure of loop3-4 is concerted to the stable binding of biotin to streptavidin. When the loop3-4 is in its open-state, biotin moves out of the binding pocket, indicating that the interactions between the loop3-4 and biotin are essential in trapping biotin in the binding pocket. (4) In the tetrameric streptavidin system, the conformational change of the loop3-4 in each monomer is independent of each other. That is, there is no cooperative binding for biotin bound to the four subunits of the tetramer. PMID:25601277

Loop conformation and dynamics of the Escherichia coli HPPK apo-enzyme and its binary complex with MgATP.

PubMed

Yang, Rong; Lee, Matthew C; Yan, Honggao; Duan, Yong

2005-07-01

Comparison of the crystallographic and NMR structures of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) suggests that the enzyme may undergo significant conformational change upon binding to its first substrate, ATP. Two of the three surface loops (loop 2 and loop 3) accounting for most of the conformational differences appear to be confined by crystal contacts, raising questions about the putative large-scale induced-fit conformational change of HPPK and the functional roles of the conserved side-chain residues on the loops. To investigate the loop dynamics in crystal-free environment, we carried out molecular dynamics and locally enhanced sampling simulations of the apo-enzyme and the HPPK.MgATP complex. Our simulations showed that the crystallographic B-factors underestimated the loop dynamics considerably. We found that the open-conformation of loop 3 in the binary complex is accessible to the apo-enzyme and is the favored conformation in solution phase. These results revise our previous view of HPPK-substrate interactions and the associated functional mechanism of conformational change. The lessons learned here offer valuable structural insights into the workings of HPPK and should be useful for structure-based drug design.

Research on the Dynamic Hysteresis Loop Model of the Residence Times Difference (RTD)-Fluxgate

PubMed Central

Wang, Yanzhang; Wu, Shujun; Zhou, Zhijian; Cheng, Defu; Pang, Na; Wan, Yunxia

2013-01-01

Based on the core hysteresis features, the RTD-fluxgate core, while working, is repeatedly saturated with excitation field. When the fluxgate simulates, the accurate characteristic model of the core may provide a precise simulation result. As the shape of the ideal hysteresis loop model is fixed, it cannot accurately reflect the actual dynamic changing rules of the hysteresis loop. In order to improve the fluxgate simulation accuracy, a dynamic hysteresis loop model containing the parameters which have actual physical meanings is proposed based on the changing rule of the permeability parameter when the fluxgate is working. Compared with the ideal hysteresis loop model, this model has considered the dynamic features of the hysteresis loop, which makes the simulation results closer to the actual output. In addition, other hysteresis loops of different magnetic materials can be explained utilizing the described model for an example of amorphous magnetic material in this manuscript. The model has been validated by the output response comparison between experiment results and fitting results using the model. PMID:24002230

Trajectory tracking control for underactuated stratospheric airship

NASA Astrophysics Data System (ADS)

Zheng, Zewei; Huo, Wei; Wu, Zhe

2012-10-01

Stratospheric airship is a new kind of aerospace system which has attracted worldwide developing interests for its broad application prospects. Based on the trajectory linearization control (TLC) theory, a novel trajectory tracking control method for an underactuated stratospheric airship is presented in this paper. Firstly, the TLC theory is described sketchily, and the dynamic model of the stratospheric airship is introduced with kinematics and dynamics equations. Then, the trajectory tracking control strategy is deduced in detail. The designed control system possesses a cascaded structure which consists of desired attitude calculation, position control loop and attitude control loop. Two sub-loops are designed for the position and attitude control loops, respectively, including the kinematics control loop and dynamics control loop. Stability analysis shows that the controlled closed-loop system is exponentially stable. Finally, simulation results for the stratospheric airship to track typical trajectories are illustrated to verify effectiveness of the proposed approach.

Well-observed dynamics of flaring and peripheral coronal magnetic loops during an M-class limb flare

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Jinhua; Zhou, Tuanhui; Ji, Haisheng

2014-08-20

In this paper, we present a variety of well-observed dynamic behaviors for the flaring and peripheral magnetic loops of the M6.6 class extreme limb flare that occurred on 2011 February 24 (SOL2011-02-24T07:20) from EUV observations by the Atmospheric Imaging Assembly on the Solar Dynamics Observatory and X-ray observations by RHESSI. The flaring loop motion confirms the earlier contraction-expansion picture. We find that the U-shaped trajectory delineated by the X-ray corona source of the flare roughly follows the direction of a filament eruption associated with the flare. Different temperature structures of the coronal source during the contraction and expansion phases stronglymore » suggest different kinds of magnetic reconnection processes. For some peripheral loops, we discover that their dynamics are closely correlated with the filament eruption. During the slow rising to abrupt, fast rising of the filament, overlying peripheral magnetic loops display different responses. Two magnetic loops on the elbow of the active region had a slow descending motion followed by an abrupt successive fast contraction, while magnetic loops on the top of the filament were pushed outward, slowly being inflated for a while and then erupting as a moving front. We show that the filament activation and eruption play a dominant role in determining the dynamics of the overlying peripheral coronal magnetic loops.« less

Molecular dynamics simulations of electrostatics and hydration distributions around RNA and DNA motifs

NASA Astrophysics Data System (ADS)

Marlowe, Ashley E.; Singh, Abhishek; Semichaevsky, Andrey V.; Yingling, Yaroslava G.

2009-03-01

Nucleic acid nanoparticles can self-assembly through the formation of complementary loop-loop interactions or stem-stem interactions. Presence and concentration of ions can significantly affect the self-assembly process and the stability of the nanostructure. In this presentation we use explicit molecular dynamics simulations to examine the variations in cationic distributions and hydration environment around DNA and RNA helices and loop-loop interactions. Our simulations show that the potassium and sodium ionic distributions are different around RNA and DNA motifs which could be indicative of ion mediated relative stability of loop-loop complexes. Moreover in RNA loop-loop motifs ions are consistently present and exchanged through a distinct electronegative channel. We will also show how we used the specific RNA loop-loop motif to design a RNA hexagonal nanoparticle.

Dynamic loop gain increases upon adopting the supine body position during sleep in patients with obstructive sleep apnoea.

PubMed

Joosten, Simon A; Landry, Shane A; Sands, Scott A; Terrill, Philip I; Mann, Dwayne; Andara, Christopher; Skuza, Elizabeth; Turton, Anthony; Berger, Philip; Hamilton, Garun S; Edwards, Bradley A

2017-11-01

Obstructive sleep apnoea (OSA) is typically worse in the supine versus lateral sleeping position. One potential factor driving this observation is a decrease in lung volume in the supine position which is expected by theory to increase a key OSA pathogenic factor: dynamic ventilatory control instability (i.e. loop gain). We aimed to quantify dynamic loop gain in OSA patients in the lateral and supine positions, and to explore the relationship between change in dynamic loop gain and change in lung volume with position. Data from 20 patients enrolled in previous studies on the effect of body position on OSA pathogenesis were retrospectively analysed. Dynamic loop gain was calculated from routinely collected polysomnographic signals using a previously validated mathematical model. Lung volumes were measured in the awake state with a nitrogen washout technique. Dynamic loop gain was significantly higher in the supine than in the lateral position (0.77 ± 0.15 vs 0.68 ± 0.14, P = 0.012). Supine functional residual capacity (FRC) was significantly lower than lateral FRC (81.0 ± 15.4% vs 87.3 ± 18.4% of the seated FRC, P = 0.021). The reduced FRC we observed on moving to the supine position was predicted by theory to increase loop gain by 10.2 (0.6, 17.1)%, a value similar to the observed increase of 8.4 (-1.5, 31.0)%. Dynamic loop gain increased by a small but statistically significant amount when moving from the lateral to supine position and this may, in part, contribute to the worsening of OSA in the supine sleeping position. © 2017 Asian Pacific Society of Respirology.

Effect of proline and glycine residues on dynamics and barriers of loop formation in polypeptide chains.

PubMed

Krieger, Florian; Möglich, Andreas; Kiefhaber, Thomas

2005-03-16

Glycine and proline residues are frequently found in turn and loop structures of proteins and are believed to play an important role during chain compaction early in folding. We investigated their effect on the dynamics of intrachain loop formation in various unstructured polypeptide chains. Loop formation is significantly slower around trans prolyl peptide bonds and faster around glycine residues compared to any other amino acid. However, short loops are formed fastest around cis prolyl bonds with a time constant of 6 ns for end-to-end contact formation in a four-residue loop. Formation of short loops encounters activation energies in the range of 15 to 30 kJ/mol. The altered dynamics around glycine and trans prolyl bonds can be mainly ascribed to their effects on the activation energy. The fast dynamics around cis prolyl bonds, in contrast, originate in a higher Arrhenius pre-exponential factor, which compensates for an increased activation energy for loop formation compared to trans isomers. All-atom simulations of proline-containing peptides indicate that the conformational space for cis prolyl isomers is largely restricted compared to trans isomers. This leads to decreased average end-to-end distances and to a smaller loss in conformational entropy upon loop formation in cis isomers. The results further show that glycine and proline residues only influence formation of short loops containing between 2 and 10 residues, which is the typical loop size in native proteins. Formation of larger loops is not affected by the presence of a single glycine or proline residue.

Dynamic restructuring drives catalytic activity on nanoporous gold–silver alloy catalysts

DOE PAGES

Zugic, Branko; Wang, Lucun; Heine, Christian; ...

2016-12-19

Bimetallic, nanostructured materials hold promise for improving catalyst activity and selectivity, yet little is known about the dynamic compositional and structural changes that these systems undergo during pretreatment that leads to efficient catalyst function. Here we use ozone-activated silver–gold alloys in the form of nanoporous gold as a case study to demonstrate the dynamic behaviour of bimetallic systems during activation to produce a functioning catalyst. We show that it is these dynamic changes that give rise to the observed catalytic activity. Advanced in situ electron microscopy and X-ray photoelectron spectroscopy are used to demonstrate that major restructuring and compositional changesmore » occur along the path to catalytic function for selective alcohol oxidation. Transient kinetic measurements correlate the restructuring to three types of oxygen on the surface. The direct influence of changes in surface silver concentration and restructuring at the nanoscale on oxidation activity is demonstrated. Finally, our results demonstrate that characterization of these dynamic changes is necessary to unlock the full potential of bimetallic catalytic materials.« less

Dynamic restructuring drives catalytic activity on nanoporous gold–silver alloy catalysts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zugic, Branko; Wang, Lucun; Heine, Christian

Bimetallic, nanostructured materials hold promise for improving catalyst activity and selectivity, yet little is known about the dynamic compositional and structural changes that these systems undergo during pretreatment that leads to efficient catalyst function. Here we use ozone-activated silver–gold alloys in the form of nanoporous gold as a case study to demonstrate the dynamic behaviour of bimetallic systems during activation to produce a functioning catalyst. We show that it is these dynamic changes that give rise to the observed catalytic activity. Advanced in situ electron microscopy and X-ray photoelectron spectroscopy are used to demonstrate that major restructuring and compositional changesmore » occur along the path to catalytic function for selective alcohol oxidation. Transient kinetic measurements correlate the restructuring to three types of oxygen on the surface. The direct influence of changes in surface silver concentration and restructuring at the nanoscale on oxidation activity is demonstrated. Finally, our results demonstrate that characterization of these dynamic changes is necessary to unlock the full potential of bimetallic catalytic materials.« less

Man-in-the-control-loop simulation of manipulators

NASA Technical Reports Server (NTRS)

Chang, J. L.; Lin, Tsung-Chieh; Yae, K. Harold

1989-01-01

A method to achieve man-in-the-control-loop simulation is presented. Emerging real-time dynamics simulation suggests a potential for creating an interactive design workstation with a human operator in the control loop. The recursive formulation for multibody dynamics simulation is studied to determine requirements for man-in-the-control-loop simulation. High speed computer graphics techniques provides realistic visual cues for the simulator. Backhoe and robot arm simulations are implemented to demonstrate the capability of man-in-the-control-loop simulation.

A molecular dynamics study of the BACE1 conformational change from Apo to closed form induced by hydroxyethylamine derived compounds.

PubMed

Gueto-Tettay, Carlos; Zuchniarz, Joshua; Fortich-Seca, Yeyson; Gueto-Tettay, Luis Roberto; Drosos-Ramirez, Juan Carlos

2016-11-01

BACE1 is an aspartyl protease which is a therapeutic target for Alzheimer's disease (AD) because of its participation in the rate-limiting step in the production of Aβ-peptide, the accumulation of which produces senile plaques and, in turn, the neurodegenerative effects associated with AD. The active site of this protease is composed in part by two aspartic residues (Asp93 and Asp289). Additionally, the catalytic site has been found to be covered by an antiparallel hairpin loop called the flap. The dynamics of this flap are fundamental to the catalytic function of the enzyme. When BACE1 is inactive (Apo), the flap adopts an open conformation, allowing a substrate or inhibitor to access the active site. Subsequent interaction with the ligand induces flap closure and the stabilization of the macromolecular complex. Further, the protonation state of the aspartic dyad is affected by the chemical nature of the species entering the active site, so that appropriate selection of protonation states for the ligand and the catalytic residues will permit the elucidation of the inhibitory pathway for BACE1. In the present study, comparative analysis of different combinations of protonation states for the BACE1-hydroxyethylamine (HEA) system is reported. HEAs are potent inhibitors of BACE1 with favorable pharmacological and kinetic properties, as well as oral bioavailability. The results of Molecular Dynamics (MD) simulations and population density calculations using 8 different parameters demonstrate that the LnAsp289 configuration (HEA with a neutral amine and the Asp289 residue protonated) is the only one which permits the expected conformational change in BACE1, from apo to closed form, after flap closure. Additionally, differences in their capacities to establish and maintain interactions with residues such as Asp93, Gly95, Thr133, Asp289, Gly291, and Asn294 during this step allow differentiation among the inhibitory activities of the HEAs. The results and methodology here reported will serve to elucidate the inhibitory pathway of other families of compounds that act as BACE1 inhibitors, as well as the design of better leader compounds for the treatment of AD. Copyright © 2016 Elsevier Inc. All rights reserved.

Spontaneous emergence of cataclysmic networks in spatially extended systems

NASA Astrophysics Data System (ADS)

Manrubia, Susanna C.; Poyatos, Juan F.; Pérez-Mercader, Juan

2002-11-01

A system of interacting chemical species able to catalyse each others' production is studied. We consider a two-dimensional surface where single molecules attach, diffuse, catalytically interact, and decay. The population of species molecules and the network of interactions among them are dynamical entities. After a short transient time, robust catalytic cycles emerge and a "stationary" state of high diversity and large population numbers settles down. Population dynamics and physical space select among possible graphs of catalytic interactions. The organization of the system is robust: parasitic invaders are short-lived, their populations are kept at low levels, and are unable to sweep away the emerging catalytic cycles.

Binding mechanism and dynamic conformational change of C subunit of PKA with different pathways

PubMed Central

Chu, Wen-Ting; Chu, Xiakun; Wang, Jin

2017-01-01

The catalytic subunit of PKA (PKAc) exhibits three major conformational states (open, intermediate, and closed) during the biocatalysis process. Both ATP and substrate/inhibitor can effectively induce the conformational changes of PKAc from open to closed states. Aiming to explore the mechanism of this allosteric regulation, we developed a coarse-grained model and analyzed the dynamics of conformational changes of PKAc during binding by performing molecular dynamics simulations for apo PKAc, binary PKAc (PKAc with ATP, PKAc with PKI), and ternary PKAc (PKAc with ATP and PKI). Our results suggest a mixed binding mechanism of induced fit and conformational selection, with the induced fit dominant. The ligands can drive the movements of Gly-rich loop as well as some regions distal to the active site in PKAc and stabilize them at complex state. In addition, there are two parallel pathways (pathway with PKAc-ATP as an intermediate and pathway PKAc-PKI as an intermediate) during the transition from open to closed states. By molecular dynamics simulations and rate constant analyses, we find that the pathway through PKAc-ATP intermediate is the main binding route from open to closed state because of the fact that the bound PKI will hamper ATP from successful binding and significantly increase the barrier for the second binding subprocess. These findings will provide fundamental insights of the mechanisms of PKAc conformational change upon binding. PMID:28855336

Binding mechanism and dynamic conformational change of C subunit of PKA with different pathways.

PubMed

Chu, Wen-Ting; Chu, Xiakun; Wang, Jin

2017-09-19

The catalytic subunit of PKA (PKAc) exhibits three major conformational states (open, intermediate, and closed) during the biocatalysis process. Both ATP and substrate/inhibitor can effectively induce the conformational changes of PKAc from open to closed states. Aiming to explore the mechanism of this allosteric regulation, we developed a coarse-grained model and analyzed the dynamics of conformational changes of PKAc during binding by performing molecular dynamics simulations for apo PKAc, binary PKAc (PKAc with ATP, PKAc with PKI), and ternary PKAc (PKAc with ATP and PKI). Our results suggest a mixed binding mechanism of induced fit and conformational selection, with the induced fit dominant. The ligands can drive the movements of Gly-rich loop as well as some regions distal to the active site in PKAc and stabilize them at complex state. In addition, there are two parallel pathways (pathway with PKAc-ATP as an intermediate and pathway PKAc-PKI as an intermediate) during the transition from open to closed states. By molecular dynamics simulations and rate constant analyses, we find that the pathway through PKAc-ATP intermediate is the main binding route from open to closed state because of the fact that the bound PKI will hamper ATP from successful binding and significantly increase the barrier for the second binding subprocess. These findings will provide fundamental insights of the mechanisms of PKAc conformational change upon binding.

Towards a Molecular Understanding of the Link between Imatinib Resistance and Kinase Conformational Dynamics

PubMed Central

Lovera, Silvia; Morando, Maria; Pucheta-Martinez, Encarna; Martinez-Torrecuadrada, Jorge L.; Saladino, Giorgio; Gervasio, Francesco L.

2015-01-01

Due to its inhibition of the Abl kinase domain in the BCR-ABL fusion protein, imatinib is strikingly effective in the initial stage of chronic myeloid leukemia with more than 90% of the patients showing complete remission. However, as in the case of most targeted anti-cancer therapies, the emergence of drug resistance is a serious concern. Several drug-resistant mutations affecting the catalytic domain of Abl and other tyrosine kinases are now known. But, despite their importance and the adverse effect that they have on the prognosis of the cancer patients harboring them, the molecular mechanism of these mutations is still debated. Here by using long molecular dynamics simulations and large-scale free energy calculations complemented by in vitro mutagenesis and microcalorimetry experiments, we model the effect of several widespread drug-resistant mutations of Abl. By comparing the conformational free energy landscape of the mutants with those of the wild-type tyrosine kinases we clarify their mode of action. It involves significant and complex changes in the inactive-to-active dynamics and entropy/enthalpy balance of two functional elements: the activation-loop and the conserved DFG motif. What is more the T315I gatekeeper mutant has a significant impact on the binding mechanism itself and on the binding kinetics. PMID:26606374

Closed Loop Vibrational Control: Theory and Applications

DTIC Science & Technology

1993-10-01

the open loop system dynamics will be close to that of Bit. However, in general, in a closed loop system with a specified feedback co-’ - oller , for...Juang, and G. Rodriguez , "Formulations and Applications of Large Structure Actuator and Sensor Placements," Second VPI & SU/AIAA Symposium on Dynamics

Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.

2012-09-17

Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg{sup 2+}-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further ourmore » understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k{sub cat} decreased 10{sup 3}- and 10{sup 5}-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp{sup 283} functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ('P-loop') provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.« less

Catalytic efficiency and thermostability improvement of Suc2 invertase through rational site-directed mutagenesis.

PubMed

Mohandesi, Nooshin; Haghbeen, Kamahldin; Ranaei, Omid; Arab, Seyed Shahriar; Hassani, Sorour

2017-01-01

Engineering of invertases has come to attention because of increasing demand for possible applications of invertases in various industrial processes. Due to the known physicochemical properties, invertases from micro-organisms such as Saccharomyces cerevisiae carrying SUC2 gene are considered as primary models. To improve thermostability and catalytic efficiency of SUC2 invertase (SInv), six influential residues with Relative Solvent Accessibility<5% were selected through multiple-sequence alignments, molecular modelling, structural and computational analyses. Consequently, SInv and 5 mutants including three mutants with single point substitution [Mut1=P152V, Mut2=S85V and Mut3=K153F)], one mutant with two points [Mut4=S305V-N463V] and one mutant with three points [Mut5=S85V-K153F-T271V] were developed via site-directed mutagenesis and produced using Pichia pastoris as the host. Physicochemical studies on these enzymes indicated that the selected amino acids which were located in the active site region mainly influenced catalytic efficiency. The best improvement belonged to Mut1 (54% increase in K cat /K m ) and Mut3 exhibited the worst effect (90% increase in K m ). These results suggest that Pro152 and Lys153 play key role in preparation of the right substrate lodging in the active site of SInv. The best thermostability improvement (16%) was observed for Mut4 in which two hydrophilic residues located on the loops, far from the active site, were replaced by Valines. These results suggest that tactful simultaneous substitution of influential hydrophilic residues in both active site region and peripheral loops with hydrophobic amino acids could result in more thermostable invertases with enhanced catalytic efficiency. Copyright © 2016 Elsevier Inc. All rights reserved.

Similarity Metrics for Closed Loop Dynamic Systems

NASA Technical Reports Server (NTRS)

Whorton, Mark S.; Yang, Lee C.; Bedrossian, Naz; Hall, Robert A.

2008-01-01

To what extent and in what ways can two closed-loop dynamic systems be said to be "similar?" This question arises in a wide range of dynamic systems modeling and control system design applications. For example, bounds on error models are fundamental to the controller optimization with modern control design methods. Metrics such as the structured singular value are direct measures of the degree to which properties such as stability or performance are maintained in the presence of specified uncertainties or variations in the plant model. Similarly, controls-related areas such as system identification, model reduction, and experimental model validation employ measures of similarity between multiple realizations of a dynamic system. Each area has its tools and approaches, with each tool more or less suited for one application or the other. Similarity in the context of closed-loop model validation via flight test is subtly different from error measures in the typical controls oriented application. Whereas similarity in a robust control context relates to plant variation and the attendant affect on stability and performance, in this context similarity metrics are sought that assess the relevance of a dynamic system test for the purpose of validating the stability and performance of a "similar" dynamic system. Similarity in the context of system identification is much more relevant than are robust control analogies in that errors between one dynamic system (the test article) and another (the nominal "design" model) are sought for the purpose of bounding the validity of a model for control design and analysis. Yet system identification typically involves open-loop plant models which are independent of the control system (with the exception of limited developments in closed-loop system identification which is nonetheless focused on obtaining open-loop plant models from closed-loop data). Moreover the objectives of system identification are not the same as a flight test and hence system identification error metrics are not directly relevant. In applications such as launch vehicles where the open loop plant is unstable it is similarity of the closed-loop system dynamics of a flight test that are relevant.

Adenosylcobinamide methyl phosphate as a pseudocoenzyme for diol dehydrase.

PubMed

Ishida, A; Toraya, T

1993-02-16

Adenosylcobinamide methyl phosphate, a novel analog of adenosylcobalamin lacking the nucleotide loop moiety, was synthesized. It did not show detectable coenzymic activity but behaved as a strong competitive inhibitor against AdoCbl with relatively high affinity (Ki = 2.5 microM). When apoenzyme was incubated at 37 degrees C with this analog in the presence of substrate, the Co-C bond of the analog was almost completely and irreversibly cleaved within 10 min, forming an enzyme-bound Co(II)-containing species. The cleavage was not observed in the absence of substrate. The Co-C bond cleavage in the presence of substrate was not catalytic but stoichiometric, implying that the Co-C bond of the analog undergoes activation when the analog binds to the active site of the enzyme. 5'-Deoxyadenosine was the only product derived from the adenosyl group of the analog upon the Co-C bond cleavage. Apoenzyme did not undergo modification during this process. Therefore, it seems likely that adenosylcobinamide methyl phosphate acts as a pseudocoenzyme or a potent suicide coenzyme. Since adenosylcobinamide neither functions as coenzyme nor binds tightly to apoenzyme, it can be concluded that the phosphodiester moiety of the nucleotide loop of adenosylcobalamin is essential for tight binding to apoenzyme and therefore for subsequent activation of the Co-C bond and catalysis. It is also evident that the nucleotide loop is obligatory for the normal progress of catalytic cycle.

Unveiling a novel transient druggable pocket in BACE-1 through molecular simulations: Conformational analysis and binding mode of multisite inhibitors

PubMed Central

Di Pietro, Ornella; Laughton, Charles A.

2017-01-01

The critical role of BACE-1 in the formation of neurotoxic ß-amyloid peptides in the brain makes it an attractive target for an efficacious treatment of Alzheimer’s disease. However, the development of clinically useful BACE-1 inhibitors has proven to be extremely challenging. In this study we examine the binding mode of a novel potent inhibitor (compound 1, with IC50 80 nM) designed by synergistic combination of two fragments—huprine and rhein—that individually are endowed with very low activity against BACE-1. Examination of crystal structures reveals no appropriate binding site large enough to accommodate 1. Therefore we have examined the conformational flexibility of BACE-1 through extended molecular dynamics simulations, paying attention to the highly flexible region shaped by loops 8–14, 154–169 and 307–318. The analysis of the protein dynamics, together with studies of pocket druggability, has allowed us to detect the transient formation of a secondary binding site, which contains Arg307 as a key residue for the interaction with small molecules, at the edge of the catalytic cleft. The formation of this druggable “floppy” pocket would enable the binding of multisite inhibitors targeting both catalytic and secondary sites. Molecular dynamics simulations of BACE-1 bound to huprine-rhein hybrid compounds support the feasibility of this hypothesis. The results provide a basis to explain the high inhibitory potency of the two enantiomeric forms of 1, together with the large dependence on the length of the oligomethylenic linker. Furthermore, the multisite hypothesis has allowed us to rationalize the inhibitory potency of a series of tacrine-chromene hybrid compounds, specifically regarding the apparent lack of sensitivity of the inhibition constant to the chemical modifications introduced in the chromene unit. Overall, these findings pave the way for the exploration of novel functionalities in the design of optimized BACE-1 multisite inhibitors. PMID:28505196

Protein arginine methyltransferase 7 has a novel homodimer-like structure formed by tandem repeats.

PubMed

Hasegawa, Morio; Toma-Fukai, Sachiko; Kim, Jun-Dal; Fukamizu, Akiyoshi; Shimizu, Toshiyuki

2014-05-21

Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to nitrogen atoms on arginine residues. Here, we describe the crystal structure of Caenorhabditis elegans PRMT7 in complex with its reaction product S-adenosyl-L-homocysteine. The structural data indicated that PRMT7 harbors two tandem repeated PRMT core domains that form a novel homodimer-like structure. S-adenosyl-L-homocysteine bound to the N-terminal catalytic site only; the C-terminal catalytic site is occupied by a loop that inhibits cofactor binding. Mutagenesis demonstrated that only the N-terminal catalytic site of PRMT7 is responsible for cofactor binding. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Molecular dynamics simulation of hepatitis C virus IRES IIId domain: structural behavior, electrostatic and energetic analysis.

PubMed

Golebiowski, Jérôme; Antonczak, Serge; Di-Giorgio, Audrey; Condom, Roger; Cabrol-Bass, Daniel

2004-02-01

The dynamic behavior of the HCV IRES IIId domain is analyzed by means of a 2.6-ns molecular dynamics simulation, starting from an NMR structure. The simulation is carried out in explicit water with Na+ counterions, and particle-mesh Ewald summation is used for the electrostatic interactions. In this work, we analyze selected patterns of the helix that are crucial for IRES activity and that could be considered as targets for the intervention of inhibitors, such as the hexanucleotide terminal loop (more particularly its three consecutive guanines) and the loop-E motif. The simulation has allowed us to analyze the dynamics of the loop substructure and has revealed a behavior among the guanine bases that might explain the different role of the third guanine of the GGG triplet upon molecular recognition. The accessibility of the loop-E motif and the loop major and minor groove is also examined, as well as the effect of Na+ or Mg2+ counterion within the simulation. The electrostatic analysis reveals several ion pockets, not discussed in the experimental structure. The positions of these ions are useful for locating specific electrostatic recognition sites for potential inhibitor binding.

A Dynamic Model of Sustainment Investment

DTIC Science & Technology

2015-02-01

Sustainment System Dynamics Model 11 Figure 7: Core Structure of Sustainment Work 12 Figure 8: Bandwagon Effect Loop 13 Figure 9: Limits to Growth Loop 14...Dynamics Model sustainment capacity sustainment performance gap Bandwagon Effect R1 Limits to Growth B1 S Work Smarter B3 Work Bigger B2 desired...which is of concern primarily when using the model as a vehicle for research. Figure 8 depicts a reinforcing loop called the “ Bandwagon Effect

Crystal structure of the human cytosolic sialidase Neu2. Evidence for the dynamic nature of substrate recognition.

PubMed

Chavas, Leonard M G; Tringali, Cristina; Fusi, Paola; Venerando, Bruno; Tettamanti, Guido; Kato, Ryuichi; Monti, Eugenio; Wakatsuki, Soichi

2005-01-07

Gangliosides play key roles in cell differentiation, cell-cell interactions, and transmembrane signaling. Sialidases hydrolyze sialic acids to produce asialo compounds, which is the first step of degradation processes of glycoproteins and gangliosides. Sialidase involvement has been implicated in some lysosomal storage disorders such as sialidosis and galactosialidosis. Neu2 is a recently identified human cytosolic sialidase. Here we report the first high resolution x-ray structures of mammalian sialidase, human Neu2, in its apo form and in complex with an inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA). The structure shows the canonical six-blade beta-propeller observed in viral and bacterial sialidases with its active site in a shallow crevice. In the complex structure, the inhibitor lies in the catalytic crevice surrounded by ten amino acids. In particular, the arginine triad, conserved among sialidases, aids in the proper positioning of the carboxylate group of DANA within the active site region. The tyrosine residue, Tyr(334), conserved among mammalian and bacterial sialidases as well as in viral neuraminidases, facilitates the enzymatic reaction by stabilizing a putative carbonium ion in the transition state. The loops containing Glu(111) and the catalytic aspartate Asp(46) are disordered in the apo form but upon binding of DANA become ordered to adopt two short alpha-helices to cover the inhibitor, illustrating the dynamic nature of substrate recognition. The N-acetyl and glycerol moieties of DANA are recognized by Neu2 residues not shared by bacterial sialidases and viral neuraminidases, which can be regarded as a key structural difference for potential drug design against bacteria, influenza, and other viruses.

Feedback control laws for highly maneuverable aircraft

NASA Technical Reports Server (NTRS)

Garrard, William L.; Balas, Gary J.

1994-01-01

During the first half of the year, the investigators concentrated their efforts on completing the design of control laws for the longitudinal axis of the HARV. During the second half of the year they concentrated on the synthesis of control laws for the lateral-directional axes. The longitudinal control law design efforts can be briefly summarized as follows. Longitudinal control laws were developed for the HARV using mu synthesis design techniques coupled with dynamic inversion. An inner loop dynamic inversion controller was used to simplify the system dynamics by eliminating the aerodynamic nonlinearities and inertial cross coupling. Models of the errors resulting from uncertainties in the principal longitudinal aerodynamic terms were developed and included in the model of the HARV with the inner loop dynamic inversion controller. This resulted in an inner loop transfer function model which was an integrator with the modeling errors characterized as uncertainties in gain and phase. Outer loop controllers were then designed using mu synthesis to provide robustness to these modeling errors and give desired response to pilot inputs. Both pitch rate and angle of attack command following systems were designed. The following tasks have been accomplished for the lateral-directional controllers: inner and outer loop dynamic inversion controllers have been designed; an error model based on a linearized perturbation model of the inner loop system was derived; controllers for the inner loop system have been designed, using classical techniques, that control roll rate and Dutch roll response; the inner loop dynamic inversion and classical controllers have been implemented on the six degree of freedom simulation; and lateral-directional control allocation scheme has been developed based on minimizing required control effort.

Analysis of the bacterial luciferase mobile loop by replica-exchange molecular dynamics.

PubMed

Campbell, Zachary T; Baldwin, Thomas O; Miyashita, Osamu

2010-12-15

Bacterial luciferase contains an extended 29-residue mobile loop. Movements of this loop are governed by binding of either flavin mononucleotide (FMNH2) or polyvalent anions. To understand this process, loop dynamics were investigated using replica-exchange molecular dynamics that yielded conformational ensembles in either the presence or absence of FMNH2. The resulting data were analyzed using clustering and network analysis. We observed the closed conformations that are visited only in the simulations with the ligand. Yet the mobile loop is intrinsically flexible, and FMNH2 binding modifies the relative populations of conformations. This model provides unique information regarding the function of a crystallographically disordered segment of the loop near the binding site. Structures at or near the fringe of this network were compatible with flavin binding or release. Finally, we demonstrate that the crystallographically observed conformation of the mobile loop bound to oxidized flavin was influenced by crystal packing. Thus, our study has revealed what we believe are novel conformations of the mobile loop and additional context for experimentally determined structures. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The Catalytic and Non-catalytic Functions of the Brahma Chromatin-Remodeling Protein Collaborate to Fine-Tune Circadian Transcription in Drosophila

PubMed Central

Kwok, Rosanna S.; Li, Ying H.; Lei, Anna J.; Edery, Isaac; Chiu, Joanna C.

2015-01-01

Daily rhythms in gene expression play a critical role in the progression of circadian clocks, and are under regulation by transcription factor binding, histone modifications, RNA polymerase II (RNAPII) recruitment and elongation, and post-transcriptional mechanisms. Although previous studies have shown that clock-controlled genes exhibit rhythmic chromatin modifications, less is known about the functions performed by chromatin remodelers in animal clockwork. Here we have identified the Brahma (Brm) complex as a regulator of the Drosophila clock. In Drosophila, CLOCK (CLK) is the master transcriptional activator driving cyclical gene expression by participating in an auto-inhibitory feedback loop that involves stimulating the expression of the main negative regulators, period (per) and timeless (tim). BRM functions catalytically to increase nucleosome density at the promoters of per and tim, creating an overall restrictive chromatin landscape to limit transcriptional output during the active phase of cycling gene expression. In addition, the non-catalytic function of BRM regulates the level and binding of CLK to target promoters and maintains transient RNAPII stalling at the per promoter, likely by recruiting repressive and pausing factors. By disentangling its catalytic versus non-catalytic functions at the promoters of CLK target genes, we uncovered a multi-leveled mechanism in which BRM fine-tunes circadian transcription. PMID:26132408

Dynamic simulation of perturbation responses in a closed-loop virtual arm model.

PubMed

Du, Yu-Fan; He, Xin; Lan, Ning

2010-01-01

A closed-loop virtual arm (VA) model has been developed in SIMULINK environment by adding spinal reflex circuits and propriospinal neural networks to the open-loop VA model developed in early study [1]. An improved virtual muscle model (VM4.0) is used to speed up simulation and to generate more precise recruitment of muscle force at low levels of muscle activation. Time delays in the reflex loops are determined by their synaptic connections and afferent transmission back to the spinal cord. Reflex gains are properly selected so that closed-loop responses are stable. With the closed-loop VA model, we are developing an approach to evaluate system behaviors by dynamic simulation of perturbation responses. Joint stiffness is calculated based on simulated perturbation responses by a least-squares algorithm in MATLAB. This method of dynamic simulation will be essential for further evaluation of feedforward and reflex control of arm movement and position.

Interaction of 〈1 0 0〉 dislocation loops with dislocations studied by dislocation dynamics in α-iron

NASA Astrophysics Data System (ADS)

Shi, X. J.; Dupuy, L.; Devincre, B.; Terentyev, D.; Vincent, L.

2015-05-01

Interstitial dislocation loops with Burgers vector of 〈1 0 0〉 type are formed in α-iron under neutron or heavy ion irradiation. As the density and size of these loops increase with radiation dose and temperature, these defects are thought to play a key role in hardening and subsequent embrittlement of iron-based steels. The aim of the present work is to study the pinning strength of the loops on mobile dislocations. Prior to run massive Dislocation Dynamics (DD) simulations involving experimentally representative array of radiation defects and dislocations, the DD code and its parameterization are validated by comparing the individual loop-dislocation reactions with those obtained from direct atomistic Molecular Dynamics (MD) simulations. Several loop-dislocation reaction mechanisms are successfully reproduced as well as the values of the unpinning stress to detach mobile dislocations from the defects.

Conformational Flexibility of Human Casein Kinase Catalytic Subunit Explored by Metadynamics

PubMed Central

Gouron, Aurélie; Milet, Anne; Jamet, Helene

2014-01-01

Casein kinase CK2 is an essential enzyme in higher organisms, catalyzing the transfer of the γ phosphate from ATP to serine and threonine residues on protein substrates. In a number of animal tumors, CK2 activity has been shown to escape normal cellular control, making it a potential target for cancer therapy. Several crystal structures of human CK2 have been published with different conformations for the CK2α catalytic subunit. This variability reflects a high flexibility for two regions of CK2α: the interdomain hinge region, and the glycine-rich loop (p-loop). Here, we present a computational study simulating the equilibrium between three conformations involving these regions. Simulations were performed using well-tempered metadynamics combined with a path collective variables approach. This provides a reference pathway describing the conformational changes being studied, based on analysis of free energy surfaces. The free energies of the three conformations were found to be close and the paths proposed had low activation barriers. Our results indicate that these conformations can exist in water. This information should be useful when designing inhibitors specific to one conformation. PMID:24606937

Active site CP-loop dynamics modulate substrate binding, catalysis, oligomerization, stability, over-oxidation and recycling of 2-Cys Peroxiredoxins.

PubMed

Kamariah, Neelagandan; Eisenhaber, Birgit; Eisenhaber, Frank; Grüber, Gerhard

2018-04-01

Peroxiredoxins (Prxs) catalyse the rapid reduction of hydrogen peroxide, organic hydroperoxide and peroxynitrite, using a fully conserved peroxidatic cysteine (C P ) located in a conserved sequence Pxxx(T/S)xxC P motif known as C P -loop. In addition, Prxs are involved in cellular signaling pathways and regulate several redox-dependent process related disease. The effective catalysis of Prxs is associated with alterations in the C P -loop between reduced, Fully Folded (FF), and oxidized, Locally Unfolded (LU) conformations, which are linked to dramatic changes in the oligomeric structure. Despite many studies, little is known about the precise structural and dynamic roles of the C P -loop on Prxs functions. Herein, the comprehensive biochemical and biophysical studies on Escherichia coli alkyl hydroperoxide reductase subunit C (EcAhpC) and the C P -loop mutants, EcAhpC-F45A and EcAhpC-F45P reveal that the reduced form of the C P -loop adopts conformational dynamics, which is essential for effective peroxide reduction. Furthermore, the point mutants alter the structure and dynamics of the reduced form of the C P -loop and, thereby, affect substrate binding, catalysis, oligomerization, stability and overoxidiation. In the oxidized form, due to restricted C P -loop dynamics, the EcAhpC-F45P mutant favours a decamer formation, which enhances the effective recycling by physiological reductases compared to wild-type EcAhpC. In addition, the study reveals that residue F45 increases the specificity of Prxs-reductase interactions. Based on these studies, we propose an evolution of the C P -loop with confined sequence conservation within Prxs subfamilies that might optimize the functional adaptation of Prxs into various physiological roles. Copyright © 2018 Elsevier Inc. All rights reserved.

Origins of stereoselectivity in evolved ketoreductases.

PubMed

Noey, Elizabeth L; Tibrewal, Nidhi; Jiménez-Osés, Gonzalo; Osuna, Sílvia; Park, Jiyong; Bond, Carly M; Cascio, Duilio; Liang, Jack; Zhang, Xiyun; Huisman, Gjalt W; Tang, Yi; Houk, Kendall N

2015-12-22

Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo- and NADP(+)-bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation.

Origins of stereoselectivity in evolved ketoreductases

PubMed Central

Noey, Elizabeth L.; Tibrewal, Nidhi; Jiménez-Osés, Gonzalo; Osuna, Sílvia; Park, Jiyong; Bond, Carly M.; Cascio, Duilio; Liang, Jack; Zhang, Xiyun; Huisman, Gjalt W.; Tang, Yi; Houk, Kendall N.

2015-01-01

Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo- and NADP+-bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation. PMID:26644568

Snapshots of Dynamics in Synthesizing N6-isopentenyladenosine at tRNA Anticodon†,‡

PubMed Central

Chimnaronk, Sarin; Forouhar, Farhad; Sakai, Junichi; Yao, Min; Tron, Cecile M.; Atta, Mohamed; Fontecave, Marc; Hunt, John F.; Tanaka, Isao

2009-01-01

Bacterial and eukaryotic transfer RNAs that decode codons starting with uridine have a hydrophobically-hypermodified adenosine at the position 37 (A37) adjacent to the 3′-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. However, it remains unclear how the corresponding tRNAs are selected to be modified by alkylation at the correct position of the adenosine base. We have determined a series of the crystal structures of bacterial tRNA isopentenyltransferase (MiaA) in apo- and tRNA-bound forms, which completely render snapshots of substrate selections during modification of RNA. A compact evolutionary inserted domain (herein ‘swinging domain’) in MiaA that exhibits as a highly mobile entity moves around the catalytic domain as likely to reach and trap the tRNA substrate. Thereby, MiaA clamps the anticodon stem loop of tRNA substrate between the catalytic and swinging domains, where the two conserved elongated residues from the swinging domain pinch the two flanking A36 and A38 together to squeeze out A37 into the reaction tunnel. The site-specific isopentenylation of RNA is thus ensured by a characteristic pinch-and-flip mechanism and by a reaction tunnel to confine the substrate selection. Furthermore, combining information from soaking experiments with structural comparisons, we propose a mechanism for the ordered substrate-binding of MiaA. PMID:19435325

Reaction Mechanism of Isopentenyl Phosphate Kinase: A QM/MM Study.

PubMed

McClory, James; Timson, David J; Singh, Warispreet; Zhang, Jian; Huang, Meilan

2017-12-14

Isopentenyl phosphate kinase (IPK) catalyzes the Mg 2+ -ATP dependent phosphorylation reactions to produce isopentenyl diphosphate, an important precursor in the synthesis of isopentenols. However, the position of the divalent metal ion in the crystal structures of IPK in complex with ATP and its native substrate IP has not been definitively resolved, and as a result ambiguity surrounds the catalytic mechanism of IP, limiting its exploitation as a biofuel and in drug design. Here we report the catalytically competent structure in complex with the metal ion Mg 2+ and elucidate the phosphorylation reaction mechanism using molecular dynamic simulations and density functional theory-based quantum mechanics/molecular mechanics calculations (B97d/AMBER99). Comparing the substrate-bound and substrate-free IPK complexes, we observed that substrate binding results in significant conformational change of three residues Lys204, Glu207, and Lys211 located on the αG helix to form a strong salt bridge network with Asp145, which in turn tethers the invariant Ser142 via H-bond interaction. The conformational change shuts the subtrate entrance channel formed between the αG and αE helices. Further, we demonstrate the phosphorylation reaction occurs with a reaction barrier of 17.58 kcal/mol, which is in agreement with the previous experimental kinetic data. We found that a highly conserved Gly8 on a glycine-rich loop, together with Lys14, stabilizes the transition state.

Structural Basis for Inactivation of the Human Pyruvate Dehydrogenase Complex by Phosphorylation: Role of Disordered Phosphorylation Loops

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kato, Masato; Wynn, R. Max; Chuang, Jacinta L.

2009-09-11

We report the crystal structures of the phosporylated pyruvate dehydrogenase (E1p) component of the human pyruvate dehydrogenase complex (PDC). The complete phosphorylation at Ser264-{alpha} (site 1) of a variant E1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the PDC core. We show that unlike its unmodified counterpart, the presence of a phosphoryl group at Ser264-{alpha} prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three phosphorylation sites. The disordering of these phosphorylation loops is caused by a previously unrecognized steric clash between the phosphoryl group at site 1 and a nearby Ser266-{alpha}, whichmore » nullifies a hydrogen-bonding network essential for maintaining the loop conformations. The disordered phosphorylation loops impede the binding of lipoyl domains of the PDC core to E1p, negating the reductive acetylation step. This results in the disruption of the substrate channeling in the PDC, leading to the inactivation of this catalytic machine.« less

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter.

PubMed

Hohl, Michael; Hürlimann, Lea M; Böhm, Simon; Schöppe, Jendrik; Grütter, Markus G; Bordignon, Enrica; Seeger, Markus A

2014-07-29

ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5'-(β,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.

Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeh, Joanne I.; Kettering, Regina; Saxl, Ruth

2009-09-11

Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft aremore » more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of glycerol kinase, the ATP molecule, may largely determine the rate of glycerol 3-phosphate production.« less

Structure and dynamics of GeoCyp: a thermophilic cyclophilin with a novel substrate binding mechanism that functions efficiently at low temperatures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holliday, Michael; Camilloni, Carlo; Armstrong, Geoffrey S.

2015-05-26

Thermophilic proteins have found extensive use in research and industrial applications due to their high stability and functionality at elevated temperatures while simultaneously providing valuable insight into our understanding of protein folding, stability, dynamics, and function. Cyclophilins, a ubiquitously expressed family of peptidyl-prolyl isomerases with a range of biological functions and disease associations, have been utilized both for conferring stress tolerances and in exploring the link between conformational dynamics and enzymatic function. To date, however, no active thermophilic cyclophilin has been fully biophysically characterized. Here, we determine the structure of a thermophilic cyclophilin (GeoCyp) from Geobacillus kaustophilus, characterize its dynamicmore » motions over several timescales using an array of methodologies that include chemical shift-based methods and relaxation experiments over a range of temperatures, and measure catalytic activity over a range of temperatures in order to compare structure, dynamics, and function to a mesophilic counterpart, human Cyclophilin A (CypA). Unlike most thermophile/mesophile pairs, GeoCyp catalysis is not substantially impaired at low temperatures as compared to CypA, retaining ~70% of the activity of its mesophilic counterpart. Examination of substrate-bound ensembles reveals a mechanism by which the two cyclophilins may have adapted to their environments through altering dynamic loop motions and a critical residue that acts as a clamp to regulate substrate binding differentially in CypA and GeoCyp. Despite subtle differences in conformational movements, dynamics over fast (ps-ns) and slow (μs) timescales are largely conserved between the two proteins.« less

Nonequilibrium phase transitions in isotropic Ashkin-Teller model

NASA Astrophysics Data System (ADS)

Akıncı, Ümit

2017-03-01

Dynamic behavior of an isotropic Ashkin-Teller model in the presence of a periodically oscillating magnetic field has been analyzed by means of the mean field approximation. The dynamic equation of motion has been constructed with the help of a Glauber type stochastic process and solved for a square lattice. After defining the possible dynamical phases of the system, phase diagrams have been given and the behavior of the hysteresis loops has been investigated in detail. The hysteresis loop for specific order parameter of isotropic Ashkin-Teller model has been defined and characteristics of this loop in different dynamical phases have been given.

Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models

NASA Astrophysics Data System (ADS)

Dodani, Sheel C.; Kiss, Gert; Cahn, Jackson K. B.; Su, Ye; Pande, Vijay S.; Arnold, Frances H.

2016-05-01

The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein stability, substrate binding and even dramatically impact enzyme function. When these flexible regions are unresolvable structurally, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here we present a collaborative approach that consists of both experiment and computation and led to the discovery of a single mutation in the F/G loop of the nitrating cytochrome P450 TxtE that simultaneously controls loop dynamics and completely shifts the enzyme's regioselectivity from the C4 to the C5 position of L-tryptophan. Furthermore, we find that this loop mutation is naturally present in a subset of homologous nitrating P450s and confirm that these uncharacterized enzymes exclusively produce 5-nitro-L-tryptophan, a previously unknown biosynthetic intermediate.

A New Concept to Reveal Protein Dynamics Based on Energy Dissipation

PubMed Central

Ma, Cheng-Wei; Xiu, Zhi-Long; Zeng, An-Ping

2011-01-01

Protein dynamics is essential for its function, especially for intramolecular signal transduction. In this work we propose a new concept, energy dissipation model, to systematically reveal protein dynamics upon effector binding and energy perturbation. The concept is applied to better understand the intramolecular signal transduction during allostery of enzymes. The E. coli allosteric enzyme, aspartokinase III, is used as a model system and special molecular dynamics simulations are designed and carried out. Computational results indicate that the number of residues affected by external energy perturbation (i.e. caused by a ligand binding) during the energy dissipation process shows a sigmoid pattern. Using the two-state Boltzmann equation, we define two parameters, the half response time and the dissipation rate constant, which can be used to well characterize the energy dissipation process. For the allostery of aspartokinase III, the residue response time indicates that besides the ACT2 signal transduction pathway, there is another pathway between the regulatory site and the catalytic site, which is suggested to be the β15-αK loop of ACT1. We further introduce the term “protein dynamical modules” based on the residue response time. Different from the protein structural modules which merely provide information about the structural stability of proteins, protein dynamical modules could reveal protein characteristics from the perspective of dynamics. Finally, the energy dissipation model is applied to investigate E. coli aspartokinase III mutations to better understand the desensitization of product feedback inhibition via allostery. In conclusion, the new concept proposed in this paper gives a novel holistic view of protein dynamics, a key question in biology with high impacts for both biotechnology and biomedicine. PMID:22022616

A class of optimum digital phase locked loops

NASA Technical Reports Server (NTRS)

Kumar, R.; Hurd, W. J.

1986-01-01

This paper presents a class of optimum digital filters for digital phase locked loops, for the important case in which the maximum update rate of the loop filter and numerically controlled oscillator (NCO) is limited. This case is typical when the loop filter is implemented in a microprocessor. In these situations, pure delay is encountered in the loop transfer function and thus the stability and gain margin of the loop are of crucial interest. The optimum filters designed for such situations are evaluated in terms of their gain margin for stability, dynamic error, and steady-state error performance. For situations involving considerably high phase dynamics an adaptive and programmable implementation is also proposed to obtain an overall optimum strategy.

Efficient dynamic coherence transfer relying on offset locking using optical phase-locked loop

NASA Astrophysics Data System (ADS)

Xie, Weilin; Dong, Yi; Bretenaker, Fabien; Shi, Hongxiao; Zhou, Qian; Xia, Zongyang; Qin, Jie; Zhang, Lin; Lin, Xi; Hu, Weisheng

2018-01-01

We design and experimentally demonstrate a highly efficient coherence transfer based on composite optical phaselocked loop comprising multiple feedback servo loops. The heterodyne offset-locking is achieved by conducting an acousto-optic frequency shifter in combination with the current tuning and the temperature controlling of the semiconductor laser. The adaptation of the composite optical phase-locked loop enables the tight coherence transfer from a frequency comb to a semiconductor laser in a fully dynamic manner.

Evolution of a designed retro-aldolase leads to complete active site remodeling

PubMed Central

Giger, Lars; Caner, Sami; Obexer, Richard; Kast, Peter; Baker, David; Ban, Nenad; Hilvert, Donald

2013-01-01

Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that dramatic molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400 fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate, and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes. PMID:23748672

The tolerance to exchanges of the Watson–Crick base pair in the hammerhead ribozyme core is determined by surrounding elements

PubMed Central

Przybilski, Rita; Hammann, Christian

2007-01-01

Tertiary interacting elements are important features of functional RNA molecules, for example, in all small nucleolytic ribozymes. The recent crystal structure of a tertiary stabilized type I hammerhead ribozyme revealed a conventional Watson–Crick base pair in the catalytic core, formed between nucleotides C3 and G8. We show that any Watson–Crick base pair between these positions retains cleavage competence in two type III ribozymes. In the Arabidopsis thaliana sequence, only moderate differences in cleavage rates are observed for the different base pairs, while the peach latent mosaic viroid (PLMVd) ribozyme exhibits a preference for a pyrimidine at position 3 and a purine at position 8. To understand these differences, we created a series of chimeric ribozymes in which we swapped sequence elements that surround the catalytic core. The kinetic characterization of the resulting ribozymes revealed that the tertiary interacting loop sequences of the PLMVd ribozyme are sufficient to induce the preference for Y3–R8 base pairs in the A. thaliana hammerhead ribozyme. In contrast to this, only when the entire stem–loops I and II of the A. thaliana sequences are grafted on the PLMVd ribozyme is any Watson–Crick base pair similarly tolerated. The data provide evidence for a complex interplay of secondary and tertiary structure elements that lead, mediated by long-range effects, to an individual modulation of the local structure in the catalytic core of different hammerhead ribozymes. PMID:17666711

De novo design, synthesis and characterisation of MP3, a new catalytic four-helix bundle hemeprotein.

PubMed

Faiella, Marina; Maglio, Ornella; Nastri, Flavia; Lombardi, Angela; Lista, Liliana; Hagen, Wilfred R; Pavone, Vincenzo

2012-12-07

A new artificial metalloenzyme, MP3 (MiniPeroxidase 3), designed by combining the excellent structural properties of four-helix bundle protein scaffolds with the activity of natural peroxidases, was synthesised and characterised. This new hemeprotein model was developed by covalently linking the deuteroporphyrin to two peptide chains of different compositions to obtain an asymmetric helix-loop-helix/heme/helix-loop-helix sandwich arrangement, characterised by 1) a His residue on one chain that acts as an axial ligand to the iron ion; 2) a vacant distal site that is able to accommodate exogenous ligands or substrates; and 3) an Arg residue in the distal site that should assist in hydrogen peroxide activation to give an HRP-like catalytic process. MP3 was synthesised and characterised as its iron complex. CD measurements revealed the high helix-forming propensity of the peptide, confirming the appropriateness of the model procedure; UV/Vis, MCD and EPR experiments gave insights into the coordination geometry and the spin state of the metal. Kinetic experiments showed that Fe(III)-MP3 possesses peroxidase-like activity comparable to R38A-hHRP, highlighting the possibility of mimicking the functional features of natural enzymes. The synergistic application of de novo design methods, synthetic procedures, and spectroscopic characterisation, described herein, demonstrates a method by which to implement and optimise catalytic activity for an enzyme mimetic. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Extended molecular dynamics of a c-kit promoter quadruplex

PubMed Central

Islam, Barira; Stadlbauer, Petr; Krepl, Miroslav; Koca, Jaroslav; Neidle, Stephen; Haider, Shozeb; Sponer, Jiri

2015-01-01

The 22-mer c-kit promoter sequence folds into a parallel-stranded quadruplex with a unique structure, which has been elucidated by crystallographic and NMR methods and shows a high degree of structural conservation. We have carried out a series of extended (up to 10 μs long, ∼50 μs in total) molecular dynamics simulations to explore conformational stability and loop dynamics of this quadruplex. Unfolding no-salt simulations are consistent with a multi-pathway model of quadruplex folding and identify the single-nucleotide propeller loops as the most fragile part of the quadruplex. Thus, formation of propeller loops represents a peculiar atomistic aspect of quadruplex folding. Unbiased simulations reveal μs-scale transitions in the loops, which emphasizes the need for extended simulations in studies of quadruplex loops. We identify ion binding in the loops which may contribute to quadruplex stability. The long lateral-propeller loop is internally very stable but extensively fluctuates as a rigid entity. It creates a size-adaptable cleft between the loop and the stem, which can facilitate ligand binding. The stability gain by forming the internal network of GA base pairs and stacks of this loop may be dictating which of the many possible quadruplex topologies is observed in the ground state by this promoter quadruplex. PMID:26245347

High frequency, high temperature specific core loss and dynamic B-H hysteresis loop characteristics of soft magnetic alloys

NASA Technical Reports Server (NTRS)

Wieserman, W. R.; Schwarze, G. E.; Niedra, J. M.

1990-01-01

Limited experimental data exists for the specific core loss and dynamic B-H loops for soft magnetic materials for the combined conditions of high frequency and high temperature. This experimental study investigates the specific core loss and dynamic B-H loop characteristics of Supermalloy and Metglas 2605SC over the frequency range of 1 to 50 kHz and temperature range of 23 to 300 C under sinusoidal voltage excitation. The experimental setup used to conduct the investigation is described. The effects of the maximum magnetic flux density, frequency, and temperature on the specific core loss and on the size and shape of the B-H loops are examined.

Flow volume loops in patients with goiters.

PubMed Central

Geraghty, J G; Coveney, E C; Kiernan, M; O'Higgins, N J

1992-01-01

Plain radiology is the standard means of assessing upper airway obstruction in patients with goiters. Flow volume loop curves will provide additional information, because they allow a quantitative assessment of airflow dynamics in the respiratory cycle. Fifty-one patients had flow volume loops performed before and after thyroidectomy. There was a significant increase in the maximum inspiratory flow rate (3.9 +/- 0.2 versus 4.9 +/- 0.2 L/second, p less than 0.01) after thyroidectomy. Eight of twelve patients with normal tracheal radiology had improved airflow dynamics in the postoperative period. The flow volume loop curve is a simple noninvasive means of assessing airflow dynamics in patients with goiters and may be superior to conventional radiology. PMID:1731653

First-principles quantum-mechanical investigations of biomass conversion at the liquid-solid interfaces

NASA Astrophysics Data System (ADS)

Dang, Hongli; Xue, Wenhua; Liu, Yingdi; Jentoft, Friederike; Resasco, Daniel; Wang, Sanwu

2014-03-01

We report first-principles density-functional calculations and ab initio molecular dynamics (MD) simulations for the reactions involving furfural, which is an important intermediate in biomass conversion, at the catalytic liquid-solid interfaces. The different dynamic processes of furfural at the water-Cu(111) and water-Pd(111) interfaces suggest different catalytic reaction mechanisms for the conversion of furfural. Simulations for the dynamic processes with and without hydrogen demonstrate the importance of the liquid-solid interface as well as the presence of hydrogen in possible catalytic reactions including hydrogenation and decarbonylation of furfural. Supported by DOE (DE-SC0004600). This research used the supercomputer resources of the XSEDE, the NERSC Center, and the Tandy Supercomputing Center.

Adaptive Sliding Mode Control of Dynamic Systems Using Double Loop Recurrent Neural Network Structure.

PubMed

Fei, Juntao; Lu, Cheng

2018-04-01

In this paper, an adaptive sliding mode control system using a double loop recurrent neural network (DLRNN) structure is proposed for a class of nonlinear dynamic systems. A new three-layer RNN is proposed to approximate unknown dynamics with two different kinds of feedback loops where the firing weights and output signal calculated in the last step are stored and used as the feedback signals in each feedback loop. Since the new structure has combined the advantages of internal feedback NN and external feedback NN, it can acquire the internal state information while the output signal is also captured, thus the new designed DLRNN can achieve better approximation performance compared with the regular NNs without feedback loops or the regular RNNs with a single feedback loop. The new proposed DLRNN structure is employed in an equivalent controller to approximate the unknown nonlinear system dynamics, and the parameters of the DLRNN are updated online by adaptive laws to get favorable approximation performance. To investigate the effectiveness of the proposed controller, the designed adaptive sliding mode controller with the DLRNN is applied to a -axis microelectromechanical system gyroscope to control the vibrating dynamics of the proof mass. Simulation results demonstrate that the proposed methodology can achieve good tracking property, and the comparisons of the approximation performance between radial basis function NN, RNN, and DLRNN show that the DLRNN can accurately estimate the unknown dynamics with a fast speed while the internal states of DLRNN are more stable.

A molecular dynamics study of loop fluctuation in human papillomavirus type 16 virus-like particles: a possible indicator of immunogenicity.

PubMed

Joshi, Harshad; Cheluvaraja, Srinath; Somogyi, Endre; Brown, Darron R; Ortoleva, Peter

2011-11-28

Immunogenicity varies between the human papillomavirus (HPV) L1 monomer assemblies of various sizes (e.g., monomers, pentamers or whole capsids). The hypothesis that this can be attributed to the intensity of fluctuations of important loops containing neutralizing epitopes for the various assemblies is proposed for HPV L1 assemblies. Molecular dynamics simulations were utilized to begin testing this hypothesis. Fluctuations of loops that contain critical neutralizing epitopes (especially FG loop) were quantified via root-mean-square fluctuation and features in the frequency spectrum of dynamic changes in loop conformation. If this fluctuation-immunogenicity hypothesis is a universal aspect of immunogenicity (i.e., immune system recognition of an epitope within a loop is more reliable when it is presented via a more stable delivery structure), then fluctuation measures can serve as one predictor of immunogenicity as part of a computer-aided vaccine design strategy. Copyright © 2011 Elsevier Ltd. All rights reserved.

Human ER Oxidoreductin-1α (Ero1α) Undergoes Dual Regulation through Complementary Redox Interactions with Protein-Disulfide Isomerase.

PubMed

Kanemura, Shingo; Okumura, Masaki; Yutani, Katsuhide; Ramming, Thomas; Hikima, Takaaki; Appenzeller-Herzog, Christian; Akiyama, Shuji; Inaba, Kenji

2016-11-11

In the mammalian endoplasmic reticulum, oxidoreductin-1α (Ero1α) generates protein disulfide bonds and transfers them specifically to canonical protein-disulfide isomerase (PDI) to sustain oxidative protein folding. This oxidative process is coupled to the reduction of O 2 to H 2 O 2 on the bound flavin adenine dinucleotide cofactor. Because excessive thiol oxidation and H 2 O 2 generation cause cell death, Ero1α activity must be properly regulated. In addition to the four catalytic cysteines (Cys 94 , Cys 99 , Cys 104 , and Cys 131 ) that are located in the flexible active site region, the Cys 208 -Cys 241 pair located at the base of another flexible loop is necessary for Ero1α regulation, although the mechanistic basis is not fully understood. The present study revealed that the Cys 208 -Cys 241 disulfide was reduced by PDI and other PDI family members during PDI oxidation. Differential scanning calorimetry and small angle X-ray scattering showed that mutation of Cys 208 and Cys 241 did not grossly affect the thermal stability or overall shape of Ero1α, suggesting that redox regulation of this cysteine pair serves a functional role. Moreover, the flexible loop flanked by Cys 208 and Cys 241 provides a platform for functional interaction with PDI, which in turn enhances the oxidative activity of Ero1α through reduction of the Cys 208 -Cys 241 disulfide. We propose a mechanism of dual Ero1α regulation by dynamic redox interactions between PDI and the two Ero1α flexible loops that harbor the regulatory cysteines. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A platform for dynamic simulation and control of movement based on OpenSim and MATLAB.

PubMed

Mansouri, Misagh; Reinbolt, Jeffrey A

2012-05-11

Numerical simulations play an important role in solving complex engineering problems and have the potential to revolutionize medical decision making and treatment strategies. In this paper, we combine the rapid model-based design, control systems and powerful numerical method strengths of MATLAB/Simulink with the simulation and human movement dynamics strengths of OpenSim by developing a new interface between the two software tools. OpenSim is integrated with Simulink using the MATLAB S-function mechanism, and the interface is demonstrated using both open-loop and closed-loop control systems. While the open-loop system uses MATLAB/Simulink to separately reproduce the OpenSim Forward Dynamics Tool, the closed-loop system adds the unique feature of feedback control to OpenSim, which is necessary for most human movement simulations. An arm model example was successfully used in both open-loop and closed-loop cases. For the open-loop case, the simulation reproduced results from the OpenSim Forward Dynamics Tool with root mean square (RMS) differences of 0.03° for the shoulder elevation angle and 0.06° for the elbow flexion angle. MATLAB's variable step-size integrator reduced the time required to generate the forward dynamic simulation from 7.1s (OpenSim) to 2.9s (MATLAB). For the closed-loop case, a proportional-integral-derivative controller was used to successfully balance a pole on model's hand despite random force disturbances on the pole. The new interface presented here not only integrates the OpenSim and MATLAB/Simulink software tools, but also will allow neuroscientists, physiologists, biomechanists, and physical therapists to adapt and generate new solutions as treatments for musculoskeletal conditions. Copyright © 2012 Elsevier Ltd. All rights reserved.

A platform for dynamic simulation and control of movement based on OpenSim and MATLAB

PubMed Central

Mansouri, Misagh; Reinbolt, Jeffrey A.

2013-01-01

Numerical simulations play an important role in solving complex engineering problems and have the potential to revolutionize medical decision making and treatment strategies. In this paper, we combine the rapid model-based design, control systems and powerful numerical method strengths of MATLAB/Simulink with the simulation and human movement dynamics strengths of OpenSim by developing a new interface between the two software tools. OpenSim is integrated with Simulink using the MATLAB S-function mechanism, and the interface is demonstrated using both open-loop and closed-loop control systems. While the open-loop system uses MATLAB/Simulink to separately reproduce the OpenSim Forward Dynamics Tool, the closed-loop system adds the unique feature of feedback control to OpenSim, which is necessary for most human movement simulations. An arm model example was successfully used in both open-loop and closed-loop cases. For the open-loop case, the simulation reproduced results from the OpenSim Forward Dynamics Tool with root mean square (RMS) differences of 0.03° for the shoulder elevation angle and 0.06° for the elbow flexion angle. MATLAB’s variable step-size integrator reduced the time required to generate the forward dynamic simulation from 7.1 s (OpenSim) to 2.9 s (MATLAB). For the closed-loop case, a proportional–integral–derivative controller was used to successfully balance a pole on model’s hand despite random force disturbances on the pole. The new interface presented here not only integrates the OpenSim and MATLAB/Simulink software tools, but also will allow neuroscientists, physiologists, biomechanists, and physical therapists to adapt and generate new solutions as treatments for musculoskeletal conditions. PMID:22464351

Solution structures and backbone dynamics of Escherichia coli rhodanese PspE in its sulfur-free and persulfide-intermediate forms: implications for the catalytic mechanism of rhodanese.

PubMed

Li, Hongwei; Yang, Fan; Kang, Xue; Xia, Bin; Jin, Changwen

2008-04-15

Rhodanese catalyzes the sulfur-transfer reaction that transfers sulfur from thiosulfate to cyanide by a double-displacement mechanism, in which an active cysteine residue plays a central role. Previous studies indicated that the phage-shock protein E (PspE) from Escherichia coli is a rhodanese composed of a single active domain and is the only accessible rhodanese among the three single-domain rhodaneses in E. coli. To understand the catalytic mechanism of rhodanese at the molecular level, we determined the solution structures of the sulfur-free and persulfide-intermediate forms of PspE by nuclear magnetic resonance (NMR) spectroscopy and identified the active site by NMR titration experiments. To obtain further insights into the catalytic mechanism, we studied backbone dynamics by NMR relaxation experiments. Our results demonstrated that the overall structures in both sulfur-free and persulfide-intermediate forms are highly similar, suggesting that no significant conformational changes occurred during the catalytic reaction. However, the backbone dynamics revealed that the motional properties of PspE in its sulfur-free form are different from the persulfide-intermediate state. The conformational exchanges are largely enhanced in the persulfide-intermediate form of PspE, especially around the active site. The present structural and biochemical studies in combination with backbone dynamics provide further insights in understanding the catalytic mechanism of rhodanese.

Predicting the effects of unmodeled dynamics on an aircraft flight control system design using eigenspace assignment

NASA Technical Reports Server (NTRS)

Johnson, Eric N.; Davidson, John B.; Murphy, Patrick C.

1994-01-01

When using eigenspace assignment to design an aircraft flight control system, one must first develop a model of the plant. Certain questions arise when creating this model as to which dynamics of the plant need to be included in the model and which dynamics can be left out or approximated. The answers to these questions are important because a poor choice can lead to closed-loop dynamics that are unpredicted by the design model. To alleviate this problem, a method has been developed for predicting the effect of not including certain dynamics in the design model on the final closed-loop eigenspace. This development provides insight as to which characteristics of unmodeled dynamics will ultimately affect the closed-loop rigid-body dynamics. What results from this insight is a guide for eigenstructure control law designers to aid them in determining which dynamics need or do not need to be included and a new way to include these dynamics in the flight control system design model to achieve a required accuracy in the closed-loop rigid-body dynamics. The method is illustrated for a lateral-directional flight control system design using eigenspace assignment for the NASA High Alpha Research Vehicle (HARV).

Properties of dynamic magnetic loss of ferrite

NASA Astrophysics Data System (ADS)

Saotome, Hideo; Azuma, Keisuke; Kizuka, Hiroki; Tanaka, Takuma

2018-05-01

The B-H loop of ferrite becomes narrower with a decrease in the excitation frequency. However, even at frequencies lower than 1 kHz, the B-H loop exhibits a certain minimum width, which is referred to as the (DC) hysteresis loop, and its area corresponds to the hysteresis loss. The dynamic magnetic loss is obtained by subtracting the hysteresis loss from the B-H loop area measured at a frequency above 1-10 kHz. The temperature characteristics of the hysteresis and dynamic magnetic losses are determined to be experimentally different, which suggests that the mechanism for the generation of dynamic magnetic loss is not exactly the same as that for the hysteresis loss. The dynamic magnetic loss is expressed using the dynamic magnetic loss parameter, which is a function of B and its time derivative, dB/dt. The dynamic magnetic loss parameter is measured under excitation with a rectangular waveform voltage. A ferrite core of TDK PC47 was used and the maximum magnetic flux density Bm, was set to 350 mT. The measured dynamic magnetic loss parameter was experimentally verified to be one of the intrinsic characteristics of ferrite and was also validated for cases of excitation with sinusoidal waveform voltages.

Flexibility of Catalytic Zinc Coordination in Thermolysin and HDAC8: A Born-Oppenheimer ab initio QM/MM Molecular Dynamics Study

PubMed Central

Wu, Ruibo; Hu, Po; Wang, Shenglong; Cao, Zexing; Zhang, Yingkai

2009-01-01

Abstracs The different coordination modes and fast ligand exchange of zinc coordination has been suggested to be one key catalytic feature of the zinc ion which makes it an invaluable metal in biological catalysis. However, partly due to the well known difficulties for zinc to be characterized by spectroscopy methods, evidence for dynamic nature of the catalytic zinc coordination has so far mainly been indirect. In this work, Born-Oppenheimer ab initio QM/MM molecular dynamics simulation has been employed, which allows for a first-principle description of the dynamics of the metal active site while properly including effects of the heterogeneous and fluctuating protein environment. Our simulations have provided direct evidence regarding inherent flexibility of the catalytic zinc coordination shell in Thermolysin (TLN) and Histone Deacetylase 8 (HDAC8). We have observed different coordination modes and fast ligand exchange during the picosecond's time-scale. For TLN, the coordination of the carboxylate group of Glu166 to Zinc is found to continuously change between monodentate and bidentate manner dynamically; while for HDAC8, the flexibility mainly comes from the coordination to a non-amino-acid ligand. Such distinct dynamics in the zinc coordination shell between two enzymes suggests that the catalytic role of Zinc in TLN and HDAC8 is likely to be different in spite of the fact that both catalyze the hydrolysis of amide bond. Meanwhile, considering that such Born-Oppenheimer ab initio QM/MM MD simulations are very much desired but are widely considered to be too computationally expensive to be feasible, our current study demonstrates the viability and powerfulness of this state-of-the-art approach in simulating metalloenzymes. PMID:20161624

DOE Office of Scientific and Technical Information (OSTI.GOV)

Joseph, J.S.; Saikatendu, K.S.; Subramanian, V.

Mature nonstructural protein-15 (nsp15) from the severe acute respiratory syndrome coronavirus (SARS-CoV) contains a novel uridylate-specific Mn{sup 2+}-dependent endoribonuclease (NendoU). Structure studies of the full-length form of the obligate hexameric enzyme from two CoVs, SARS-CoV and murine hepatitis virus, and its monomeric homologue, XendoU from Xenopus laevis, combined with mutagenesis studies have implicated several residues in enzymatic activity and the N-terminal domain as the major determinant of hexamerization. However, the tight link between hexamerization and enzyme activity in NendoUs has remained an enigma. Here, we report the structure of a trimmed, monomeric form of SARS-CoV nsp15 (residues 28 to 335)more » determined to a resolution of 2.9 Angstroms. The catalytic loop (residues 234 to 249) with its two reactive histidines (His 234 and His 249) is dramatically flipped by {approx}120 degrees into the active site cleft. Furthermore, the catalytic nucleophile Lys 289 points in a diametrically opposite direction, a consequence of an outward displacement of the supporting loop (residues 276 to 295). In the full-length hexameric forms, these two loops are packed against each other and are stabilized by intimate intersubunit interactions. Our results support the hypothesis that absence of an adjacent monomer due to deletion of the hexamerization domain is the most likely cause for disruption of the active site, offering a structural basis for why only the hexameric form of this enzyme is active.« less

Structural and Dynamic Insights into the Mechanism of Allosteric Signal Transmission in ERK2-Mediated MKP3 Activation.

PubMed

Lu, Chang; Liu, Xin; Zhang, Chen-Song; Gong, Haipeng; Wu, Jia-Wei; Wang, Zhi-Xin

2017-11-21

The mitogen-activated protein kinases (MAPKs) are key components of cellular signal transduction pathways, which are down-regulated by the MAPK phosphatases (MKPs). Catalytic activity of the MKPs is controlled both by their ability to recognize selective MAPKs and by allosteric activation upon binding to MAPK substrates. Here, we use a combination of experimental and computational techniques to elucidate the molecular mechanism for the ERK2-induced MKP3 activation. Mutational and kinetic study shows that the 334 FNFM 337 motif in the MKP3 catalytic domain is essential for MKP3-mediated ERK2 inactivation and is responsible for ERK2-mediated MKP3 activation. The long-term molecular dynamics (MD) simulations further reveal a complete dynamic process in which the catalytic domain of MKP3 gradually changes to a conformation that resembles an active MKP catalytic domain over the time scale of the simulation, providing a direct time-dependent observation of allosteric signal transmission in ERK2-induced MKP3 activation.

The mitochondrial intermembrane loop region of rat carnitine palmitoyltransferase 1A is a major determinant of its malonyl-CoA sensitivity.

PubMed

Borthwick, Karen; Jackson, Vicky N; Price, Nigel T; Zammit, Victor A

2006-11-03

Carnitine palmitoyltransferase (CPT) 1A adopts a polytopic conformation within the mitochondrial outer membrane, having both the N- and C-terminal segments on the cytosolic aspect of the membrane and a loop region connecting the two transmembrane (TM) segments protruding into the inter membrane space. In this study we demonstrate that the loop exerts major effects on the sensitivity of the enzyme to its inhibitor, malonyl-CoA. Insertion of a 16-residue spacer between the C-terminal part of the loop sequence (i.e. between residues 100 and 101) and TM2 (which is predicted to start at residue 102) increased the sensitivity to malonyl-CoA inhibition of the resultant mutant protein by more than 10-fold. By contrast, the same insertion made between TM1 and the loop had no effects on the kinetic properties of the enzyme, indicating that effects on the catalytic C-terminal segment were specifically induced by loop-TM2 interactions. Enhanced sensitivity was also observed in all mutants in which the native TM2-loop pairing was disrupted either by making chimeras in which the loops and TM2 segments of CPT 1A and CPT 1B were exchanged or by deleting successive 9-residue segments from the loop sequence. The data suggest that the sequence spanning the loop-TM2 boundary determines the disposition of this TM in the membrane so as to alter the conformation of the C-terminal segment and thus affect its interaction with malonyl-CoA.

Ab initio study on the dynamics of furfural at the liquid-solid interfaces

NASA Astrophysics Data System (ADS)

Dang, Hongli; Xue, Wenhua; Shields, Darwin; Liu, Yingdi; Jentoft, Friederike; Resasco, Daniel; Wang, Sanwu

2013-03-01

Catalytic biomass conversion sometimes occurs at the liquid-solid interfaces. We report ab initio molecular dynamics simulations at finite temperatures for the catalytic reactions involving furfural at the water-Pd and water-Cu interfaces. We found that, during the dynamic process, the furan ring of furfural prefers to be parallel to the Pd surface and the aldehyde group tends to be away from the Pd surface. On the other hand, at the water-Cu(111) interface, furfural prefers to be tilted to the Cu surface while the aldehyde group is bonded to the surface. In both cases, interaction of liquid water and furfural is identified. The difference of dynamic process of furfural at the two interfaces suggests different catalytic reaction mechanisms for the conversion of furfural, consistent with the experimental investigations. Supported by DOE (DE-SC0004600). Simulations and calculations were performed on XSED's and NERSC's supercomputers

The role of protein dynamics in the evolution of new enzyme function.

PubMed

Campbell, Eleanor; Kaltenbach, Miriam; Correy, Galen J; Carr, Paul D; Porebski, Benjamin T; Livingstone, Emma K; Afriat-Jurnou, Livnat; Buckle, Ashley M; Weik, Martin; Hollfelder, Florian; Tokuriki, Nobuhiko; Jackson, Colin J

2016-11-01

Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview.

PubMed

Šponer, Jiří; Bussi, Giovanni; Krepl, Miroslav; Banáš, Pavel; Bottaro, Sandro; Cunha, Richard A; Gil-Ley, Alejandro; Pinamonti, Giovanni; Poblete, Simón; Jurečka, Petr; Walter, Nils G; Otyepka, Michal

2018-04-25

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA-ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field.

Conformational trapping of mismatch recognition complex MSH2/MSH3 on repair-resistant DNA loops.

PubMed

Lang, Walter H; Coats, Julie E; Majka, Jerzy; Hura, Greg L; Lin, Yuyen; Rasnik, Ivan; McMurray, Cynthia T

2011-10-18

Insertion and deletion of small heteroduplex loops are common mutations in DNA, but why some loops are prone to mutation and others are efficiently repaired is unknown. Here we report that the mismatch recognition complex, MSH2/MSH3, discriminates between a repair-competent and a repair-resistant loop by sensing the conformational dynamics of their junctions. MSH2/MSH3 binds, bends, and dissociates from repair-competent loops to signal downstream repair. Repair-resistant Cytosine-Adenine-Guanine (CAG) loops adopt a unique DNA junction that traps nucleotide-bound MSH2/MSH3, and inhibits its dissociation from the DNA. We envision that junction dynamics is an active participant and a conformational regulator of repair signaling, and governs whether a loop is removed by MSH2/MSH3 or escapes to become a precursor for mutation.

Heating and dynamics of two flare loop systems observed by AIA and EIS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Y.; Ding, M. D.; Qiu, J., E-mail: yingli@nju.edu.cn

2014-02-01

We investigate heating and evolution of flare loops in a C4.7 two-ribbon flare on 2011 February 13. From Solar Dynamics Observatory/Atmospheric Imaging Assembly (AIA) imaging observations, we can identify two sets of loops. Hinode/EUV Imaging Spectrometer (EIS) spectroscopic observations reveal blueshifts at the feet of both sets of loops. The evolution and dynamics of the two sets are quite different. The first set of loops exhibits blueshifts for about 25 minutes followed by redshifts, while the second set shows stronger blueshifts, which are maintained for about one hour. The UV 1600 observation by AIA also shows that the feet ofmore » the second set of loops brighten twice. These suggest that continuous heating may be present in the second set of loops. We use spatially resolved UV light curves to infer heating rates in the few tens of individual loops comprising the two loop systems. With these heating rates, we then compute plasma evolution in these loops with the 'enthalpy-based thermal evolution of loops' model. The results show that, for the first set of loops, the synthetic EUV light curves from the model compare favorably with the observed light curves in six AIA channels and eight EIS spectral lines, and the computed mean enthalpy flow velocities also agree with the Doppler shift measurements by EIS. For the second set of loops modeled with twice-heating, there are some discrepancies between modeled and observed EUV light curves in low-temperature bands, and the model does not fully produce the prolonged blueshift signatures as observed. We discuss possible causes for the discrepancies.« less

Loop-quantum-gravity vertex amplitude.

PubMed

Engle, Jonathan; Pereira, Roberto; Rovelli, Carlo

2007-10-19

Spin foam models are hoped to provide the dynamics of loop-quantum gravity. However, the most popular of these, the Barrett-Crane model, does not have the good boundary state space and there are indications that it fails to yield good low-energy n-point functions. We present an alternative dynamics that can be derived as a quantization of a Regge discretization of Euclidean general relativity, where second class constraints are imposed weakly. Its state space matches the SO(3) loop gravity one and it yields an SO(4)-covariant vertex amplitude for Euclidean loop gravity.

Analysis of flexible aircraft longitudinal dynamics and handling qualities. Volume 2: Data

NASA Technical Reports Server (NTRS)

Waszak, M. R.; Schmidt, D. K.

1985-01-01

Two analysis methods are applied to a family of flexible aircraft in order to investigate how and when structural (especially dynamic aeroelastic) effects affect the dynamic characteristics of aircraft. The first type of analysis is an open loop modal analysis technique. This method considers the effect of modal residue magnitudes on determining vehicle handling qualities. The second method is a pilot in the loop analysis procedure that considers several closed loop system characteristics. Both analyses indicated that dynamic aeroelastic effects caused a degradation in vehicle tracking performance, based on the evaluation of some simulation results. Volume 2 consists of the presentation of the state variable models of the flexible aircraft configurations used in the analysis applications mode shape plots for the structural modes, numerical results from the modal analysis frequency response plots from the pilot in the loop analysis and a listing of the modal analysis computer program.

Optimal control of coupled parabolic-hyperbolic non-autonomous PDEs: infinite-dimensional state-space approach

NASA Astrophysics Data System (ADS)

Aksikas, I.; Moghadam, A. Alizadeh; Forbes, J. F.

2018-04-01

This paper deals with the design of an optimal state-feedback linear-quadratic (LQ) controller for a system of coupled parabolic-hypebolic non-autonomous partial differential equations (PDEs). The infinite-dimensional state space representation and the corresponding operator Riccati differential equation are used to solve the control problem. Dynamical properties of the coupled system of interest are analysed to guarantee the existence and uniqueness of the solution of the LQ-optimal control problem and also to guarantee the exponential stability of the closed-loop system. Thanks to the eigenvalues and eigenfunctions of the parabolic operator and also the fact that the hyperbolic-associated operator Riccati differential equation can be converted to a scalar Riccati PDE, an algorithm to solve the LQ control problem has been presented. The results are applied to a non-isothermal packed-bed catalytic reactor. The LQ optimal controller designed in the early portion of the paper is implemented for the original non-linear model. Numerical simulations are performed to show the controller performances.

Exploring the Dynamics of Propeller Loops in Human Telomeric DNA Quadruplexes Using Atomistic Simulations.

PubMed

Islam, Barira; Stadlbauer, Petr; Gil-Ley, Alejandro; Pérez-Hernández, Guillermo; Haider, Shozeb; Neidle, Stephen; Bussi, Giovanni; Banas, Pavel; Otyepka, Michal; Sponer, Jiri

2017-06-13

We have carried out a series of extended unbiased molecular dynamics (MD) simulations (up to 10 μs long, ∼162 μs in total) complemented by replica-exchange with the collective variable tempering (RECT) approach for several human telomeric DNA G-quadruplex (GQ) topologies with TTA propeller loops. We used different AMBER DNA force-field variants and also processed simulations by Markov State Model (MSM) analysis. The slow conformational transitions in the propeller loops took place on a scale of a few μs, emphasizing the need for long simulations in studies of GQ dynamics. The propeller loops sampled similar ensembles for all GQ topologies and for all force-field dihedral-potential variants. The outcomes of standard and RECT simulations were consistent and captured similar spectrum of loop conformations. However, the most common crystallographic loop conformation was very unstable with all force-field versions. Although the loss of canonical γ-trans state of the first propeller loop nucleotide could be related to the indispensable bsc0 α/γ dihedral potential, even supporting this particular dihedral by a bias was insufficient to populate the experimentally dominant loop conformation. In conclusion, while our simulations were capable of providing a reasonable albeit not converged sampling of the TTA propeller loop conformational space, the force-field description still remained far from satisfactory.

Exploring the Dynamics of Propeller Loops in Human Telomeric DNA Quadruplexes Using Atomistic Simulations

PubMed Central

2017-01-01

We have carried out a series of extended unbiased molecular dynamics (MD) simulations (up to 10 μs long, ∼162 μs in total) complemented by replica-exchange with the collective variable tempering (RECT) approach for several human telomeric DNA G-quadruplex (GQ) topologies with TTA propeller loops. We used different AMBER DNA force-field variants and also processed simulations by Markov State Model (MSM) analysis. The slow conformational transitions in the propeller loops took place on a scale of a few μs, emphasizing the need for long simulations in studies of GQ dynamics. The propeller loops sampled similar ensembles for all GQ topologies and for all force-field dihedral-potential variants. The outcomes of standard and RECT simulations were consistent and captured similar spectrum of loop conformations. However, the most common crystallographic loop conformation was very unstable with all force-field versions. Although the loss of canonical γ-trans state of the first propeller loop nucleotide could be related to the indispensable bsc0 α/γ dihedral potential, even supporting this particular dihedral by a bias was insufficient to populate the experimentally dominant loop conformation. In conclusion, while our simulations were capable of providing a reasonable albeit not converged sampling of the TTA propeller loop conformational space, the force-field description still remained far from satisfactory. PMID:28475322

Bandwidth controller for phase-locked-loop

NASA Technical Reports Server (NTRS)

Brockman, Milton H. (Inventor)

1992-01-01

A phase locked loop utilizing digital techniques to control the closed loop bandwidth of the RF carrier phase locked loop in a receiver provides high sensitivity and a wide dynamic range for signal reception. After analog to digital conversion, a digital phase locked loop bandwidth controller provides phase error detection with automatic RF carrier closed loop tracking bandwidth control to accommodate several modes of transmission.

Simulating Coronal Loop Implosion and Compressible Wave Modes in a Flare Hit Active Region

NASA Astrophysics Data System (ADS)

Sarkar, Aveek; Vaidya, Bhargav; Hazra, Soumitra; Bhattacharyya, Jishnu

2017-12-01

There is considerable observational evidence of implosion of magnetic loop systems inside solar coronal active regions following high-energy events like solar flares. In this work, we propose that such collapse can be modeled in three dimensions quite accurately within the framework of ideal magnetohydrodynamics. We furthermore argue that the dynamics of loop implosion is only sensitive to the transmitted disturbance of one or more of the system variables, e.g., velocity generated at the event site. This indicates that to understand loop implosion, it is sensible to leave the event site out of the simulated active region. Toward our goal, a velocity pulse is introduced to model the transmitted disturbance generated at the event site. Magnetic field lines inside our simulated active region are traced in real time, and it is demonstrated that the subsequent dynamics of the simulated loops closely resemble observed imploding loops. Our work highlights the role of plasma β in regards to the rigidity of the loop systems and how that might affect the imploding loops’ dynamics. Compressible magnetohydrodynamic modes such as kink and sausage are also shown to be generated during such processes, in accordance with observations.

Substrate Induced Structural and Dynamics Changes in Human Phosphomevalonate Kinase and Implications for Mechanism

PubMed Central

Olson, Andrew L.; Yao, Huili; Herdendorf, Timothy J.; Miziorko, Henry M.; Hannongbua, Supa; Saparpakorn, Patchareenart; Cai, Sheng; Sem, Daniel S.

2008-01-01

Phosphomevalonate kinase (PMK) catalyzes an essential step in the mevalonate pathway, which is the only pathway for synthesis of isoprenoids and steroids in humans. PMK catalyzes transfer of the γ-phosphate of ATP to mevalonate 5-phosphate (M5P) to form mevalonate 5-diphosphate. Bringing these phosphate groups in proximity to react is especially challenging, given the high negative charge density on the four phosphate groups in the active site. As such, conformational and dynamics changes needed to form the Michaelis complex are of mechanistic interest. Herein, we report the characterization of substrate induced changes (Mg-ADP, M5P, and the ternary complex) in PMK, using NMR-based dynamics and chemical shift perturbation measurements. Mg-ADP and M5P Kd's were 6-60 μM in all complexes, consistent with there being little binding synergy. Binding of M5P causes the PMK structure to compress (τc= 13.5 nsec), while subsequent binding of Mg-ADP opens the structure up (τc= 17.6 nsec). The overall complex seems to stay very rigid on the psec-nsec timescale with an average NMR order parameter of S2∼0.88. Data are consistent with addition of M5P causing movement around a hinge region to permit domain closure, which would bring the M5P domain close to ATP to permit catalysis. Dynamics data identify potential hinge residues as H55 and R93, based on their low order parameters and their location in extended regions that connect the M5P and ATP domains in the PMK homology model. Likewise, D163 may be a hinge residue for the lid region that is homologous to the adenylate kinase lid, covering the “Walker-A” catalytic loop. Binding of ATP or ADP appears to cause similar conformational changes; but, these observations do not indicate an obvious role for γ-phosphate binding interactions. Indeed, the role of γ-phosphate interactions may be more subtle than suggested by ATP/ADP comparisons, since the conservative O to NH substitution in the β-γ bridge of ATP causes a dramatic decrease in affinity and induces few chemical shift perturbations. In terms of positioning of catalytic residues, binding of M5P induces a rigidification of Gly21 (adjacent to the catalytically important Lys22), although exchange broadening in the ternary complex suggests some motion on a slower timescale does still occur. Finally, the first 9 residues of the N-terminus are highly disordered, suggesting they may be part of a cleavable signal or regulatory peptide sequence. PMID:18798562

Protein kinase D displays intrinsic Tyr autophosphorylation activity: insights into mechanism and regulation.

PubMed

Cobbaut, Mathias; Derua, Rita; Parker, Peter J; Waelkens, Etienne; Janssens, Veerle; Van Lint, Johan

2018-06-22

The protein kinase D (PKD) family is regulated through multi-site phosphorylation, including autophosphorylation. For example, PKD displays in vivo autophosphorylation on Ser-742 (and Ser-738 in vitro) in the activation loop and Ser-910 in the C-tail (hPKD1 numbering). In this paper, we describe the surprising observation that PKD also displays in vitro autocatalytic activity towards a Tyr residue in the P+1 loop of the activation segment. We define the molecular determinants for this unusual activity and identify a Cys residue (C705 in PKD1) in the catalytic loop as of utmost importance. In cells, PKD Tyr autophosphorylation is suppressed through the association of an inhibitory factor. Our findings provide important novel insights into PKD (auto)regulation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Isolation and characterization of two cDNA clones encoding for glutamate dehydrogenase in Nicotiana plumbaginifolia.

PubMed

Ficarelli, A; Tassi, F; Restivo, F M

1999-03-01

We have isolated two full length cDNA clones encoding Nicotiana plumbaginifolia NADH-glutamate dehydrogenase. Both clones share amino acid boxes of homology corresponding to conserved GDH catalytic domains and putative mitochondrial targeting sequence. One clone shows a putative EF-hand loop. The level of the two transcripts is affected differently by carbon source.

Effects of Single Nucleotide Polymorphisms on Human N-Acetyltransferase 2 Structure and Dynamics by Molecular Dynamics Simulation

PubMed Central

Rajasekaran, M.; Abirami, Santhanam; Chen, Chinpan

2011-01-01

Background Arylamine N-acetyltransferase 2 (NAT2) is an important catalytic enzyme that metabolizes the carcinogenic arylamines, hydrazine drugs and chemicals. This enzyme is highly polymorphic in different human populations. Several polymorphisms of NAT2, including the single amino acid substitutions R64Q, I114T, D122N, L137F, Q145P, R197Q, and G286E, are classified as slow acetylators, whereas the wild-type NAT2 is classified as a fast acetylator. The slow acetylators are often associated with drug toxicity and efficacy as well as cancer susceptibility. The biological functions of these 7 mutations have previously been characterized, but the structural basis behind the reduced catalytic activity and reduced protein level is not clear. Methodology/Principal Findings We performed multiple molecular dynamics simulations of these mutants as well as NAT2 to investigate the structural and dynamical effects throughout the protein structure, specifically the catalytic triad, cofactor binding site, and the substrate binding pocket. None of these mutations induced unfolding; instead, their effects were confined to the inter-domain, domain 3 and 17-residue insert region, where the flexibility was significantly reduced relative to the wild-type. Structural effects of these mutations propagate through space and cause a change in catalytic triad conformation, cofactor binding site, substrate binding pocket size/shape and electrostatic potential. Conclusions/Significance Our results showed that the dynamical properties of all the mutant structures, especially in inter-domain, domain 3 and 17-residue insert region were affected in the same manner. Similarly, the electrostatic potential of all the mutants were altered and also the functionally important regions such as catalytic triad, cofactor binding site, and substrate binding pocket adopted different orientation and/or conformation relative to the wild-type that may affect the functions of the mutants. Overall, our study may provide the structural basis for reduced catalytic activity and protein level, as was experimentally observed for these polymorphisms. PMID:21980537

Conformational flexibility of human casein kinase catalytic subunit explored by metadynamics.

PubMed

Gouron, Aurélie; Milet, Anne; Jamet, Helene

2014-03-04

Casein kinase CK2 is an essential enzyme in higher organisms, catalyzing the transfer of the γ phosphate from ATP to serine and threonine residues on protein substrates. In a number of animal tumors, CK2 activity has been shown to escape normal cellular control, making it a potential target for cancer therapy. Several crystal structures of human CK2 have been published with different conformations for the CK2α catalytic subunit. This variability reflects a high flexibility for two regions of CK2α: the interdomain hinge region, and the glycine-rich loop (p-loop). Here, we present a computational study simulating the equilibrium between three conformations involving these regions. Simulations were performed using well-tempered metadynamics combined with a path collective variables approach. This provides a reference pathway describing the conformational changes being studied, based on analysis of free energy surfaces. The free energies of the three conformations were found to be close and the paths proposed had low activation barriers. Our results indicate that these conformations can exist in water. This information should be useful when designing inhibitors specific to one conformation. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Delineating Substrate Diversity of Disparate Short-Chain Dehydrogenase Reductase from Debaryomyces hansenii.

PubMed

Ghatak, Arindam; Bharatham, Nagakumar; Shanbhag, Anirudh P; Datta, Santanu; Venkatraman, Janani

2017-01-01

Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from Debaryomyces hansenii and compared it with structurally and functionally characterized SDR Synechococcus elongatus. PCC 7942 FabG to delineate its industrial significance. It was observed that DHK was significantly more efficient than FabG, reducing a diverse set of ketones albeit at higher conversion rates. Comparison of the FabG structure with a homology model of DHK and a docking of substrate to both structures revealed the presence of additional flexible loops near the substrate binding site of DHK. The comparative elasticity of the cofactor and substrate binding site of FabG and DHK was experimentally substantiated using differential scanning fluorimetry. It is postulated that the loop flexibility may account for the superior catalytic efficiency of DHK although the positioning of the catalytic triad is conserved.

Delineating Substrate Diversity of Disparate Short-Chain Dehydrogenase Reductase from Debaryomyces hansenii

PubMed Central

Ghatak, Arindam; Bharatham, Nagakumar; Shanbhag, Anirudh P.; Datta, Santanu; Venkatraman, Janani

2017-01-01

Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from Debaryomyces hansenii and compared it with structurally and functionally characterized SDR Synechococcus elongatus. PCC 7942 FabG to delineate its industrial significance. It was observed that DHK was significantly more efficient than FabG, reducing a diverse set of ketones albeit at higher conversion rates. Comparison of the FabG structure with a homology model of DHK and a docking of substrate to both structures revealed the presence of additional flexible loops near the substrate binding site of DHK. The comparative elasticity of the cofactor and substrate binding site of FabG and DHK was experimentally substantiated using differential scanning fluorimetry. It is postulated that the loop flexibility may account for the superior catalytic efficiency of DHK although the positioning of the catalytic triad is conserved. PMID:28107498

Robust Adaptive Flight Control Design of Air-breathing Hypersonic Vehicles

DTIC Science & Technology

2016-12-07

dynamic inversion controller design for a non -minimum phase hypersonic vehicle is derived by Kuipers et al. [2008]. Moreover, integrated guidance and...stabilization time for inner loop variables is lesser than the intermediate loop variables because of the three-loop-control design methodology . The control...adaptive design . Control Engineering Practice, 2016. Michael A Bolender and David B Doman. A non -linear model for the longitudinal dynamics of a

A molecular mechanism of P-loop pliability of Rho-kinase investigated by molecular dynamic simulation

NASA Astrophysics Data System (ADS)

Gohda, Keigo; Hakoshima, Toshio

2008-11-01

Rho-kinase is a leading player in the regulation of cytoskeletal events involving smooth muscle contraction and neurite growth-cone collapse and retraction, and is a promising drug target in the treatment of both vascular and neurological disorders. Recent crystal structure of Rho-kinase complexed with a small-molecule inhibitor fasudil has revealed structural details of the ATP-binding site, which represents the target site for the inhibitor, and showed that the conserved phenylalanine on the P-loop occupies the pocket, resulting in an increase of protein-ligand contacts. Thus, the P-loop pliability is considered to play an important role in inhibitor binding affinity and specificity. In this study, we carried out a molecular dynamic simulation for Rho-kinase-fasudil complexes with two different P-loop conformations, i.e., the extended and folded conformations, in order to understand the P-loop pliability and dynamics at atomic level. A PKA-fasudil complex was also used for comparison. In the MD simulation, the flip-flop movement of the P-loop conformation starting either from the extended or folded conformation was not able to be observed. However, a significant conformational change in a long loop region covering over the P-loop, and also alteration of ionic interaction-manner of fasudil with acidic residues in the ATP binding site were shown only in the Rho-kinase-fasudil complex with the extended P-loop conformation, while Rho-kinase with the folded P-loop conformation and PKA complexes did not show large fluctuations, suggesting that the Rho-kinase-fasudil complex with the extended P-loop conformation represents a meta-stable state. The information of the P-loop pliability at atomic level obtained in this study could provide valuable clues to designing potent and/or selective inhibitors for Rho-kinase.

A simplified dynamic model of the T700 turboshaft engine

NASA Technical Reports Server (NTRS)

Duyar, Ahmet; Gu, Zhen; Litt, Jonathan S.

1992-01-01

A simplified open-loop dynamic model of the T700 turboshaft engine, valid within the normal operating range of the engine, is developed. This model is obtained by linking linear state space models obtained at different engine operating points. Each linear model is developed from a detailed nonlinear engine simulation using a multivariable system identification and realization method. The simplified model may be used with a model-based real time diagnostic scheme for fault detection and diagnostics, as well as for open loop engine dynamics studies and closed loop control analysis utilizing a user generated control law.

Closed-Loop and Activity-Guided Optogenetic Control

PubMed Central

Grosenick, Logan; Marshel, James H.; Deisseroth, Karl

2016-01-01

Advances in optical manipulation and observation of neural activity have set the stage for widespread implementation of closed-loop and activity-guided optical control of neural circuit dynamics. Closing the loop optogenetically (i.e., basing optogenetic stimulation on simultaneously observed dynamics in a principled way) is a powerful strategy for causal investigation of neural circuitry. In particular, observing and feeding back the effects of circuit interventions on physiologically relevant timescales is valuable for directly testing whether inferred models of dynamics, connectivity, and causation are accurate in vivo. Here we highlight technical and theoretical foundations as well as recent advances and opportunities in this area, and we review in detail the known caveats and limitations of optogenetic experimentation in the context of addressing these challenges with closed-loop optogenetic control in behaving animals. PMID:25856490

Unraveling HIV protease flaps dynamics by Constant pH Molecular Dynamics simulations.

PubMed

Soares, Rosemberg O; Torres, Pedro H M; da Silva, Manuela L; Pascutti, Pedro G

2016-08-01

The active site of HIV protease (HIV-PR) is covered by two flaps. These flaps are known to be essential for the catalytic activity of the HIV-PR, but their exact conformations at the different stages of the enzymatic pathway remain subject to debate. Understanding the correct functional dynamics of the flaps might aid the development of new HIV-PR inhibitors. It is known that, the HIV-PR catalytic efficiency is pH-dependent, likely due to the influence of processes such as charge transfer and protonation/deprotonation of ionizable residues. Several Molecular Dynamics (MD) simulations have reported information about the HIV-PR flaps. However, in MD simulations the protonation of a residue is fixed and thus it is not possible to study the correlation between conformation and protonation state. To address this shortcoming, this work attempts to capture, through Constant pH Molecular Dynamics (CpHMD), the conformations of the apo, substrate-bound and inhibitor-bound HIV-PR, which differ drastically in their flap arrangements. The results show that the HIV-PR flaps conformations are defined by the protonation of the catalytic residues Asp25/Asp25' and that these residues are sensitive to pH changes. This study suggests that the catalytic aspartates can modulate the opening of the active site and substrate binding. Copyright © 2016 Elsevier Inc. All rights reserved.

Charge neutralization in the active site of the catalytic trimer of aspartate transcarbamoylase promotes diverse structural changes.

PubMed

Endrizzi, James A; Beernink, Peter T

2017-11-01

A classical model for allosteric regulation of enzyme activity posits an equilibrium between inactive and active conformations. An alternative view is that allosteric activation is achieved by increasing the potential for conformational changes that are essential for catalysis. In the present study, substitution of a basic residue in the active site of the catalytic (C) trimer of aspartate transcarbamoylase with a non-polar residue results in large interdomain hinge changes in the three chains of the trimer. One conformation is more open than the chains in both the wild-type C trimer and the catalytic chains in the holoenzyme, the second is closed similar to the bisubstrate-analog bound conformation and the third hinge angle is intermediate to the other two. The active-site 240s loop conformation is very different between the most open and closed chains, and is disordered in the third chain, as in the holoenzyme. We hypothesize that binding of anionic substrates may promote similar structural changes. Further, the ability of the three catalytic chains in the trimer to access the open and closed active-site conformations simultaneously suggests a cyclic catalytic mechanism, in which at least one of the chains is in an open conformation suitable for substrate binding whereas another chain is closed for catalytic turnover. Based on the many conformations observed for the chains in the isolated catalytic trimer to date, we propose that allosteric activation of the holoenzyme occurs by release of quaternary constraint into an ensemble of active-site conformations. © 2017 The Protein Society.

The Ω-loop lid domain of phosphoenolpyruvate carboxykinase is essential for catalytic function

PubMed Central

Johnson, Troy A.; Holyoak, Todd

2012-01-01

Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Recent studies have demonstrated that the enzyme contains a mobile active site lid domain that transitions between an open/disorded conformation to a closed/ordered conformation as the enzyme progresses through the catalytic cycle. The understanding of how this mobile domain functions in catalysis is incomplete. Previous studies show that the closure of the lid domain stabilizes the reaction intermediate and protects the reactive intermediate from spurious protonation and thus contributes to the fidelity of the enzyme. In order to more fully investigate the roles of the lid domain in PEPCK function we created three mutations that replaced the 11-residue lid domain with one, two or three glycine residues. Kinetic analysis of the mutant enzymes demonstrates that none of the enzyme constructs exhibit any measurable kinetic activity resulting in a decrease in the catalytic parameters by at least 106. Structural characterization of the mutants in complexes representing the catalytic cycle suggest that the inactivity is due to a role for the lid domain in the formation of the fully closed state of the enzyme that is required for catalytic function. In the absence of the lid domain, the enzyme is unable to achieve the fully closed state and is rendered inactive despite possessing all of the residues and substrates required for catalytic function. This work demonstrates how enzyme catalytic function can be abolished through the alteration of conformational equilibria despite all elements required for chemical conversion of substrates to products remaining intact. PMID:23127136

Diversity of Secondary Structure in Catalytic Peptides with β-Turn-Biased Sequences

PubMed Central

2016-01-01

X-ray crystallography has been applied to the structural analysis of a series of tetrapeptides that were previously assessed for catalytic activity in an atroposelective bromination reaction. Common to the series is a central Pro-Xaa sequence, where Pro is either l- or d-proline, which was chosen to favor nucleation of canonical β-turn secondary structures. Crystallographic analysis of 35 different peptide sequences revealed a range of conformational states. The observed differences appear not only in cases where the Pro-Xaa loop-region is altered, but also when seemingly subtle alterations to the flanking residues are introduced. In many instances, distinct conformers of the same sequence were observed, either as symmetry-independent molecules within the same unit cell or as polymorphs. Computational studies using DFT provided additional insight into the analysis of solid-state structural features. Select X-ray crystal structures were compared to the corresponding solution structures derived from measured proton chemical shifts, 3J-values, and 1H–1H-NOESY contacts. These findings imply that the conformational space available to simple peptide-based catalysts is more diverse than precedent might suggest. The direct observation of multiple ground state conformations for peptides of this family, as well as the dynamic processes associated with conformational equilibria, underscore not only the challenge of designing peptide-based catalysts, but also the difficulty in predicting their accessible transition states. These findings implicate the advantages of low-barrier interconversions between conformations of peptide-based catalysts for multistep, enantioselective reactions. PMID:28029251

Closed-loop spontaneous baroreflex transfer function is inappropriate for system identification of neural arc but partly accurate for peripheral arc: predictability analysis

PubMed Central

Kamiya, Atsunori; Kawada, Toru; Shimizu, Shuji; Sugimachi, Masaru

2011-01-01

Abstract Although the dynamic characteristics of the baroreflex system have been described by baroreflex transfer functions obtained from open-loop analysis, the predictability of time-series output dynamics from input signals, which should confirm the accuracy of system identification, remains to be elucidated. Moreover, despite theoretical concerns over closed-loop system identification, the accuracy and the predictability of the closed-loop spontaneous baroreflex transfer function have not been evaluated compared with the open-loop transfer function. Using urethane and α-chloralose anaesthetized, vagotomized and aortic-denervated rabbits (n = 10), we identified open-loop baroreflex transfer functions by recording renal sympathetic nerve activity (SNA) while varying the vascularly isolated intracarotid sinus pressure (CSP) according to a binary random (white-noise) sequence (operating pressure ± 20 mmHg), and using a simplified equation to calculate closed-loop-spontaneous baroreflex transfer function while matching CSP with systemic arterial pressure (AP). Our results showed that the open-loop baroreflex transfer functions for the neural and peripheral arcs predicted the time-series SNA and AP outputs from measured CSP and SNA inputs, with r2 of 0.8 ± 0.1 and 0.8 ± 0.1, respectively. In contrast, the closed-loop-spontaneous baroreflex transfer function for the neural arc was markedly different from the open-loop transfer function (enhanced gain increase and a phase lead), and did not predict the time-series SNA dynamics (r2; 0.1 ± 0.1). However, the closed-loop-spontaneous baroreflex transfer function of the peripheral arc partially matched the open-loop transfer function in gain and phase functions, and had limited but reasonable predictability of the time-series AP dynamics (r2, 0.7 ± 0.1). A numerical simulation suggested that a noise predominantly in the neural arc under resting conditions might be a possible mechanism responsible for our findings. Furthermore, the predictabilities of the neural arc transfer functions obtained in open-loop and closed-loop conditions were validated by closed-loop pharmacological (phenylephrine and nitroprusside infusions) pressure interventions. Time-series SNA responses to drug-induced AP changes predicted by the open-loop transfer function matched closely the measured responses (r2, 0.9 ± 0.1), whereas SNA responses predicted by closed-loop-spontaneous transfer function deviated greatly and were the inverse of measured responses (r, −0.8 ± 0.2). These results indicate that although the spontaneous baroreflex transfer function obtained by closed-loop analysis has been believed to represent the neural arc function, it is inappropriate for system identification of the neural arc but is essentially appropriate for the peripheral arc under resting conditions, when compared with open-loop analysis. PMID:21486839

Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation

NASA Astrophysics Data System (ADS)

Lira-Navarrete, Erandi; de Las Rivas, Matilde; Compañón, Ismael; Pallarés, María Carmen; Kong, Yun; Iglesias-Fernández, Javier; Bernardes, Gonçalo J. L.; Peregrina, Jesús M.; Rovira, Carme; Bernadó, Pau; Bruscolini, Pierpaolo; Clausen, Henrik; Lostao, Anabel; Corzana, Francisco; Hurtado-Guerrero, Ramon

2015-05-01

Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.

Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes

DOE PAGES

Li, Y.; Zakharov, D.; Zhao, S.; ...

2015-06-29

Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction—ethylene hydrogenation. This work exploits a microfabricated catalytic reactor compatible with both probes. The results demonstrate dynamic transformations of the ensemble of Pt clusters that spans a broad size range throughout changing reaction conditions. Lastly,more » this method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes.« less

Dynamic formation of single-atom catalytic active sites on ceria-supported gold nanoparticles

DOE PAGES

Wang, Yanggang; Mei, Donghai; Glezakou, Vassiliki Alexandra; ...

2015-03-04

Ab initio Molecular Dynamics simulations and static Density Functional Theory calculations have been performed to investigate the reaction mechanism of CO oxidation on Au/CeO 2 catalyst. It is found that under reaction condition CO adsorption significantly labializes the surface atoms of the Au cluster and leads to the formation of isolated Au+-CO species that resides on the support in the vicinity of the Au particle. In this context, we identified a dynamic single-atom catalytic mechanism at the interfacial area for CO oxidation on Au/CeO 2 catalyst, which is a lower energy pathway than that of CO oxidation at the interfacemore » with the metal particle. This results from the ability of the single atom site to strongly couple with the redox properties of the support in a synergistic manner thereby lowering the barrier for redox reactions. We find that the single Au+ ion, which only exists under reaction conditions, breaks away from the Au cluster to catalyze CO oxidation and returns to the Au cluster after the catalytic cycle is completed. Generally, our study highlights the importance of the dynamic creation of active sites under reaction conditions and their essential role in a catalytic process.« less

Improvement in the thermostability of chitosanase from Bacillus ehimensis by introducing artificial disulfide bonds.

PubMed

Sheng, Jun; Ji, Xiaofeng; Zheng, Yuan; Wang, Zhipeng; Sun, Mi

2016-10-01

To determine the effects of artificial disulfide bridges on the thermostability and catalytic efficiency of chitosanase EAG1. Five artificial disulfide bridges were designed based on the structural information derived from the three-dimensional (3-D) model of chitosanase EAG1. Two beneficial mutants (G113C/D116C, A207C-L286C) were located in the flexible surface loop region, whereas the similar substitutions introduced in α-helices regions had a negligible effect. Mut5, the most active mutant, had a longer half-life at 50 °C (from 10.5 to 69.3 min) and a 200 % higher catalytic efficiency (K cat/K m) than that of the original EAG1. The contribution of disulfide bridges to enzyme thermostability is mainly dependent on its location within the polypeptide chain. Strategical placement of a disulfide bridge in flexible regions provides a rigid support and creation of a protected microenvironment, which is effective in improving enzyme's thermostability and catalytic efficiency.

CTCF and cohesin regulate chromatin loop stability with distinct dynamics

PubMed Central

Hansen, Anders S; Pustova, Iryna; Cattoglio, Claudia; Tjian, Robert; Darzacq, Xavier

2017-01-01

Folding of mammalian genomes into spatial domains is critical for gene regulation. The insulator protein CTCF and cohesin control domain location by folding domains into loop structures, which are widely thought to be stable. Combining genomic and biochemical approaches we show that CTCF and cohesin co-occupy the same sites and physically interact as a biochemically stable complex. However, using single-molecule imaging we find that CTCF binds chromatin much more dynamically than cohesin (~1–2 min vs. ~22 min residence time). Moreover, after unbinding, CTCF quickly rebinds another cognate site unlike cohesin for which the search process is long (~1 min vs. ~33 min). Thus, CTCF and cohesin form a rapidly exchanging 'dynamic complex' rather than a typical stable complex. Since CTCF and cohesin are required for loop domain formation, our results suggest that chromatin loops are dynamic and frequently break and reform throughout the cell cycle. DOI: http://dx.doi.org/10.7554/eLife.25776.001 PMID:28467304

SILHIL Replication of Electric Aircraft Powertrain Dynamics and Inner-Loop Control for V&V of System Health Management Routines

NASA Technical Reports Server (NTRS)

Bole, Brian; Teubert, Christopher Allen; Cuong Chi, Quach; Hogge, Edward; Vazquez, Sixto; Goebel, Kai; George, Vachtsevanos

2013-01-01

Software-in-the-loop and Hardware-in-the-loop testing of failure prognostics and decision making tools for aircraft systems will facilitate more comprehensive and cost-effective testing than what is practical to conduct with flight tests. A framework is described for the offline recreation of dynamic loads on simulated or physical aircraft powertrain components based on a real-time simulation of airframe dynamics running on a flight simulator, an inner-loop flight control policy executed by either an autopilot routine or a human pilot, and a supervisory fault management control policy. The creation of an offline framework for verifying and validating supervisory failure prognostics and decision making routines is described for the example of battery charge depletion failure scenarios onboard a prototype electric unmanned aerial vehicle.

A control system design approach for flexible spacecraft

NASA Technical Reports Server (NTRS)

Silverberg, L. M.

1985-01-01

A control system design approach for flexible spacecraft is presented. The control system design is carried out in two steps. The first step consists of determining the ideal control system in terms of a desirable dynamic performance. The second step consists of designing a control system using a limited number of actuators that possess a dynamic performance that is close to the ideal dynamic performance. The effects of using a limited number of actuators is that the actual closed-loop eigenvalues differ from the ideal closed-loop eigenvalues. A method is presented to approximate the actual closed-loop eigenvalues so that the calculation of the actual closed-loop eigenvalues can be avoided. Depending on the application, it also may be desirable to apply the control forces as impulses. The effect of digitizing the control to produce the appropriate impulses is also examined.

Substrate Binding Process and Mechanistic Functioning of Type 1 11β-Hydroxysteroid Dehydrogenase from Enhanced Sampling Methods

PubMed Central

Recanatini, Maurizio; Cavalli, Andrea

2011-01-01

In humans, type 1 11β-hydroxysteroid dehydrogenase (11β-HSD-1) plays a key role in the regulation of the glucocorticoids balance by converting the inactive hormone cortisone into cortisol. Numerous functional aspects of 11β-HSD-1 have been understood thanks to the availability at the Worldwide Protein Data Bank of a number of X-ray structures of the enzyme either alone or in complex with inhibitors, and to several experimental data. However at present, a complete description of the dynamic behaviour of 11β-HSD-1 upon substrate binding is missing. To this aim we firstly docked cortisone into the catalytic site of 11β-HSD-1 (both wild type and Y177A mutant), and then we used steered molecular dynamics and metadynamics to simulate its undocking. This methodology helped shedding light at molecular level on the complex relationship between the enzyme and its natural substrate. In particular, the work highlights a) the reason behind the functional dimerisation of 11β-HSD-1, b) the key role of Y177 in the cortisone binding event, c) the fine tuning of the active site degree of solvation, and d) the role of the S228-P237 loop in ligand recognition. PMID:21966510

Understanding the interactions of different substrates with wild-type and mutant acylaminoacyl peptidase using molecular dynamics simulations.

PubMed

Zhu, Jingxuan; Wang, Yan; Li, Xin; Han, Weiwei; Zhao, Li

2017-12-20

Acylaminoacylpeptidase (AAP) belongs to peptidase protein family, which can degrade amyloid β-peptide forms in the brains of patients, and hence leads to Alzheimer's disease. And so, AAP is considered to be a novel target in the design of drugs against Alzheimer's disease. In this investigation, six molecular dynamics simulations were used to find that the interaction between the wild-type and R526V AAP with two different substrates (p-nitrophenylcaprylate and Ac-Leu-p-nitroanilide). Our results were as follows: firstly, Ac-Leu-p-nitroanilide bound to R526V AAP to form a more disordered loop (residues 552-562) in the α/β-hydrolase fold like of AAP, which caused an open and inactive AAP domain form, secondly, binding p-nitrophenylcaprylate and Ac-Leu-p-nitroanilide to AAP can decrease the flexibility of residues 225-250, 260-270, and 425-450, in which the ordered secondary structures may contain the suitable geometrical structure and so it is useful to serine attack. Our theoretical results showed that the binding of the two substrates can induce specific conformational changes responsible for the diverse AAP catalytic specificity. These theoretical substrate-induced structural diversities can help explain the abilities of AAPs to recognize and hydrolyze extremely different substrates.

Crystallographic and Molecular Dynamics Simulation Analysis of Escherichia Coli Dihydroneopterin Aldolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blaszczyk, Jaroslaw; Lu, Zhenwei; Li, Yue

2014-09-01

To understand the structural basis for the biochemical differences and further investigate the catalytic mechanism of DHNA, we have determined the structure of EcDHNA complexed with NP at 1.07-Å resolution [PDB:2O90], built an atomic model of EcDHNA complexed with the substrate DHNP, and performed molecular dynamics (MD) simulation analysis of the substrate complex. EcDHNA has the same fold as SaDHNA and also forms an octamer that consists of two tetramers, but the packing of one tetramer with the other is significantly different between the two enzymes. Furthermore, the structures reveal significant differences in the vicinity of the active site, particularlymore » in the loop that connects strands β3 and β4, mainly due to the substitution of nearby residues. The building of an atomic model of the complex of EcDHNA and the substrate DHNP and the MD simulation of the complex show that some of the hydrogen bonds between the substrate and the enzyme are persistent, whereas others are transient. The substrate binding model and MD simulation provide the molecular basis for the biochemical behaviors of the enzyme, including noncooperative substrate binding, indiscrimination of a pair of epimers as the substrates, proton wire switching during catalysis, and formation of epimerization product.« less

A dynamic flare with anomalously dense flare loops

NASA Technical Reports Server (NTRS)

Svestka, Z.; Fontenla, J. M.; Machado, M. E.; Martin, S. F.; Neidig, D. F.

1986-01-01

The dynamic flare of November 6, 1980 developed a rich system of growing loops which could be followed in H-alpha for 1.5 hours. Throughout the flare, these loops, near the limb, were seen in emission against the disk. Theoretical computations of b-values for a hydrogen atom reveal that this requires electron densities in the loops to be close to 10 to the 12th per cu cm. From measured widths of higher Balmer lines the density at the tops of the loops was found to be 4 x 10 to the 12th per cu cm if no nonthermal motions were present. It is now general knowledge that flare loops are initially observed in X-rays and become visible in H-alpha only after cooling. For such a high density a loop would cool through radiation from 10 to the 7th K to 10 to the 4th K within a few minutes so that the dense H-alpha loops should have heights very close to the heights of the X-ray loops. This, however, contradicts the observations obtained by the HXIS and FCS instruments on board SMM which show the X-ray loops at much higher altitudes than the loops in H-alpha. Therefore, the density must have been significantly smaller when the loops were formed and the flare loops were apparently both shrinking and becoming denser while cooling.

Ensembler: Enabling High-Throughput Molecular Simulations at the Superfamily Scale.

PubMed

Parton, Daniel L; Grinaway, Patrick B; Hanson, Sonya M; Beauchamp, Kyle A; Chodera, John D

2016-06-01

The rapidly expanding body of available genomic and protein structural data provides a rich resource for understanding protein dynamics with biomolecular simulation. While computational infrastructure has grown rapidly, simulations on an omics scale are not yet widespread, primarily because software infrastructure to enable simulations at this scale has not kept pace. It should now be possible to study protein dynamics across entire (super)families, exploiting both available structural biology data and conformational similarities across homologous proteins. Here, we present a new tool for enabling high-throughput simulation in the genomics era. Ensembler takes any set of sequences-from a single sequence to an entire superfamily-and shepherds them through various stages of modeling and refinement to produce simulation-ready structures. This includes comparative modeling to all relevant PDB structures (which may span multiple conformational states of interest), reconstruction of missing loops, addition of missing atoms, culling of nearly identical structures, assignment of appropriate protonation states, solvation in explicit solvent, and refinement and filtering with molecular simulation to ensure stable simulation. The output of this pipeline is an ensemble of structures ready for subsequent molecular simulations using computer clusters, supercomputers, or distributed computing projects like Folding@home. Ensembler thus automates much of the time-consuming process of preparing protein models suitable for simulation, while allowing scalability up to entire superfamilies. A particular advantage of this approach can be found in the construction of kinetic models of conformational dynamics-such as Markov state models (MSMs)-which benefit from a diverse array of initial configurations that span the accessible conformational states to aid sampling. We demonstrate the power of this approach by constructing models for all catalytic domains in the human tyrosine kinase family, using all available kinase catalytic domain structures from any organism as structural templates. Ensembler is free and open source software licensed under the GNU General Public License (GPL) v2. It is compatible with Linux and OS X. The latest release can be installed via the conda package manager, and the latest source can be downloaded from https://github.com/choderalab/ensembler.

Dynamics of catalytic tubular microjet engines: Dependence on geometry and chemical environment

NASA Astrophysics Data System (ADS)

LiJ. X. L.; G. S. H. Contributed Equally To This Work., Jinxing; Huang, Gaoshan; Ye, Mengmeng; Li, Menglin; Liu, Ran; Mei, Yongfeng

2011-12-01

Strain-engineered tubular microjet engines with various geometric dimensions hold interesting autonomous motions in an aqueous fuel solution when propelled by catalytic decomposition of hydrogen peroxide to oxygen and water. The catalytically-generated oxygen bubbles expelled from microtubular cavities propel the microjet step by step in discrete increments. We focus on the dynamics of our tubular microjets in one step and build up a body deformation model to elucidate the interaction between tubular microjets and the bubbles they produce. The average microjet velocity is calculated analytically based on our model and the obtained results demonstrate that the velocity of the microjet increases linearly with the concentration of hydrogen peroxide. The geometric dimensions of the microjet, such as length and radius, also influence its dynamic characteristics significantly. A close consistency between experimental and calculated results is achieved despite a small deviation due to the existence of an approximation in the model. The results presented in this work improve our understanding regarding catalytic motions of tubular microjets and demonstrate the controllability of the microjet which may have potential applications in drug delivery and biology.Strain-engineered tubular microjet engines with various geometric dimensions hold interesting autonomous motions in an aqueous fuel solution when propelled by catalytic decomposition of hydrogen peroxide to oxygen and water. The catalytically-generated oxygen bubbles expelled from microtubular cavities propel the microjet step by step in discrete increments. We focus on the dynamics of our tubular microjets in one step and build up a body deformation model to elucidate the interaction between tubular microjets and the bubbles they produce. The average microjet velocity is calculated analytically based on our model and the obtained results demonstrate that the velocity of the microjet increases linearly with the concentration of hydrogen peroxide. The geometric dimensions of the microjet, such as length and radius, also influence its dynamic characteristics significantly. A close consistency between experimental and calculated results is achieved despite a small deviation due to the existence of an approximation in the model. The results presented in this work improve our understanding regarding catalytic motions of tubular microjets and demonstrate the controllability of the microjet which may have potential applications in drug delivery and biology. Electronic supplementary information (ESI) available: I. Video of the catalytic motion of a typical microjet moving in a linear way. II. Detailed numerical analyses: Reynolds number calculation, displacement of the microjet and the bubble after separation, and example of experimental velocity calculation. See DOI: 10.1039/c1nr10840a

Coronal Loops: Observations and Modeling of Confined Plasma.

PubMed

Reale, Fabio

Coronal loops are the building blocks of the X-ray bright solar corona. They owe their brightness to the dense confined plasma, and this review focuses on loops mostly as structures confining plasma. After a brief historical overview, the review is divided into two separate but not independent parts: the first illustrates the observational framework, the second reviews the theoretical knowledge. Quiescent loops and their confined plasma are considered and, therefore, topics such as loop oscillations and flaring loops (except for non-solar ones, which provide information on stellar loops) are not specifically addressed here. The observational section discusses the classification, populations, and the morphology of coronal loops, its relationship with the magnetic field, and the loop stranded structure. The section continues with the thermal properties and diagnostics of the loop plasma, according to the classification into hot, warm, and cool loops. Then, temporal analyses of loops and the observations of plasma dynamics, hot and cool flows, and waves are illustrated. In the modeling section, some basics of loop physics are provided, supplying fundamental scaling laws and timescales, a useful tool for consultation. The concept of loop modeling is introduced and models are divided into those treating loops as monolithic and static, and those resolving loops into thin and dynamic strands. More specific discussions address modeling the loop fine structure and the plasma flowing along the loops. Special attention is devoted to the question of loop heating, with separate discussion of wave (AC) and impulsive (DC) heating. Large-scale models including atmosphere boxes and the magnetic field are also discussed. Finally, a brief discussion about stellar coronal loops is followed by highlights and open questions.

Generalization of a model of hysteresis for dynamical systems.

PubMed

Piquette, Jean C; McLaughlin, Elizabeth A; Ren, Wei; Mukherjee, Binu K

2002-06-01

A previously described model of hysteresis [J. C. Piquette and S. E. Forsythe, J. Acoust. Soc. Am. 106, 3317-3327 (1999); 106, 3328-3334 (1999)] is generalized to apply to a dynamical system. The original model produces theoretical hysteresis loops that agree well with laboratory measurements acquired under quasi-static conditions. The loops are produced using three-dimensional rotation matrices. An iterative procedure, which allows the model to be applied to a dynamical system, is introduced here. It is shown that, unlike the quasi-static case, self-crossing of the loops is a realistic possibility when inertia and viscous friction are taken into account.

Enzymatic function of loop movement in enolase: preparation and some properties of H159N, H159A, H159F, and N207A enolases.

PubMed

Brewer, John M; Glover, Claiborne V C; Holland, Michael J; Lebioda, Lukasz

2003-05-01

The hypothesis that His159 in yeast enolase moves on a polypeptide loop to protonate the phosphoryl of 2-phosphoglycerate to initiate its conversion to phosphoenolpyruvate was tested by preparing H159N, H159A, and H159F enolases. These have 0.07%-0.25% of the native activity under standard assay conditions and the pH dependence of maximum velocities of H159A and H159N mutants is markedly altered. Activation by Mg2+ is biphasic, with the smaller Mg2+ activation constant closer to that of the "catalytic" Mg2+ binding site of native enolase and the larger in the mM range in which native enolase is inhibited. A third Mg2+ may bind to the phosphoryl, functionally replacing proton donation by His159. N207A enolase lacks an intersubunit interaction that stabilizes the closed loop(s) conformation when 2-phosphoglycerate binds. It has 21% of the native activity, also exhibits biphasic Mg2+ activation, and its reaction with the aldehyde analogue of the substrate is more strongly inhibited than is its normal enzymatic reaction. Polypeptide loop(s) closure may keep a proton from His159 interacting with the substrate phosphoryl oxygen long enough to stabilize a carbanion intermediate.

Dynamic Disorder in Quasi-Equilibrium Enzymatic Systems

PubMed Central

Chaudhury, Srabanti; Igoshin, Oleg A.

2010-01-01

Conformations and catalytic rates of enzymes fluctuate over a wide range of timescales. Despite these fluctuations, there exist some limiting cases in which the enzymatic catalytic rate follows the macroscopic rate equation such as the Michaelis-Menten law. In this paper we investigate the applicability of macroscopic rate laws for fluctuating enzyme systems in which catalytic transitions are slower than ligand binding-dissociation reactions. In this quasi-equilibrium limit, for an arbitrary reaction scheme we show that the catalytic rate has the same dependence on ligand concentrations as obtained from mass-action kinetics even in the presence of slow conformational fluctuations. These results indicate that the timescale of conformational dynamics – no matter how slow – will not affect the enzymatic rate in quasi-equilibrium limit. Our numerical results for two enzyme-catalyzed reaction schemes involving multiple substrates and inhibitors further support our general theory. PMID:20808776

Motor-sensory confluence in tactile perception.

PubMed

Saig, Avraham; Gordon, Goren; Assa, Eldad; Arieli, Amos; Ahissar, Ehud

2012-10-03

Perception involves motor control of sensory organs. However, the dynamics underlying emergence of perception from motor-sensory interactions are not yet known. Two extreme possibilities are as follows: (1) motor and sensory signals interact within an open-loop scheme in which motor signals determine sensory sampling but are not affected by sensory processing and (2) motor and sensory signals are affected by each other within a closed-loop scheme. We studied the scheme of motor-sensory interactions in humans using a novel object localization task that enabled monitoring the relevant overt motor and sensory variables. We found that motor variables were dynamically controlled within each perceptual trial, such that they gradually converged to steady values. Training on this task resulted in improvement in perceptual acuity, which was achieved solely by changes in motor variables, without any change in the acuity of sensory readout. The within-trial dynamics is captured by a hierarchical closed-loop model in which lower loops actively maintain constant sensory coding, and higher loops maintain constant sensory update flow. These findings demonstrate interchangeability of motor and sensory variables in perception, motor convergence during perception, and a consistent hierarchical closed-loop perceptual model.

Investigation of Inner Loop Flight Control Strategies for High-Speed Research

NASA Technical Reports Server (NTRS)

Newman, Brett; Kassem, Ayman

1999-01-01

This report describes the activities and findings conducted under contract NAS1-19858 with NASA Langley Research Center. Subject matter is the investigation of suitable flight control design methodologies and solutions for large, flexible high-speed vehicles. Specifically, methodologies are to address the inner control loops used for stabilization and augmentation of a highly coupled airframe system possibly involving rigid-body motion, structural vibrations, unsteady aerodynamics, and actuator dynamics. Techniques considered in this body of work are primarily conventional-based, and the vehicle of interest is the High-Speed Civil Transport (HSCT). Major findings include 1) current aeroelastic vehicle modeling procedures require further emphasis and refinement, 2) traditional and nontraditional inner loop flight control strategies employing a single feedback loop do not appear sufficient for highly flexible HSCT class vehicles, 3) inner loop flight control systems will, in all likelihood, require multiple interacting feedback loops, and 4) Ref. H HSCT configuration presents major challenges to designing acceptable closed-loop flight dynamics.

HOW GAS-DYNAMIC FLARE MODELS POWERED BY PETSCHEK RECONNECTION DIFFER FROM THOSE WITH AD HOC ENERGY SOURCES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Longcope, D. W.; Klimchuk, J. A.

Aspects of solar flare dynamics, such as chromospheric evaporation and flare light curves, have long been studied using one-dimensional models of plasma dynamics inside a static flare loop, subjected to some energy input. While extremely successful at explaining the observed characteristics of flares, all such models so far have specified energy input ad hoc, rather than deriving it self-consistently. There is broad consensus that flares are powered by magnetic energy released through reconnection. Recent work has generalized Petschek’s basic reconnection scenario, topological change followed by field line retraction and shock heating, to permit its inclusion in a one-dimensional flare loop model. Heremore » we compare the gas dynamics driven by retraction and shocking to those from more conventional static loop models energized by ad hoc source terms. We find significant differences during the first minute, when retraction leads to larger kinetic energies and produces higher densities at the loop top, while ad hoc heating tends to rarify the loop top. The loop-top density concentration is related to the slow magnetosonic shock, characteristic of Petschek’s model, but persists beyond the retraction phase occurring in the outflow jet. This offers an explanation for observed loop-top sources of X-ray and EUV emission, with advantages over that provided by ad hoc heating scenarios. The cooling phases of the two models are, however, notably similar to one another, suggesting that observations at that stage will yield little information on the nature of energy input.« less

Crystal structure of NTPDase2 in complex with the sulfoanthraquinone inhibitor PSB-071.

PubMed

Zebisch, Matthias; Baqi, Younis; Schäfer, Petra; Müller, Christa E; Sträter, Norbert

2014-03-01

In many vertebrate tissues CD39-like ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) act in concert with ecto-5'-nucleotidase (e5NT, CD73) to convert extracellular ATP to adenosine. Extracellular ATP is a cytotoxic, pro-inflammatory signalling molecule whereas its product adenosine constitutes a universal and potent immune suppressor. Interference with these ectonucleotidases by use of small molecule inhibitors or inhibitory antibodies appears to be an effective strategy to enhance anti-tumour immunity and suppress neoangiogenesis. Here we present the first crystal structures of an NTPDase catalytic ectodomain in complex with the Reactive Blue 2 (RB2)-derived inhibitor PSB-071. In both of the two crystal forms presented the inhibitor binds as a sandwich of two molecules at the nucleoside binding site. One of the molecules is well defined in its orientation. Specific hydrogen bonds are formed between the sulfonyl group and the nucleoside binding loop. The methylphenyl side chain functionality that improved NTPDase2-specificity is sandwiched between R245 and R394, the latter of which is exclusively found in NTPDase2. The second molecule exhibits great in-plane rotational freedom and could not be modelled in a specific orientation. In addition to this structural insight into NTPDase inhibition, the observation of the putative membrane interaction loop (MIL) in two different conformations related by a 10° rotation identifies the MIL as a dynamic section of NTPDases that is potentially involved in regulation of catalysis. Copyright © 2014 Elsevier Inc. All rights reserved.

Three critical hydrogen bonds determine the catalytic activity of the Diels–Alderase ribozyme

PubMed Central

Kraut, Stefanie; Bebenroth, Dirk; Nierth, Alexander; Kobitski, Andrei Y.; Nienhaus, G. Ulrich; Jäschke, Andres

2012-01-01

Compared to protein enzymes, our knowledge about how RNA accelerates chemical reactions is rather limited. The crystal structures of a ribozyme that catalyzes Diels–Alder reactions suggest a rich tertiary architecture responsible for catalysis. In this study, we systematically probe the relevance of crystallographically observed ground-state interactions for catalytic function using atomic mutagenesis in combination with various analytical techniques. The largest energetic contribution apparently arises from the precise shape complementarity between transition state and catalytic pocket: A single point mutant that folds correctly into the tertiary structure but lacks one H-bond that normally stabilizes the pocket is completely inactive. In the rate-limiting chemical step, the dienophile is furthermore activated by two weak H-bonds that contribute ∼7–8 kJ/mol to transition state stabilization, as indicated by the 25-fold slower reaction rates of deletion mutants. These H-bonds are also responsible for the tight binding of the Diels–Alder product by the ribozyme that causes product inhibition. For high catalytic activity, the ribozyme requires a fine-tuned balance between rigidity and flexibility that is determined by the combined action of one inter-strand H-bond and one magnesium ion. A sharp 360° turn reminiscent of the T-loop motif observed in tRNA is found to be important for catalytic function. PMID:21976731

Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity

PubMed Central

Yang, Guang; Yao, Hua; Mozzicafreddo, Matteo; Ballarini, Patrizia; Pucciarelli, Sandra

2017-01-01

ABSTRACT The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel EfAmy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications. PMID:28455329

Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity.

PubMed

Yang, Guang; Yao, Hua; Mozzicafreddo, Matteo; Ballarini, Patrizia; Pucciarelli, Sandra; Miceli, Cristina

2017-07-01

The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii ( Ef Amy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, Ef Amy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active Ef Amy with improved thermostability and catalytic efficiency at low temperatures. We engineered two Ef Amy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of Ef Amy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named Ef Amy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel Ef Amy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications. Copyright © 2017 Yang et al.

Photonic Activation of Plasminogen Induced by Low Dose UVB

PubMed Central

Correia, Manuel; Snabe, Torben; Thiagarajan, Viruthachalam; Petersen, Steffen Bjørn; Campos, Sara R. R.; Baptista, António M.; Neves-Petersen, Maria Teresa

2015-01-01

Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity was observed after 10 min illumination of human plasminogen. Irradiance levels used are in the same order of magnitude of the UVB solar irradiance. Activation is correlated with light induced disruption of disulphide bridges upon UVB excitation of the aromatic residues and with the formation of photochemical products, e.g. dityrosine and N-formylkynurenine. Most of the protein fold is maintained after 10 min illumination since no major changes are observed in the near-UV CD spectrum. Far-UV CD shows loss of secondary structure after illumination (33.4% signal loss at 206 nm). Thermal unfolding CD studies show that plasminogen retains a native like cooperative transition at ~70 ºC after UV-illumination. We propose that UVB activation of plasminogen occurs upon photo-cleavage of a functional allosteric disulphide bond, Cys737-Cys765, located in the catalytic domain and in van der Waals contact with Trp761 (4.3 Å). Such proximity makes its disruption very likely, which may occur upon electron transfer from excited Trp761. Reduction of Cys737-Cys765 will result in likely conformational changes in the catalytic site. Molecular dynamics simulations reveal that reduction of Cys737-Cys765 in plasminogen leads to an increase of the fluctuations of loop 760–765, the S1-entrance frame located close to the active site. These fluctuations affect the range of solvent exposure of the catalytic triad, particularly of Asp646 and Ser74, which acquire an exposure profile similar to the values in plasmin. The presented photonic mechanism of plasminogen activation has the potential to be used in clinical applications, possibly together with other enzymatic treatments for the elimination of blood clots. PMID:25635856

Site-Specific Measurement of Water Dynamics in the Substrate Pocket of Ketosteroid Isomerase Using Time-Resolved Vibrational Spectroscopy

PubMed Central

Jha, Santosh Kumar; Ji, Minbiao; Gaffney, Kelly J.; Boxer, Steven G.

2012-01-01

Little is known about the reorganization capacity of water molecules at the active sites of enzymes and how this couples to the catalytic reaction. Here, we study the dynamics of water molecules at the active site of a highly proficient enzyme, Δ5-3-ketosteroid isomerase (KSI), during a light-activated mimic of its catalytic cycle. Photo-excitation of a nitrile containing photo-acid, coumarin183 (C183), mimics the change in charge density that occurs at the active site of KSI during the first step of the catalytic reaction. The nitrile of C183 is exposed to water when bound to the KSI active site, and we used time-resolved vibrational spectroscopy as a site-specific probe to study the solvation dynamics of water molecules in the vicinity of the nitrile. We observed that water molecules at the active site of KSI are highly rigid, during the light-activated catalytic cycle, compared to the solvation dynamics observed in bulk water. Based upon this result we hypothesize that rigid water dipoles at the active site might help in the maintenance of the pre-organized electrostatic environment required for efficient catalysis. The results also demonstrate the utility of nitrile probes in measuring the dynamics of local (H-bonded) water molecules in contrast to the commonly used fluorescence methods which measure the average behavior of primary and subsequent spheres of solvation. PMID:22931297

Phase structure of the Polyakov-quark-meson model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schaefer, B.-J.; Pawlowski, J. M.; Wambach, J.

2007-10-01

The relation between the deconfinement and chiral phase transition is explored in the framework of a Polyakov-loop-extended two-flavor quark-meson (PQM) model. In this model the Polyakov loop dynamics is represented by a background temporal gauge field which also couples to the quarks. As a novelty an explicit quark chemical potential and N{sub f}-dependence in the Polyakov loop potential is proposed by using renormalization group arguments. The behavior of the Polyakov loop as well as the chiral condensate as function of temperature and quark chemical potential is obtained by minimizing the grand canonical thermodynamic potential of the system. The effect ofmore » the Polyakov loop dynamics on the chiral phase diagram and on several thermodynamic bulk quantities is presented.« less

A Culture-Behavior-Brain Loop Model of Human Development.

PubMed

Han, Shihui; Ma, Yina

2015-11-01

Increasing evidence suggests that cultural influences on brain activity are associated with multiple cognitive and affective processes. These findings prompt an integrative framework to account for dynamic interactions between culture, behavior, and the brain. We put forward a culture-behavior-brain (CBB) loop model of human development that proposes that culture shapes the brain by contextualizing behavior, and the brain fits and modifies culture via behavioral influences. Genes provide a fundamental basis for, and interact with, the CBB loop at both individual and population levels. The CBB loop model advances our understanding of the dynamic relationships between culture, behavior, and the brain, which are crucial for human phylogeny and ontogeny. Future brain changes due to cultural influences are discussed based on the CBB loop model. Copyright © 2015 Elsevier Ltd. All rights reserved.

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

PubMed Central

2018-01-01

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA–ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field. PMID:29297679

A method for reducing sampling jitter in digital control systems

NASA Technical Reports Server (NTRS)

Anderson, T. O.; HURBD W. J.; Hurd, W. J.

1969-01-01

Digital phase lock loop system is designed by smoothing the proportional control with a low pass filter. This method does not significantly affect the loop dynamics when the smoothing filter bandwidth is wide compared to loop bandwidth.

DNA looping by FokI: the impact of twisting and bending rigidity on protein-induced looping dynamics

PubMed Central

Laurens, Niels; Rusling, David A.; Pernstich, Christian; Brouwer, Ineke; Halford, Stephen E.; Wuite, Gijs J. L.

2012-01-01

Protein-induced DNA looping is crucial for many genetic processes such as transcription, gene regulation and DNA replication. Here, we use tethered-particle motion to examine the impact of DNA bending and twisting rigidity on loop capture and release, using the restriction endonuclease FokI as a test system. To cleave DNA efficiently, FokI bridges two copies of an asymmetric sequence, invariably aligning the sites in parallel. On account of the fixed alignment, the topology of the DNA loop is set by the orientation of the sites along the DNA. We show that both the separation of the FokI sites and their orientation, altering, respectively, the twisting and the bending of the DNA needed to juxtapose the sites, have profound effects on the dynamics of the looping interaction. Surprisingly, the presence of a nick within the loop does not affect the observed rigidity of the DNA. In contrast, the introduction of a 4-nt gap fully relaxes all of the torque present in the system but does not necessarily enhance loop stability. FokI therefore employs torque to stabilise its DNA-looping interaction by acting as a ‘torsional’ catch bond. PMID:22373924

Closing loop base pairs in RNA loop-loop complexes: structural behavior, interaction energy and solvation analysis through molecular dynamics simulations.

PubMed

Golebiowski, Jérôme; Antonczak, Serge; Fernandez-Carmona, Juan; Condom, Roger; Cabrol-Bass, Daniel

2004-12-01

Nanosecond molecular dynamics using the Ewald summation method have been performed to elucidate the structural and energetic role of the closing base pair in loop-loop RNA duplexes neutralized by Mg2+ counterions in aqueous phases. Mismatches GA, CU and Watson-Crick GC base pairs have been considered for closing the loop of an RNA in complementary interaction with HIV-1 TAR. The simulations reveal that the mismatch GA base, mediated by a water molecule, leads to a complex that presents the best compromise between flexibility and energetic contributions. The mismatch CU base pair, in spite of the presence of an inserted water molecule, is too short to achieve a tight interaction at the closing-loop junction and seems to force TAR to reorganize upon binding. An energetic analysis has allowed us to quantify the strength of the interactions of the closing and the loop-loop pairs throughout the simulations. Although the water-mediated GA closing base pair presents an interaction energy similar to that found on fully geometry-optimized structure, the water-mediated CU closing base pair energy interaction reaches less than half the optimal value.

Structural and mechanistic insights into Mps1 kinase activation.

PubMed

Wang, Wei; Yang, Yuting; Gao, Yuefeng; Xu, Quanbin; Wang, Feng; Zhu, Songcheng; Old, William; Resing, Katheryn; Ahn, Natalie; Lei, Ming; Liu, Xuedong

2009-08-01

Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signalling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7-A-resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the C helix of the N-terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation. Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices EF and F that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.

A dynamically reconfigurable multi-functional PLL for SRAM-based FPGA in 65nm CMOS technology

NASA Astrophysics Data System (ADS)

Yang, Mingqian; Chen, Lei; Li, Xuewu; Zhang, Yanlong

2018-04-01

Phase-locked loops (PLL) have been widely utilized in FPGA as an important module for clock management. PLL with dynamic reconfiguration capability is always welcomed in FPGA design as it is able to decrease power consumption and simultaneously improve flexibility. In this paper, a multi-functional PLL with dynamic reconfiguration capability for 65nm SRAM-based FPGA is proposed. Firstly, configurable charge pump and loop filter are utilized to optimize the loop bandwidth. Secondly, the PLL incorporates a VCO with dual control voltages to accelerate the adjustment of oscillation frequency. Thirdly, three configurable dividers are presented for flexible frequency synthesis. Lastly, a configuration block with dynamic reconfiguration function is proposed. Simulation results demonstrate that the proposed multi-functional PLL can output clocks with configurable division ratio, phase shift and duty cycle. The PLL can also be dynamically reconfigured without affecting other parts' running or halting the FPGA device.

Identification of the gamma subunit-interacting residues on photoreceptor cGMP phosphodiesterase, PDE6alpha '.

PubMed

Granovsky, A E; Artemyev, N O

2000-12-29

Photoreceptor cGMP phosphodiesterase (PDE6) is the effector enzyme in the G protein-mediated visual transduction cascade. In the dark, the activity of PDE6 is shut off by the inhibitory gamma subunit (Pgamma). Chimeric proteins between cone PDE6alpha' and cGMP-binding and cGMP-specific PDE (PDE5) have been constructed and expressed in Sf9 cells to study the mechanism of inhibition of PDE6 catalytic activity by Pgamma. Substitution of the segment PDE5-(773-820) by the corresponding PDE6alpha'-(737-784) sequence in the wild-type PDE5 or in a PDE5/PDE6alpha' chimera containing the catalytic domain of PDE5 results in chimeric enzymes capable of inhibitory interaction with Pgamma. The catalytic properties of the chimeric PDEs remained similar to those of PDE5. Ala-scanning mutational analysis of the Pgamma-binding region, PDE6alpha'-(750-760), revealed PDE6alpha' residues essential for the interaction. The M758A mutation markedly impaired and the Q752A mutation moderately impaired the inhibition of chimeric PDE by Pgamma. The analysis of the catalytic properties of mutant PDEs and a model of the PDE6 catalytic domain suggest that residues Met(758) and Gln(752) directly bind Pgamma. A model of the PDE6 catalytic site shows that PDE6alpha'-(750-760) forms a loop at the entrance to the cGMP-binding pocket. Binding of Pgamma to Met(758) would effectively block access of cGMP to the catalytic cavity, providing a structural basis for the mechanism of PDE6 inhibition.

Exploration of alternate catalytic mechanisms and optimization strategies for retroaldolase design.

PubMed

Bjelic, Sinisa; Kipnis, Yakov; Wang, Ling; Pianowski, Zbigniew; Vorobiev, Sergey; Su, Min; Seetharaman, Jayaraman; Xiao, Rong; Kornhaber, Gregory; Hunt, John F; Tong, Liang; Hilvert, Donald; Baker, David

2014-01-09

Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches. © 2013.

A rationale for human operator pulsive control behavior

NASA Technical Reports Server (NTRS)

Hess, R. A.

1979-01-01

When performing tracking tasks which involve demanding controlled elements such as those with K/s-squared dynamics, the human operator often develops discrete or pulsive control outputs. A dual-loop model of the human operator is discussed, the dominant adaptive feature of which is the explicit appearance of an internal model of the manipulator-controlled element dynamics in an inner feedback loop. Using this model, a rationale for pulsive control behavior is offered which is based upon the assumption that the human attempts to reduce the computational burden associated with time integration of sensory inputs. It is shown that such time integration is a natural consequence of having an internal representation of the K/s-squared-controlled element dynamics in the dual-loop model. A digital simulation is discussed in which a modified form of the dual-loop model is shown to be capable of producing pulsive control behavior qualitively comparable to that obtained in experiment.

Structure and Dynamics Analysis on Plexin-B1 Rho GTPase Binding Domain as a Monomer and Dimer

PubMed Central

2015-01-01

Plexin-B1 is a single-pass transmembrane receptor. Its Rho GTPase binding domain (RBD) can associate with small Rho GTPases and can also self-bind to form a dimer. In total, more than 400 ns of NAMD molecular dynamics simulations were performed on RBD monomer and dimer. Different analysis methods, such as root mean squared fluctuation (RMSF), order parameters (S2), dihedral angle correlation, transfer entropy, principal component analysis, and dynamical network analysis, were carried out to characterize the motions seen in the trajectories. RMSF results show that after binding, the L4 loop becomes more rigid, but the L2 loop and a number of residues in other regions become slightly more flexible. Calculating order parameters (S2) for CH, NH, and CO bonds on both backbone and side chain shows that the L4 loop becomes essentially rigid after binding, but part of the L1 loop becomes slightly more flexible. Backbone dihedral angle cross-correlation results show that loop regions such as the L1 loop including residues Q25 and G26, the L2 loop including residue R61, and the L4 loop including residues L89–R91, are highly correlated compared to other regions in the monomer form. Analysis of the correlated motions at these residues, such as Q25 and R61, indicate two signal pathways. Transfer entropy calculations on the RBD monomer and dimer forms suggest that the binding process should be driven by the L4 loop and C-terminal. However, after binding, the L4 loop functions as the motion responder. The signal pathways in RBD were predicted based on a dynamical network analysis method using the pathways predicted from the dihedral angle cross-correlation calculations as input. It is found that the shortest pathways predicted from both inputs can overlap, but signal pathway 2 (from F90 to R61) is more dominant and overlaps all of the routes of pathway 1 (from F90 to P111). This project confirms the allosteric mechanism in signal transmission inside the RBD network, which was in part proposed in the previous experimental study. PMID:24901636

Proline Restricts Loop I Conformation of the High Affinity WW Domain from Human Nedd4-1 to a Ligand Binding-Competent Type I β-Turn.

PubMed

Schulte, Marianne; Panwalkar, Vineet; Freischem, Stefan; Willbold, Dieter; Dingley, Andrew J

2018-04-19

Sequence alignment of the four WW domains from human Nedd4-1 (neuronal precursor cell expressed developmentally down-regulated gene 4-1) reveals that the highest sequence diversity exists in loop I. Three residues in this type I β-turn interact with the PPxY motif of the human epithelial Na + channel (hENaC) subunits, indicating that peptide affinity is defined by the loop I sequence. The third WW domain (WW3*) has the highest ligand affinity and unlike the other three hNedd4-1 WW domains or other WW domains studied contains the highly statistically preferred proline at the ( i + 1) position found in β-turns. In this report, molecular dynamics simulations and experimental data were combined to characterize loop I stability and dynamics. Exchange of the proline to the equivalent residue in WW4 (Thr) results in the presence of a predominantly open seven residue Ω loop rather than the type I β-turn conformation for the wild-type apo-WW3*. In the presence of the ligand, the structure of the mutated loop I is locked into a type I β-turn. Thus, proline in loop I ensures a stable peptide binding-competent β-turn conformation, indicating that amino acid sequence modulates local flexibility to tune binding preferences and stability of dynamic interaction motifs.

Experimental test of an eco-evolutionary dynamic feedback loop between evolution and population density in the green peach aphid.

PubMed

Turcotte, Martin M; Reznick, David N; Daniel Hare, J

2013-05-01

An eco-evolutionary feedback loop is defined as the reciprocal impacts of ecology on evolutionary dynamics and evolution on ecological dynamics on contemporary timescales. We experimentally tested for an eco-evolutionary feedback loop in the green peach aphid, Myzus persicae, by manipulating initial densities and evolution. We found strong evidence that initial aphid density alters the rate and direction of evolution, as measured by changes in genotype frequencies through time. We also found that evolution of aphids within only 16 days, or approximately three generations, alters the rate of population growth and predicts density compared to nonevolving controls. The impact of evolution on population dynamics also depended on density. In one evolution treatment, evolution accelerated population growth by up to 10.3% at high initial density or reduced it by up to 6.4% at low initial density. The impact of evolution on population growth was as strong as or stronger than that caused by a threefold change in intraspecific density. We found that, taken together, ecological condition, here intraspecific density, alters evolutionary dynamics, which in turn alter concurrent population growth rate (ecological dynamics) in an eco-evolutionary feedback loop. Our results suggest that ignoring evolution in studies predicting population dynamics might lead us to over- or underestimate population density and that we cannot predict the evolutionary outcome within aphid populations without considering population size.

Impact of oxidation on protein therapeutics: Conformational dynamics of intact and oxidized acid-β-glucocerebrosidase at near-physiological pH

PubMed Central

Bobst, Cedric E; Thomas, John J; Salinas, Paul A; Savickas, Philip; Kaltashov, Igor A

2010-01-01

The solution dynamics of an enzyme acid-β-glucocerebrosidase (GCase) probed at a physiologically relevant (lysosomal) pH by hydrogen/deuterium exchange mass spectrometry (HDX-MS) reveals very uneven distribution of backbone amide protection across the polypeptide chain. Highly mobile segments are observed even within the catalytic cavity alongside highly protective segments, highlighting the importance of the balance between conformational stability and flexibility for enzymatic activity. Forced oxidation of GCase that resulted in a 40–60% reduction in in vitro biological activity affects the stability of some key structural elements within the catalytic site. These changes in dynamics occur on a longer time scale that is irrelevant for catalysis, effectively ruling out loss of structure in the catalytic site as a major factor contributing to the reduction of the catalytic activity. Oxidation also leads to noticeable destabilization of conformation in remote protein segments on a much larger scale, which is likely to increase the aggregation propensity of GCase and affect its bioavailability. Therefore, it appears that oxidation exerts its negative impact on the biological activity of GCase indirectly, primarily through accelerated aggregation and impaired trafficking. PMID:20945356

Crystal structure of the zymogen form of the group A Streptococcus virulence factor SpeB: an integrin-binding cysteine protease.

PubMed

Kagawa, T F; Cooney, J C; Baker, H M; McSweeney, S; Liu, M; Gubba, S; Musser, J M; Baker, E N

2000-02-29

Pathogenic bacteria secrete protein toxins that weaken or disable their host, and thereby act as virulence factors. We have determined the crystal structure of streptococcal pyrogenic exotoxin B (SpeB), a cysteine protease that is a major virulence factor of the human pathogen Streptococcus pyogenes and participates in invasive disease episodes, including necrotizing fasciitis. The structure, determined for the 40-kDa precursor form of SpeB at 1.6-A resolution, reveals that the protein is a distant homologue of the papain superfamily that includes the mammalian cathepsins B, K, L, and S. Despite negligible sequence identity, the protease portion has the canonical papain fold, albeit with major loop insertions and deletions. The catalytic site differs from most other cysteine proteases in that it lacks the Asn residue of the Cys-His-Asn triad. The prosegment has a unique fold and inactivation mechanism that involves displacement of the catalytically essential His residue by a loop inserted into the active site. The structure also reveals the surface location of an integrin-binding Arg-Gly-Asp (RGD) motif that is a feature unique to SpeB among cysteine proteases and is linked to the pathogenesis of the most invasive strains of S. pyogenes.

Structural and biochemical characterization of novel bacterial α-galactosidases belonging to glycoside hydrolase family 31.

PubMed

Miyazaki, Takatsugu; Ishizaki, Yuichi; Ichikawa, Megumi; Nishikawa, Atsushi; Tonozuka, Takashi

2015-07-01

Glycoside hydrolase family 31 (GH31) proteins have been reportedly identified as exo-α-glycosidases with activity for α-glucosides and α-xylosides. We focused on a GH31 subfamily, which contains proteins with low sequence identity (<24%) to the previously reported GH31 glycosidases and characterized two enzymes from Pedobacter heparinus and Pedobacter saltans. The enzymes unexpectedly exhibited α-galactosidase activity, but were not active on α-glucosides and α-xylosides. The crystal structures of one of the enzymes, PsGal31A, in unliganded form and in complexes with D-galactose or L-fucose and the catalytic nucleophile mutant in unliganded form and in complex with p-nitrophenyl-α-D-galactopyranoside, were determined at 1.85-2.30 Å (1 Å=0.1 nm) resolution. The overall structure of PsGal31A contains four domains and the catalytic domain adopts a (β/α)8-barrel fold that resembles the structures of other GH31 enzymes. Two catalytic aspartic acid residues are structurally conserved in the enzymes, whereas most residues forming the active site differ from those of GH31 α-glucosidases and α-xylosidases. PsGal31A forms a dimer via a unique loop that is not conserved in other reported GH31 enzymes; this loop is involved in its aglycone specificity and in binding L-fucose. Considering potential genes for α-L-fucosidases and carbohydrate-related proteins within the vicinity of Pedobacter Gal31, the identified Gal31 enzymes are likely to function in a novel sugar degradation system. This is the first report of α-galactosidases which belong to GH31 family. © 2015 Authors; published by Portland Press Limited.

Stopped-in-loop flow analysis system for successive determination of trace vanadium and iron in drinking water using their catalytic reactions.

PubMed

Ayala Quezada, Alejandro; Ohara, Keisuke; Ratanawimarnwong, Nuanlaor; Nacapricha, Duangjai; Murakami, Hiroya; Teshima, Norio; Sakai, Tadao

2015-11-01

An automated stopped-in-loop flow analysis (SILFA) system is proposed for the successive catalytic determination of vanadium and iron. The determination of vanadium was based on the p-anisidine oxidation by potassium bromate in the presence of Tiron as an activator to form a reddish dye, which has an absorption maximum at 510 nm. The selectivity of the vanadium determination was greatly improved by adding diphosphate as a masking agent of iron. For the iron determination, an iron-catalyzed oxidative reaction of p-anisidine by hydrogen peroxide with 1,10-phenanthroline as an activator to produce a reddish dye (510 nm) was employed. The SILFA system consisted of two peristaltic pumps, two six-port injection valves, a four-port selection valve, a heater device, a spectrophotometric detector and a data acquisition device. One six-port injection valve was used for the isolation of a mixed solution of standard/sample and reagent to promote each catalytic reaction, and another six-port injection valve was used for switching the reagent for vanadium or iron to achieve selective determination of each analyte. The above mentioned four-port selection valve was used to select standard solutions or sample. These three valves and the two peristaltic pumps were controlled by a built-in programmable logic controller in a touchscreen controller. The obtained results showed that the proposed SILFA monitoring system constituted an effective approach for the selective determination of vanadium and iron. The limits of detection, 0.052 and 0.55 µg L(-1), were obtained for vanadium and iron, respectively. The proposed system was successfully applied to drinking water samples without any preconcentration procedures. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of maneuver dynamics on drag polars of the X-29A forward-swept-wing aircraft with automatic wing camber control

NASA Technical Reports Server (NTRS)

Hicks, John W.; Moulton, Bryan J.

1988-01-01

The camber control loop of the X-29A FSW aircraft was designed to furnish the optimum L/D for trimmed, stabilized flight. A marked difference was noted between automatic wing camber control loop behavior in dynamic maneuvers and in stabilized flight conditions, which in turn affected subsonic aerodynamic performance. The degree of drag level increase was a direct function of maneuver rate. Attention is given to the aircraft flight drag polar effects of maneuver dynamics in light of wing camber control loop schedule. The effect of changing camber scheduling to better track the optimum automatic camber control L/D schedule is discussed.

Two AFC Loops For Low CNR And High Dynamics

NASA Technical Reports Server (NTRS)

Hinedi, Sami M.; Aguirre, Sergio

1992-01-01

Two alternative digital automatic-frequency-control (AFC) loops proposed to acquire (or reacquire) and track frequency of received carrier radio signal. Intended for use where carrier-to-noise ratios (CNR's) low and carrier frequency characterized by high Doppler shift and Doppler rate because of high relative speed and acceleration, respectively, between transmitter and receiver. Either AFC loops used in place of phase-locked loop. New loop concepts integrate ideas from classical spectrum-estimation, digital-phase-locked-loop, and Kalman-Filter theories.

Thermodynamics and kinetics of RNA tertiary structure formation in the junctionless hairpin ribozyme.

PubMed

White, Neil A; Hoogstraten, Charles G

2017-09-01

The hairpin ribozyme consists of two RNA internal loops that interact to form the catalytically active structure. This docking transition is a rare example of intermolecular formation of RNA tertiary structure without coupling to helix annealing. We have used temperature-dependent surface plasmon resonance (SPR) to characterize the thermodynamics and kinetics of RNA tertiary structure formation for the junctionless form of the ribozyme, in which loops A and B reside on separate molecules. We find docking to be strongly enthalpy-driven and to be accompanied by substantial activation barriers for association and dissociation, consistent with the structural reorganization of both internal loops upon complex formation. Comparisons with the parallel analysis of a ribozyme variant carrying a 2'-O-methyl modification at the self-cleavage site and with published data in other systems reveal a surprising diversity of thermodynamic signatures, emphasizing the delicate balance of contributions to the free energy of formation of RNA tertiary structure. Copyright © 2017 Elsevier B.V. All rights reserved.

Solution structure of a GAAA tetraloop receptor RNA.

PubMed Central

Butcher, S E; Dieckmann, T; Feigon, J

1997-01-01

The GAAA tetraloop receptor is an 11-nucleotide RNA sequence that participates in the tertiary folding of a variety of large catalytic RNAs by providing a specific binding site for GAAA tetraloops. Here we report the solution structure of the isolated tetraloop receptor as solved by multidimensional, heteronuclear magnetic resonance spectroscopy. The internal loop of the tetraloop receptor has three adenosines stacked in a cross-strand or zipper-like fashion. This arrangement produces a high degree of base stacking within the asymmetric internal loop without extrahelical bases or kinking the helix. Additional interactions within the internal loop include a U. U mismatch pair and a G.U wobble pair. A comparison with the crystal structure of the receptor RNA bound to its tetraloop shows that a conformational change has to occur upon tetraloop binding, which is in good agreement with previous biochemical data. A model for an alternative binding site within the receptor is proposed based on the NMR structure, phylogenetic data and previous crystallographic structures of tetraloop interactions. PMID:9405377

Probing the dynamics of restriction endonuclease NgoMIV-DNA interaction by single-molecule FRET.

PubMed

Tutkus, Marijonas; Sasnauskas, Giedrius; Rutkauskas, Danielis

2017-12-01

Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single-molecule Förster resonance energy transfer (smFRET) of surface-immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein-induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA-NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA-protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites ("slow" trajectories) or by semi-specific interactions of two DNA-bound NgoMIV tetramers ("fast" trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules. © 2017 Wiley Periodicals, Inc.

High-resolution reversible folding of hyperstable RNA tetraloops using molecular dynamics simulations

PubMed Central

Chen, Alan A.; García, Angel E.

2013-01-01

We report the de novo folding of three hyperstable RNA tetraloops to 1–3 Å rmsd from their experimentally determined structures using molecular dynamics simulations initialized in the unfolded state. RNA tetraloops with loop sequences UUCG, GCAA, or CUUG are hyperstable because of the formation of noncanonical loop-stabilizing interactions, and they are all faithfully reproduced to angstrom-level accuracy in replica exchange molecular dynamics simulations, including explicit solvent and ion molecules. This accuracy is accomplished using unique RNA parameters, in which biases that favor rigid, highly stacked conformations are corrected to accurately capture the inherent flexibility of ssRNA loops, accurate base stacking energetics, and purine syn-anti interconversions. In a departure from traditional quantum chemistrycentric approaches to force field optimization, our parameters are calibrated directly from thermodynamic and kinetic measurements of intra- and internucleotide structural transitions. The ability to recapitulate the signature noncanonical interactions of the three most abundant hyperstable stem loop motifs represents a significant milestone to the accurate prediction of RNA tertiary structure using unbiased all-atom molecular dynamics simulations. PMID:24043821

Phosphoenolpyruvate carboxylase: a new era of structural biology.

PubMed

Izui, Katsura; Matsumura, Hiroyoshi; Furumoto, Tsuyoshi; Kai, Yasushi

2004-01-01

There have been remarkable advances in our knowledge of this important enzyme in the last decade. This review focuses on three recent topics: the three-dimensional structure of the protein, molecular mechanisms of catalytic and regulatory functions, and the molecular cloning and characterization of PEPC kinases, which are Ser/Thr kinases involved specifically in regulatory phosphorylation of vascular plant PEPC. Analysis by X-ray crystallography and site-directed mutagenesis for E. coli and maize PEPC identified the catalytic site and allosteric effector binding sites, and revealed the functional importance of mobile loops. We present the reaction mechanism of PEPC in which we assign the roles of individual amino acid residues. We discuss the unique molecular property of PEPC kinase and its possible regulation at the post-translational level.

Study of the post-flare loops on 29 July 1973. I - Dynamics of the X-ray loops

NASA Technical Reports Server (NTRS)

Nolte, J. T.; Gerassimenko, M.; Krieger, A. S.; Petrasso, R. D.; Svestka, Z.

1979-01-01

We derive an empirical model of the X-ray emitting post-flare loops observed during the decay phase of the 29 July 1973 flare. We find that the loops are elliptical, with the brightest emitting region at the tops. We determine the height, velocity of growth, and ratio of height to width of the loops at times from 3 to 12 hr after the flare onset.

Review article: closed-loop systems in anesthesia: is there a potential for closed-loop fluid management and hemodynamic optimization?

PubMed

Rinehart, Joseph; Liu, Ngai; Alexander, Brenton; Cannesson, Maxime

2012-01-01

Closed-loop (automated) controllers are encountered in all aspects of modern life in applications ranging from air-conditioning to spaceflight. Although these systems are virtually ubiquitous, they are infrequently used in anesthesiology because of the complexity of physiologic systems and the difficulty in obtaining reliable and valid feedback data from the patient. Despite these challenges, closed-loop systems are being increasingly studied and improved for medical use. Two recent developments have made fluid administration a candidate for closed-loop control. First, the further description and development of dynamic predictors of fluid responsiveness provides a strong parameter for use as a control variable to guide fluid administration. Second, rapid advances in noninvasive monitoring of cardiac output and other hemodynamic variables make goal-directed therapy applicable for a wide range of patients in a variety of clinical care settings. In this article, we review the history of closed-loop controllers in clinical care, discuss the current understanding and limitations of the dynamic predictors of fluid responsiveness, and examine how these variables might be incorporated into a closed-loop fluid administration system.

Atomistic Simulations of the pH Induced Functional Rearrangement of Influenza Hemagglutinin

NASA Astrophysics Data System (ADS)

Lin, Xingcheng; Noel, Jeffrey; Wang, Qinghua; Ma, Jianpeng; Onuchic, Jose

Influenza hemagglutinin (HA), a surface glycoprotein responsible for the entry and replication of flu viruses in their host cells, functions by starting a dramatic conformational rearrangement, which leads to a fusion of the viral and endosomal membranes. It has been claimed that a loop-to-coiled-coil transition of the B-loop domain of HA drives the HA-induced membrane fusion. On the lack of dynamical details, however, the microscopic picture for this proposed ``spring-loaded'' movement is missing. To elaborate on the transition of the B-loop, we performed a set of unbiased all-atom molecular dynamics simulations of the full B-loop structure with the CHARMM36 force field. The complete free-energy profile constructed from our simulations reveals a slow transition rate for the B-loop that is incompatible with a downhill process. Additionally, our simulations indicate two potential sources of kinetic traps in the structural switch of the B-loop: Desolvation barriers and non-native secondary structure formation. The slow timescale of the B-loop transition also confirms our previous discovery from simulations using a coarse-grained structure-based model, which identified two competitive pathways both with a slow B-loop transition for HA to guide the membrane fusion.

Phase transitions in single macromolecules: Loop-stretch transition versus loop adsorption transition in end-grafted polymer chains

NASA Astrophysics Data System (ADS)

Zhang, Shuangshuang; Qi, Shuanhu; Klushin, Leonid I.; Skvortsov, Alexander M.; Yan, Dadong; Schmid, Friederike

2018-01-01

We use Brownian dynamics simulations and analytical theory to compare two prominent types of single molecule transitions. One is the adsorption transition of a loop (a chain with two ends bound to an attractive substrate) driven by an attraction parameter ɛ and the other is the loop-stretch transition in a chain with one end attached to a repulsive substrate, driven by an external end-force F applied to the free end. Specifically, we compare the behavior of the respective order parameters of the transitions, i.e., the mean number of surface contacts in the case of the adsorption transition and the mean position of the chain end in the case of the loop-stretch transition. Close to the transition points, both the static behavior and the dynamic behavior of chains with different length N are very well described by a scaling ansatz with the scaling parameters (ɛ - ɛ*)Nϕ (adsorption transition) and (F - F*)Nν (loop-stretch transition), respectively, where ϕ is the crossover exponent of the adsorption transition and ν is the Flory exponent. We show that both the loop-stretch and the loop adsorption transitions provide an exceptional opportunity to construct explicit analytical expressions for the crossover functions which perfectly describe all simulation results on static properties in the finite-size scaling regime. Explicit crossover functions are based on the ansatz for the analytical form of the order parameter distributions at the respective transition points. In contrast to the close similarity in equilibrium static behavior, the dynamic relaxation at the two transitions shows qualitative differences, especially in the strongly ordered regimes. This is attributed to the fact that the surface contact dynamics in a strongly adsorbed chain is governed by local processes, whereas the end height relaxation of a strongly stretched chain involves the full spectrum of Rouse modes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brandao, T.; Robinson, H; Johnson, S

Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presencemore » of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 Angstroms structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO]2 core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.« less

GROWING TRANSVERSE OSCILLATIONS OF A MULTISTRANDED LOOP OBSERVED BY SDO/AIA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Tongjiang; Ofman, Leon; Su, Yang

The first evidence of transverse oscillations of a multistranded loop with growing amplitudes and internal coupling observed by the Atmospheric Imaging Assembly on board the Solar Dynamics Observatory is presented. The loop oscillation event occurred on 2011 March 8, triggered by a coronal mass ejection (CME). The multiwavelength analysis reveals the presence of multithermal strands in the oscillating loop, whose dynamic behaviors are temperature-dependent, showing differences in their oscillation amplitudes, phases, and emission evolution. The physical parameters of growing oscillations of two strands in 171 A are measured and the three-dimensional loop geometry is determined using STEREO-A/EUVI data. These strandsmore » have very similar frequencies, and between two 193 A strands a quarter-period phase delay sets up. These features suggest the coupling between kink oscillations of neighboring strands and the interpretation by the collective kink mode as predicted by some models. However, the temperature dependence of the multistranded loop oscillations was not studied previously and needs further investigation. The transverse loop oscillations are associated with intensity and loop width variations. We suggest that the amplitude-growing kink oscillations may be a result of continuous non-periodic driving by magnetic deformation of the CME, which deposits energy into the loop system at a rate faster than its loss.« less

Dressed Wilson loops as dual condensates in response to magnetic and electric fields

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bruckmann, Falk; Endroedi, Gergely

2011-10-01

We introduce dressed Wilson loops as a novel confinement observable. It consists of closed planar loops of arbitrary geometry but fixed area, and its expectation values decay with the latter. The construction of dressed Wilson loops is based on chiral condensates in response to magnetic and electric fields, thus linking different physical concepts. We present results for generalized condensates and dressed Wilson loops on dynamical lattice configurations and confirm the agreement with conventional Wilson loops in the limit of large probe mass. We comment on the renormalization of dressed Wilson loops.

Ferroelectrics: A pathway to switchable surface chemistry and catalysis

NASA Astrophysics Data System (ADS)

Kakekhani, Arvin; Ismail-Beigi, Sohrab; Altman, Eric I.

2016-08-01

It has been known for more than six decades that ferroelectricity can affect a material's surface physics and chemistry thereby potentially enhancing its catalytic properties. Ferroelectrics are a class of materials with a switchable electrical polarization that can affect surface stoichiometry and electronic structure and thus adsorption energies and modes; e.g., molecular versus dissociative. Therefore, ferroelectrics may be utilized to achieve switchable surface chemistry whereby surface properties are not fixed but can be dynamically controlled by, for example, applying an external electric field or modulating the temperature. Several important examples of applications of ferroelectric and polar materials in photocatalysis and heterogeneous catalysis are discussed. In photocatalysis, the polarization direction can control band bending at water/ferroelectric and ferroelectric/semiconductor interfaces, thereby facilitating charge separation and transfer to the electrolyte and enhancing photocatalytic activity. For gas-surface interactions, available results suggest that using ferroelectrics to support catalytically active transition metals and oxides is another way to enhance catalytic activity. Finally, the possibility of incorporating ferroelectric switching into the catalytic cycle itself is described. In this scenario, a dynamic collaboration of two polarization states can be used to drive reactions that have been historically challenging to achieve on surfaces with fixed chemical properties (e.g., direct NOx decomposition and the selective partial oxidation of methane). These predictions show that dynamic modulation of the polarization can help overcome some of the fundamental limitations on catalytic activity imposed by the Sabatier principle.

Conformation-selective inhibitors reveal differences in the activation and phosphate-binding loops of the tyrosine kinases Abl and Src

PubMed Central

Hari, Sanjay B.; Perera, B. Gayani K.; Ranjitkar, Pratistha; Seeliger, Markus A.; Maly, Dustin J.

2013-01-01

Over the last decade, an increasingly diverse array of potent and selective inhibitors that target the ATP-binding sites of protein kinases have been developed. Many of these inhibitors, like the clinically approved drug imatinib (Gleevec), stabilize a specific catalytically inactive ATP-binding site conformation of their kinases targets. Imatinib is notable in that it is highly selective for its kinase target, Abl, over other closely-related tyrosine kinases, like Src. In addition, imatinib is highly sensitive to the phosphorylation state of Abl's activation loop, which is believed to be a general characteristic of all inhibitors that stabilize a similar inactive ATP-binding site conformation. In this report, we perform a systematic analysis of a diverse series of ATP-competitive inhibitors that stabilize a similar inactive ATP-binding site conformation as imatinib with the tyrosine kinases Src and Abl. In contrast to imatinib, many of these inhibitors have very similar potencies against Src and Abl. Furthermore, only a subset of this class of inhibitors is sensitive to the phosphorylation state of the activation loop of these kinases. In attempting to explain this observation, we have uncovered an unexpected correlation between Abl's activation loop and another flexible active site feature, called the phosphate-binding loop (p-loop). These studies shed light on how imatinib is able to obtain its high target selectivity and reveal how the conformational preference of flexible active site regions can vary between closely related kinases. PMID:24106839

Behaviour of fractional loop delay zero crossing digital phase locked loop (FR-ZCDPLL)

NASA Astrophysics Data System (ADS)

Nasir, Qassim

2018-01-01

This article analyses the performance of the first-order zero crossing digital phase locked loops (FR-ZCDPLL) when fractional loop delay is added to loop. The non-linear dynamics of the loop is presented, analysed and examined through bifurcation behaviour. Numerical simulation of the loop is conducted to proof the mathematical analysis of the loop operation. The results of the loop simulation show that the proposed FR-ZCDPLL has enhanced the performance compared to the conventional zero crossing DPLL in terms of wider lock range, captured range and stable operation region. In addition, extensive experimental simulation was conducted to find the optimum loop parameters for different loop environmental conditions. The addition of the fractional loop delay network in the conventional loop also reduces the phase jitter and its variance especially when the signal-to-noise ratio is low.

Multi-thermal observations of newly formed loops in a dynamic flare

NASA Technical Reports Server (NTRS)

Svestka, Zdenek F.; Fontenla, Juan M.; Machado, Marcos E.; Martin, Sara F.; Neidig, Donald F.

1987-01-01

The dynamic flare of November 6, 1980 (max at about 15:26 UT) developed a rich system of growing loops which could be followed in H-alpha for 1.5 hr. Throughout the flare, these loops, near the limb, were seen in emission against the disk. Theoretical computations of deviations from LTE populations for a hydrogen atom reveal that this requires electron densities in the loops close to, or in excess of 10 to the 12th/cu cm. From measured widths of higher Balmer lines the density at the tops of the loops was found to be 4 x 10 to the 12th/cu cm if no nonthermal motions were present, or 5 x 10 to the 11th/cu cm for a turbulent velocity of about 12 km/s. It is now general knowledge that flare loops are initially observed in X-rays and become visible in H-alpha only after cooling. For such a high density, a loop would cool through radiation from 10 to the 7th to 10 to the 4th K within a few minutes so that the dense H-alpha loops should have heights very close to the heights of the X-ray loops. This, however, contradicts the observations obtained by the HXIS and FCS instruments on board SMM which show the X-ray loops at much higher altitudes than the loops in H-alpha. Therefore, it is suggested that the density must have been significantly lower when the loops were formed, and that the flare loops were apparently both shrinking and increasing in density while cooling.

Radiation from quantum weakly dynamical horizons in loop quantum gravity.

PubMed

Pranzetti, Daniele

2012-07-06

We provide a statistical mechanical analysis of quantum horizons near equilibrium in the grand canonical ensemble. By matching the description of the nonequilibrium phase in terms of weakly dynamical horizons with a local statistical framework, we implement loop quantum gravity dynamics near the boundary. The resulting radiation process provides a quantum gravity description of the horizon evaporation. For large black holes, the spectrum we derive presents a discrete structure which could be potentially observable.

Hidden attractors in dynamical models of phase-locked loop circuits: Limitations of simulation in MATLAB and SPICE

NASA Astrophysics Data System (ADS)

Kuznetsov, N. V.; Leonov, G. A.; Yuldashev, M. V.; Yuldashev, R. V.

2017-10-01

During recent years it has been shown that hidden oscillations, whose basin of attraction does not overlap with small neighborhoods of equilibria, may significantly complicate simulation of dynamical models, lead to unreliable results and wrong conclusions, and cause serious damage in drilling systems, aircrafts control systems, electromechanical systems, and other applications. This article provides a survey of various phase-locked loop based circuits (used in satellite navigation systems, optical, and digital communication), where such difficulties take place in MATLAB and SPICE. Considered examples can be used for testing other phase-locked loop based circuits and simulation tools, and motivate the development and application of rigorous analytical methods for the global analysis of phase-locked loop based circuits.

Hydrogen bonds between the alpha and beta subunits of the F1-ATPase allow communication between the catalytic site and the interface of the beta catch loop and the gamma subunit.

PubMed

Boltz, Kathryn W; Frasch, Wayne D

2006-09-19

F(1)-ATPase mutations in Escherichia coli that changed the strength of hydrogen bonds between the alpha and beta subunits in a location that links the catalytic site to the interface between the beta catch loop and the gamma subunit were examined. Loss of the ability to form the hydrogen bonds involving alphaS337, betaD301, and alphaD335 lowered the k(cat) of ATPase and decreased its susceptibility to Mg(2+)-ADP-AlF(n) inhibition, while mutations that maintain or strengthen these bonds increased the susceptibility to Mg(2+)-ADP-AlF(n) inhibition and lowered the k(cat) of ATPase. These data suggest that hydrogen bonds connecting alphaS337 to betaD301 and betaR323 and connecting alphaD335 to alphaS337 are important to transition state stabilization and catalytic function that may result from the proper alignment of catalytic site residues betaR182 and alphaR376 through the VISIT sequence (alpha344-348). Mutations betaD301E, betaR323K, and alphaR282Q changed the rate-limiting step of the reaction as determined by an isokinetic plot. Hydrophobic mutations of betaR323 decreased the susceptibility to Mg(2+)-ADP-AlF(n)() inhibition and lowered the number of interactions required in the rate-limiting step yet did not affect the k(cat) of ATPase, suggesting that betaR323 is important to transition state formation. The decreased rate of ATP synthase-dependent growth and decreased level of lactate-dependent quenching observed with alphaD335, betaD301, and alphaE283 mutations suggest that these residues may be important to the formation of an alternative set of hydrogen bonds at the interface of the alpha and beta subunits that permits the release of intersubunit bonds upon the binding of ATP, allowing gamma rotation in the escapement mechanism.

Replacement of Val3 in human thymidylate synthase affects its kinetic properties and intracellular stability .

PubMed

Huang, Xiao; Gibson, Lydia M; Bell, Brittnaie J; Lovelace, Leslie L; Peña, Maria Marjorette O; Berger, Franklin G; Berger, Sondra H; Lebioda, Lukasz

2010-03-23

Human and other mammalian thymidylate synthase (TS) enzymes have an N-terminal extension of approximately 27 amino acids that is not present in bacterial TSs. The extension, which is disordered in all reported crystal structures of TSs, has been considered to play a primary role in protein turnover but not in catalytic activity. In mammalian cells, the variant V3A has a half-life similar to that of wild-type human TS (wt hTS) while V3T is much more stable; V3L, V3F, and V3Y have half-lives approximately half of that for wt hTS. Catalytic turnover rates for most Val3 mutants are only slightly diminished, as expected. However, two mutants, V3L and V3F, have strongly compromised dUMP binding, with K(m,app) values increased by factors of 47 and 58, respectively. For V3L, this observation can be explained by stabilization of the inactive conformation of the loop of residues 181-197, which prevents substrate binding. In the crystal structure of V3L, electron density corresponding to a leucine residue is present in a position that stabilizes the loop of residues 181-197 in the inactive conformation. Since this density is not observed in other mutants and all other leucine residues are ordered in this structure, it is likely that this density represents Leu3. In the crystal structure of a V3F.FdUMP binary complex, the nucleotide is bound in an alternative mode to that proposed for the catalytic complex, indicating that the high K(m,app) value is caused not by stabilization of the inactive conformer but by substrate binding in a nonproductive, inhibitory site. These observations show that the N-terminal extension affects the conformational state of the hTS catalytic region. Each of the mechanisms leading to the high K(m,app) values can be exploited to facilitate design of compounds acting as allosteric inhibitors of hTS.

Kinetic and Binding Analysis of the Catalytic Involvement of Ribose Moieties of a trans-Acting δ Ribozyme*

PubMed Central

Fiola, Karine; Perreault, Jean-Pierre

2010-01-01

We have identified ribose 2′-hydroxyl groups (2′-OHs) that are critical for the activity of a trans-cleaving δ ribozyme derived from the antigenomic strand of the hepatitis δ virus. Initially, an RNA-DNA mixed ribozyme composed of 26 deoxyribo- (specifically the nucleotides forming the P2 stem and the P4 stem-loop) and 31 ribonucleotides (those forming the catalytic center) was engineered. This mixed ribozyme catalyzed the cleavage of a small substrate with kinetic parameters virtually identical to those of the all-RNA ribozyme. The further substitution of deoxyribose for ribose residues permitted us to investigate the contribution of all 2′-OHs to catalysis. Determination of the kinetic parameters for the cleavage reaction of the resulting ribozymes revealed (i) 10 2′-OH groups appear to be important in supporting the formation of several hydrogen bonds within the catalytic core, (ii) none of the important 2′-OHs seem to coordinate a magnesium cation, and (iii) 1 of the tested RNA-DNA mixed polymers appeared to stabilize the ribozyme-substrate transition-state complex, resulting in an improvement over the all-RNA counterpart. The contribution of the 2′-OHs to the catalytic mechanism is discussed, and differences with the crystal structure of a genomic δ self-cleaved product are explained. Clearly, the 2′-OHs are essential components of the network of interactions involved in the formation of the catalytic center of the δ ribozyme. PMID:12015324

Studying the role of protein dynamics in an SN2 enzyme reaction using free-energy surfaces and solvent coordinates

NASA Astrophysics Data System (ADS)

García-Meseguer, Rafael; Martí, Sergio; Ruiz-Pernía, J. Javier; Moliner, Vicent; Tuñón, Iñaki

2013-07-01

Conformational changes are known to be able to drive an enzyme through its catalytic cycle, allowing, for example, substrate binding or product release. However, the influence of protein motions on the chemical step is a controversial issue. One proposal is that the simple equilibrium fluctuations incorporated into transition-state theory are insufficient to account for the catalytic effect of enzymes and that protein motions should be treated dynamically. Here, we propose the use of free-energy surfaces, obtained as a function of both a chemical coordinate and an environmental coordinate, as an efficient way to elucidate the role of protein structure and motions during the reaction. We show that the structure of the protein provides an adequate environment for the progress of the reaction, although a certain degree of flexibility is needed to attain the full catalytic effect. However, these motions do not introduce significant dynamical corrections to the rate constant and can be described as equilibrium fluctuations.

System Dynamics

NASA Astrophysics Data System (ADS)

Morecroft, John

System dynamics is an approach for thinking about and simulating situations and organisations of all kinds and sizes by visualising how the elements fit together, interact and change over time. This chapter, written by John Morecroft, describes modern system dynamics which retains the fundamentals developed in the 1950s by Jay W. Forrester of the MIT Sloan School of Management. It looks at feedback loops and time delays that affect system behaviour in a non-linear way, and illustrates how dynamic behaviour depends upon feedback loop structures. It also recognises improvements as part of the ongoing process of managing a situation in order to achieve goals. Significantly it recognises the importance of context, and practitioner skills. Feedback systems thinking views problems and solutions as being intertwined. The main concepts and tools: feedback structure and behaviour, causal loop diagrams, dynamics, are practically illustrated in a wide variety of contexts from a hot water shower through to a symphony orchestra and the practical application of the approach is described through several real examples of its use for strategic planning and evaluation.

Dynamic response of a fiber-optic ring resonator: Analysis with influences of light-source parameters

NASA Astrophysics Data System (ADS)

Seraji, Faramarz E.

2009-03-01

In practice, dynamic behavior of fiber-optic ring resonator (FORR) appears as a detrimental factor to influence the transmission response of the FORR. This paper presents dynamic response analysis of the FORR by considering phase modulation of the FORR loop and sinewave modulation of input signal applied to the FORR from a laser diode. The analysis investigates the influences of modulation frequency and amplitude modulation index of laser diode, loop delay time of the FORR, phase angle between FM and AM response of laser diode, and laser diode line-width on dynamic response of the FORR. The analysis shows that the transient response of the FORR strongly depends on the product of modulation frequency and loop delay time, coupling and transmission coefficients of the FORR. The analyses presented here may have applications in optical systems employing an FORR with a laser diode source.

Thrust Control Loop Design for Electric-Powered UAV

NASA Astrophysics Data System (ADS)

Byun, Heejae; Park, Sanghyuk

2018-04-01

This paper describes a process of designing a thrust control loop for an electric-powered fixed-wing unmanned aerial vehicle equipped with a propeller and a motor. In particular, the modeling method of the thrust system for thrust control is described in detail and the propeller thrust and torque force are modeled using blade element theory. A relation between current and torque of the motor is obtained using an experimental setup. Another relation between current, voltage and angular velocity is also obtained. The electric motor and the propeller dynamics are combined to model the thrust dynamics. The associated trim and linearization equations are derived. Then, the thrust dynamics are coupled with the flight dynamics to allow a proper design for the thrust loop in the flight control. The proposed method is validated by an application to a testbed UAV through simulations and flight test.

Dual-loop model of the human controller

NASA Technical Reports Server (NTRS)

Hess, R. A.

1978-01-01

A dual-loop model of the human controller in single-axis compensatory tracking tasks is introduced. This model possesses an inner-loop closure that involves feeding back that portion of controlled element output rate that is due to control activity. A novel feature of the model is the explicit appearance of the human's internal representation of the manipulator-controlled element dynamics in the inner loop. The sensor inputs to the human controller are assumed to be system error and control force. The former can be sensed via visual, aural, or tactile displays, whereas the latter is assumed to be sensed in kinesthetic fashion. A set of general adaptive characteristics for the model is hypothesized, including a method for selecting simplified internal models of the manipulator-controlled element dynamics. It is demonstrated that the model can produce controller describing functions that closely approximate those measured in four laboratory tracking tasks in which the controlled element dynamics vary considerably in terms of ease of control. An empirically derived expression for the normalized injected error remnant spectrum is introduced.

Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis.

PubMed

Han, S; Arvai, A S; Clancy, S B; Tainer, J A

2001-01-05

Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 A resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding beta-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation. Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins. Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an alpha-helix, alpha3. Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join beta5 and beta6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family. Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket. This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties. Turn 2 centrally anchors the catalytic Glu214 within the ARTT motif, and furthermore distinguishes the C3 toxin class by a conserved turn 2 Gln and the VIP2 binary toxin class by a conserved turn 2 Glu for appropriate target side-chain hydrogen-bonding recognition. Taken together, these structural results provide a molecular basis for understanding the coupled activity and recognition specificity for C3 and for the newly defined ARTT toxin family, which acts in the depolymerization of the actin cytoskeleton. This beta5 to beta6 region of the toxin fold represents an experimentally testable and potentially general recognition motif region for other ADP-ribosylating toxins that have a similar beta-structure framework. Copyright 2001 Academic Press.

Structural and mechanistic insights into Mps1 kinase activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Yang, Yuting; Gao, Yuefeng

2010-11-05

Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signalling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7-{angstrom}-resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the {alpha}C helix of the N-terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation.more » Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices {alpha}EF and {alpha}F that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.« less

Spatial operator algebra framework for multibody system dynamics

NASA Technical Reports Server (NTRS)

Rodriguez, G.; Jain, Abhinandan; Kreutz, K.

1989-01-01

The Spatial Operator Algebra framework for the dynamics of general multibody systems is described. The use of a spatial operator-based methodology permits the formulation of the dynamical equations of motion of multibody systems in a concise and systematic way. The dynamical equations of progressively more complex grid multibody systems are developed in an evolutionary manner beginning with a serial chain system, followed by a tree topology system and finally, systems with arbitrary closed loops. Operator factorizations and identities are used to develop novel recursive algorithms for the forward dynamics of systems with closed loops. Extensions required to deal with flexible elements are also discussed.

Spatial Operator Algebra for multibody system dynamics

NASA Technical Reports Server (NTRS)

Rodriguez, G.; Jain, A.; Kreutz-Delgado, K.

1992-01-01

The Spatial Operator Algebra framework for the dynamics of general multibody systems is described. The use of a spatial operator-based methodology permits the formulation of the dynamical equations of motion of multibody systems in a concise and systematic way. The dynamical equations of progressively more complex grid multibody systems are developed in an evolutionary manner beginning with a serial chain system, followed by a tree topology system and finally, systems with arbitrary closed loops. Operator factorizations and identities are used to develop novel recursive algorithms for the forward dynamics of systems with closed loops. Extensions required to deal with flexible elements are also discussed.

Super energy saver heat pump with dynamic hybrid phase change material

DOEpatents

Ally, Moonis Raza [Oak Ridge, TN; Tomlinson, John Jager [Knoxville, TN; Rice, Clifford Keith [Clinton, TN

2010-07-20

A heat pump has a refrigerant loop, a compressor in fluid communication with the refrigerant loop, at least one indoor heat exchanger in fluid communication with the refrigerant loop, and at least one outdoor heat exchanger in fluid communication with the refrigerant loop. The at least one outdoor heat exchanger has a phase change material in thermal communication with the refrigerant loop and in fluid communication with an outdoor environment. Other systems, devices, and methods are described.

Shortening a loop can increase protein native state entropy.

PubMed

Gavrilov, Yulian; Dagan, Shlomi; Levy, Yaakov

2015-12-01

Protein loops are essential structural elements that influence not only function but also protein stability and folding rates. It was recently reported that shortening a loop in the AcP protein may increase its native state conformational entropy. This effect on the entropy of the folded state can be much larger than the lower entropic penalty of ordering a shorter loop upon folding, and can therefore result in a more pronounced stabilization than predicted by polymer model for loop closure entropy. In this study, which aims at generalizing the effect of loop length shortening on native state dynamics, we use all-atom molecular dynamics simulations to study how gradual shortening a very long or solvent-exposed loop region in four different proteins can affect their stability. For two proteins, AcP and Ubc7, we show an increase in native state entropy in addition to the known effect of the loop length on the unfolded state entropy. However, for two permutants of SH3 domain, shortening a loop results only with the expected change in the entropy of the unfolded state, which nicely reproduces the observed experimental stabilization. Here, we show that an increase in the native state entropy following loop shortening is not unique to the AcP protein, yet nor is it a general rule that applies to all proteins following the truncation of any loop. This modification of the loop length on the folded state and on the unfolded state may result with a greater effect on protein stability. © 2015 Wiley Periodicals, Inc.

The Sugar Model: Catalytic Flow Reactor Dynamics of Pyruvaldehyde Synthesis from Triose Catalyzed by Poly-L-Lysine Contained in a Dialyzer

NASA Technical Reports Server (NTRS)

Weber, Arthur L.; DeVincenzi, Donald (Technical Monitor)

2000-01-01

The formation of pyruvaldehyde from triose sugars was catalyzed by poly-L-lysine contained in a small dialyzer (100 MWCO) suspended in a much larger triose substrate reservoir. The polylysine confined in the dialyzer functioned as a catalytic flow reactor that constantly brought in triose from the substrate reservoir by diffusion to offset the drop in triose concentration within the reactor caused by its conversion to pyruvaldehyde. A 400 mM solution of poly-L-lysine contained in a 0.35 ml dialyzer placed in a 120 ml solution of triose substrate (pH 5.5, 40 C) generated pyruvaldehyde 11 -times faster than an a control reaction without the catalytic dialyzer. However, since the catalytic dialyzer's volume was 343-times smaller than the control reaction, the synthetic intensity (rate/volume) of pyruvaldehyde synthesis within the catalytic dialyzer was 3400-times greater than that of the control reaction and substrate solution. A similar result was obtained using a dialyzer with a 500 MWCO value. Acting as a catalytic flow reactor the polylysine catalytic dialyzer synthesized about 3.5 molecules of pyruvaldehyde per lysine residue in 7 days -- an amount of triose equal to twice the weight of the catalyst. At 7 days the catalytic activity of polylysine was 16% of its initial value, a result indicating catalyst-poisoning caused by reaction of pyruvaldehyde with the e-amino groups of polylysine. The dialyzer method of catalyst containment was selected it provides a simple, flexible, and easily manipulated experimental system for studying the dynamics and evolutionary development of confined autocatalytic processes related to the origin of life under anaerobic conditions.

Experimental Observation of Classical Dynamical Monodromy

NASA Astrophysics Data System (ADS)

Nerem, M. P.; Salmon, D.; Aubin, S.; Delos, J. B.

2018-03-01

A Hamiltonian system is said to have nontrivial monodromy if its fundamental action-angle loops do not return to their initial topological state at the end of a closed circuit in angular momentum-energy space. This process has been predicted to have consequences which can be seen in dynamical systems, called dynamical monodromy. Using an apparatus consisting of a spherical pendulum subject to magnetic potentials and torques, we observe nontrivial monodromy by the associated topological change in the evolution of a loop of trajectories.

Dynamic formation and magnetic support of loop or arcade prominences

NASA Technical Reports Server (NTRS)

Vanhoven, Gerard; Mok, Y.; Drake, J. F.

1992-01-01

The results of model dynamic simulations of the formation and support of a narrow prominence at the apex of a coronal magnetic loop or arcade are described. The condensation process proceeds via an initial radiative cooling and pressure drop, and a secondary siphon flow from the dense chromospheric ends. The antibuoyancy effect as the prominence forms causes a bending of a confining magnetic field, which propagates toward the semirigid ends of the magnetic loop. Thus, a wide magnetic 'hammock' or well (of a normal polarity Kippenhahn-Schlueter type) is formed, which supports the prominence at or near the field apex.

Influence of the feedback loops in the trp operon of B. subtilis on the system dynamic response and noise amplitude.

PubMed

Zamora-Chimal, Criseida; Santillán, Moisés; Rodríguez-González, Jesús

2012-10-07

In this paper we introduce a mathematical model for the tryptophan operon regulatory pathway in Bacillus subtilis. This model considers the transcription-attenuation, and the enzyme-inhibition regulatory mechanisms. Special attention is paid to the estimation of all the model parameters from reported experimental data. With the aid of this model we investigate, from a mathematical-modeling point of view, whether the existing multiplicity of regulatory feedback loops is advantageous in some sense, regarding the dynamic response and the biochemical noise in the system. The tryptophan operon dynamic behavior is studied by means of deterministic numeric simulations, while the biochemical noise is analyzed with the aid of stochastic simulations. The model feasibility is tested comparing its stochastic and deterministic results with experimental reports. Our results for the wildtype and for a couple of mutant bacterial strains suggest that the enzyme-inhibition feedback loop, dynamically accelerates the operon response, and plays a major role in the reduction of biochemical noise. Also, the transcription-attenuation feedback loop makes the trp operon sensitive to changes in the endogenous tryptophan level, and increases the amplitude of the biochemical noise. Copyright © 2012 Elsevier Ltd. All rights reserved.

Correlating structural dynamics and catalytic activity of AgAu nanoparticles with ultrafast spectroscopy and all-atom molecular dynamics simulations.

PubMed

Ferbonink, G F; Rodrigues, T S; Dos Santos, D P; Camargo, P H C; Albuquerque, R Q; Nome, R A

2018-05-29

In this study, we investigated hollow AgAu nanoparticles with the goal of improving our understanding of the composition-dependent catalytic activity of these nanoparticles. AgAu nanoparticles were synthesized via the galvanic replacement method with controlled size and nanoparticle compositions. We studied extinction spectra with UV-Vis spectroscopy and simulations based on Mie theory and the boundary element method, and ultrafast spectroscopy measurements to characterize decay constants and the overall energy transfer dynamics as a function of AgAu composition. Electron-phonon coupling times for each composition were obtained from pump-power dependent pump-probe transients. These spectroscopic studies showed how nanoscale surface segregation, hollow interiors and porosity affect the surface plasmon resonance wavelength and fundamental electron-phonon coupling times. Analysis of the spectroscopic data was used to correlate electron-phonon coupling times to AgAu composition, and thus to surface segregation and catalytic activity. We have performed all-atom molecular dynamics simulations of model hollow AgAu core-shell nanoparticles to characterize nanoparticle stability and equilibrium structures, besides providing atomic level views of nanoparticle surface segregation. Overall, the basic atomistic and electron-lattice dynamics of core-shell AgAu nanoparticles characterized here thus aid the mechanistic understanding and performance optimization of AgAu nanoparticle catalysts.

Dynameomics: data-driven methods and models for utilizing large-scale protein structure repositories for improving fragment-based loop prediction.

PubMed

Rysavy, Steven J; Beck, David A C; Daggett, Valerie

2014-11-01

Protein function is intimately linked to protein structure and dynamics yet experimentally determined structures frequently omit regions within a protein due to indeterminate data, which is often due protein dynamics. We propose that atomistic molecular dynamics simulations provide a diverse sampling of biologically relevant structures for these missing segments (and beyond) to improve structural modeling and structure prediction. Here we make use of the Dynameomics data warehouse, which contains simulations of representatives of essentially all known protein folds. We developed novel computational methods to efficiently identify, rank and retrieve small peptide structures, or fragments, from this database. We also created a novel data model to analyze and compare large repositories of structural data, such as contained within the Protein Data Bank and the Dynameomics data warehouse. Our evaluation compares these structural repositories for improving loop predictions and analyzes the utility of our methods and models. Using a standard set of loop structures, containing 510 loops, 30 for each loop length from 4 to 20 residues, we find that the inclusion of Dynameomics structures in fragment-based methods improves the quality of the loop predictions without being dependent on sequence homology. Depending on loop length, ∼ 25-75% of the best predictions came from the Dynameomics set, resulting in lower main chain root-mean-square deviations for all fragment lengths using the combined fragment library. We also provide specific cases where Dynameomics fragments provide better predictions for NMR loop structures than fragments from crystal structures. Online access to these fragment libraries is available at http://www.dynameomics.org/fragments. © 2014 The Protein Society.

Dynameomics: Data-driven methods and models for utilizing large-scale protein structure repositories for improving fragment-based loop prediction

PubMed Central

Rysavy, Steven J; Beck, David AC; Daggett, Valerie

2014-01-01

Protein function is intimately linked to protein structure and dynamics yet experimentally determined structures frequently omit regions within a protein due to indeterminate data, which is often due protein dynamics. We propose that atomistic molecular dynamics simulations provide a diverse sampling of biologically relevant structures for these missing segments (and beyond) to improve structural modeling and structure prediction. Here we make use of the Dynameomics data warehouse, which contains simulations of representatives of essentially all known protein folds. We developed novel computational methods to efficiently identify, rank and retrieve small peptide structures, or fragments, from this database. We also created a novel data model to analyze and compare large repositories of structural data, such as contained within the Protein Data Bank and the Dynameomics data warehouse. Our evaluation compares these structural repositories for improving loop predictions and analyzes the utility of our methods and models. Using a standard set of loop structures, containing 510 loops, 30 for each loop length from 4 to 20 residues, we find that the inclusion of Dynameomics structures in fragment-based methods improves the quality of the loop predictions without being dependent on sequence homology. Depending on loop length, ∼25–75% of the best predictions came from the Dynameomics set, resulting in lower main chain root-mean-square deviations for all fragment lengths using the combined fragment library. We also provide specific cases where Dynameomics fragments provide better predictions for NMR loop structures than fragments from crystal structures. Online access to these fragment libraries is available at http://www.dynameomics.org/fragments. PMID:25142412

Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications.

PubMed Central

Williams, D M; Pieken, W A; Eckstein, F

1992-01-01

The importance of the 2'-hydroxyl group of several guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. Five ribozymes in which single guanosine residues were substituted with 2'-amino-, 2'-fluoro-, or 2'-deoxyguanosine were chemically synthesized. The comparison of the catalytic activity of the three 2' modifications at a specific position allows conclusions about the functional role of the parent 2'-hydroxyl group. Substitutions of nonconserved nucleotides within the ribozyme caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, when either of the guanosines within the single-stranded loop between stem I and stem II of the ribozyme was replaced by 2'-deoxyguanosine or 2'-fluoro-2'-deoxyguanosine, the catalytic activities of the resulting ribozymes were reduced by factors of at least 150. The catalytic activities of the corresponding ribozymes containing 2'-amino-2'-deoxyguanosine substitutions at these positions, however, were both reduced by factors of 15. These effects resulted from decreases in the respective kcat values, whereas variations in the Km values were comparatively small. A different pattern of reactivity of the three 2' modifications was observed at the guanosine immediately 3' to stem II of the ribozyme. Whereas both 2'-deoxyguanosine and 2'-amino-2'-deoxyguanosine at this position showed catalytic activity similar to that of the unmodified ribozyme, the activity of the corresponding 2'-fluoro-2'-deoxyguanosine-containing ribozyme was reduced by a factor of 15. The implications of these substitution-specific reactivities on the functional role of the native 2'-hydroxyl groups are discussed. Images PMID:1736306

Instant Integrated Ultradeep Quantitative-structural Membrane Proteomics Discovered Post-translational Modification Signatures for Human Cys-loop Receptor Subunit Bias*

PubMed Central

Zhang, Xi

2016-01-01

Neurotransmitter ligand-gated ion channels (LGICs) are widespread and pivotal in brain functions. Unveiling their structure-function mechanisms is crucial to drive drug discovery, and demands robust proteomic quantitation of expression, post-translational modifications (PTMs) and dynamic structures. Yet unbiased digestion of these modified transmembrane proteins—at high efficiency and peptide reproducibility—poses the obstacle. Targeting both enzyme-substrate contacts and PTMs for peptide formation and detection, we devised flow-and-detergent-facilitated protease and de-PTM digestions for deep sequencing (FDD) method that combined omni-compatible detergent, tandem immobilized protease/PNGase columns, and Cys-selective reduction/alkylation, to achieve streamlined ultradeep peptide preparation within minutes not days, at high peptide reproducibility and low abundance-bias. FDD transformed enzyme-protein contacts into equal catalytic travel paths through enzyme-excessive columns regardless of protein abundance, removed products instantly preventing inhibition, tackled intricate structures via sequential multiple micro-digestions along the flow, and precisely controlled peptide formation by flow rate. Peptide-stage reactions reduced steric bias; low contamination deepened MS/MS scan; distinguishing disulfide from M oxidation and avoiding gain/loss artifacts unmasked protein-endogenous oxidation states. Using a recent interactome of 285-kDa human GABA type A receptor, this pilot study validated FDD platform's applicability to deep sequencing (up to 99% coverage), H/D-exchange and TMT-based structural mapping. FDD discovered novel subunit-specific PTM signatures, including unusual nontop-surface N-glycosylations, that may drive subunit biases in human Cys-loop LGIC assembly and pharmacology, by redefining subunit/ligand interfaces and connecting function domains. PMID:27073180

Triangular Diagrams Teach Steady and Dynamic Behaviour of Catalytic Reactions.

ERIC Educational Resources Information Center

Klusacek, K.; And Others

1989-01-01

Illustrates how triangular diagrams can aid in presenting some of the rather complex transient interactions that occur among gas and surface species during heterogeneous catalytic reactions. The basic equations and numerical examples are described. Classroom use of the triangular diagram is discussed. Several diagrams and graphs are provided. (YP)

Treadmilling of a prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis

PubMed Central

Larsen, Rachel A.; Cusumano, Christina; Fujioka, Akina; Lim-Fong, Grace; Patterson, Paula; Pogliano, Joe

2007-01-01

Prokaryotes rely on a distant tubulin homolog, FtsZ, for assembling the cytokinetic ring essential for cell division, but are otherwise generally thought to lack tubulin-like polymers that participate in processes such as DNA segregation. Here we characterize a protein (TubZ) from the Bacillus thuringiensis virulence plasmid pBtoxis, which is a member of the tubulin/FtsZ GTPase superfamily but is only distantly related to both FtsZ and tubulin. TubZ assembles dynamic, linear polymers that exhibit directional polymerization with plus and minus ends, movement by treadmilling, and a critical concentration for assembly. A point mutation (D269A) that alters a highly conserved catalytic residue within the T7 loop completely eliminates treadmilling and allows the formation of stable polymers at a much lower protein concentration than the wild-type protein. When expressed in trans, TubZ(D269A) coassembles with wild-type TubZ and significantly reduces the stability of pBtoxis, demonstrating a direct correlation between TubZ dynamics and plasmid maintenance. The tubZ gene is in an operon with tubR, which encodes a putative DNA-binding protein that regulates TubZ levels. Our results suggest that TubZ is representative of a novel class of prokaryotic cytoskeletal proteins important for plasmid stability that diverged long ago from the ancient tubulin/FtsZ ancestor. PMID:17510284

Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for -1 ribosomal frameshifting stimulation

NASA Astrophysics Data System (ADS)

Zhong, Zhensheng; Yang, Lixia; Zhang, Haiping; Shi, Jiahao; Vandana, J. Jeya; Lam, Do Thuy Uyen Ha; Olsthoorn, René C. L.; Lu, Lanyuan; Chen, Gang

2016-12-01

Minus-one ribosomal frameshifting is a translational recoding mechanism widely utilized by many RNA viruses to generate accurate ratios of structural and catalytic proteins. An RNA pseudoknot structure located in the overlapping region of the gag and pro genes of Simian Retrovirus type 1 (SRV-1) stimulates frameshifting. However, the experimental characterization of SRV-1 pseudoknot (un)folding dynamics and the effect of the base triple formation is lacking. Here, we report the results of our single-molecule nanomanipulation using optical tweezers and theoretical simulation by steered molecular dynamics. Our results directly reveal that the energetic coupling between loop 2 and stem 1 via minor-groove base triple formation enhances the mechanical stability. The terminal base pair in stem 1 (directly in contact with a translating ribosome at the slippery site) also affects the mechanical stability of the pseudoknot. The -1 frameshifting efficiency is positively correlated with the cooperative one-step unfolding force and inversely correlated with the one-step mechanical unfolding rate at zero force. A significantly improved correlation was observed between -1 frameshifting efficiency and unfolding rate at forces of 15-35 pN, consistent with the fact that the ribosome is a force-generating molecular motor with helicase activity. No correlation was observed between thermal stability and -1 frameshifting efficiency.

The Trade-Off Mechanism in Mammalian Circadian Clock Model with Two Time Delays

NASA Astrophysics Data System (ADS)

Yan, Jie; Kang, Xiaxia; Yang, Ling

Circadian clock is an autonomous oscillator which orchestrates the daily rhythms of physiology and behaviors. This study is devoted to explore how a positive feedback loop affects the dynamics of mammalian circadian clock. We simplify an experimentally validated mathematical model in our previous work, to a nonlinear differential equation with two time delays. This simplified mathematical model incorporates the pacemaker of mammalian circadian clock, a negative primary feedback loop, and a critical positive auxiliary feedback loop, Rev-erbα/Cry1 loop. We perform analytical studies of the system. Delay-dependent conditions for the asymptotic stability of the nontrivial positive steady state of the model are investigated. We also prove the existence of Hopf bifurcation, which leads to self-sustained oscillation of mammalian circadian clock. Our theoretical analyses show that the oscillatory regime is reduced upon the participation of the delayed positive auxiliary loop. However, further simulations reveal that the auxiliary loop can enable the circadian clock gain widely adjustable amplitudes and robust period. Thus, the positive auxiliary feedback loop may provide a trade-off mechanism, to use the small loss in the robustness of oscillation in exchange for adaptable flexibility in mammalian circadian clock. The results obtained from the model may gain new insights into the dynamics of biological oscillators with interlocked feedback loops.

Creating stable stem regions for loop elongation in Fcabs — Insights from combining yeast surface display, in silico loop reconstruction and molecular dynamics simulations

PubMed Central

Hasenhindl, Christoph; Lai, Balder; Delgado, Javier; Traxlmayr, Michael W.; Stadlmayr, Gerhard; Rüker, Florian; Serrano, Luis; Oostenbrink, Chris; Obinger, Christian

2014-01-01

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules. PMID:24792385

Creating stable stem regions for loop elongation in Fcabs - insights from combining yeast surface display, in silico loop reconstruction and molecular dynamics simulations.

PubMed

Hasenhindl, Christoph; Lai, Balder; Delgado, Javier; Traxlmayr, Michael W; Stadlmayr, Gerhard; Rüker, Florian; Serrano, Luis; Oostenbrink, Chris; Obinger, Christian

2014-09-01

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules. Copyright © 2014. Published by Elsevier B.V.

Design of a colicin E7 based chimeric zinc-finger nuclease

NASA Astrophysics Data System (ADS)

Németh, Eszter; Schilli, Gabriella K.; Nagy, Gábor; Hasenhindl, Christoph; Gyurcsik, Béla; Oostenbrink, Chris

2014-08-01

Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity—offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region.

Analysis of flexible aircraft longitudinal dynamics and handling qualities. Volume 1: Analysis methods

NASA Technical Reports Server (NTRS)

Waszak, M. R.; Schmidt, D. S.

1985-01-01

As aircraft become larger and lighter due to design requirements for increased payload and improved fuel efficiency, they will also become more flexible. For highly flexible vehicles, the handling qualities may not be accurately predicted by conventional methods. This study applies two analysis methods to a family of flexible aircraft in order to investigate how and when structural (especially dynamic aeroelastic) effects affect the dynamic characteristics of aircraft. The first type of analysis is an open loop model analysis technique. This method considers the effects of modal residue magnitudes on determining vehicle handling qualities. The second method is a pilot in the loop analysis procedure that considers several closed loop system characteristics. Volume 1 consists of the development and application of the two analysis methods described above.

The Response of Ω-Loop D Dynamics to Truncation of Trimethyllysine 72 of Yeast Iso-1-cytochrome c Depends on the Nature of Loop Deformation

PubMed Central

McClelland, Levi J.; Seagraves, Sean M.; Khan, Khurshid Alam; Cherney, Melisa M.; Bandi, Swati; Culbertson, Justin E.; Bowler, Bruce E.

2015-01-01

Trimethyllysine 72 (tmK72) has been suggested to play a role in sterically constraining the heme crevice dynamics of yeast iso-1-cytochrome c mediated by the Ω-loop D cooperative substructure (residues 70 to 85). A tmK72A mutation causes a gain in peroxidase activity, a function of cytochrome c that is important early in apoptosis. More than one higher energy state is accessible for the Ω-loop D substructure via tier 0 dynamics. Two of these are alkaline conformers mediated by Lys73 and Lys79. In the current work, the effect of the tmK72A mutation on the thermodynamic and kinetic properties of wild type iso-1-cytochrome c (yWT versus WT*) and on variants carrying a K73H mutation (yWT/K73H versus WT*/K73H) is studied. Whereas the tmK72A mutation confers increased peroxidase activity in wild type yeast iso-1-cytochrome c and increased dynamics for formation of a previously studied His79-heme alkaline conformer, the tmK72A mutation speeds return of the His73-heme alkaline conformer to the native state through destabilization of the His73-heme alkaline conformer relative to the native conformer. These opposing behaviors demonstrate that the response of the dynamics of a protein substructure to mutation depends on the nature of the perturbation to the substructure. For a protein substructure which mediates more than one function of a protein through multiple non-native structures, a mutation could change the partitioning between these functions. The current results suggest that the tier 0 dynamics of Ω-loop D that mediates peroxidase activity has similarities to the tier 0 dynamics required to form the His79-heme alkaline conformer. PMID:25948392

Structure and Dynamics of Cool Flare Loops Observed by the Interface Region Imaging Spectrograph

NASA Astrophysics Data System (ADS)

Mikuła, K.; Heinzel, P.; Liu, W.; Berlicki, A.

2017-08-01

Flare loops were well observed with the Interface Region Imaging Spectrograph (IRIS) during the gradual phase of two solar flares on 2014 March 29 and 2015 June 22. Cool flare loops are visible in various spectral lines formed at chromospheric and transition-region temperatures and exhibit large downflows which correspond to the standard scenario. The principal aim of this work is to analyze the structure and dynamics of cool flare loops observed in Mg II lines. Synthetic profiles of the Mg II h line are computed using the classical cloud model and assuming a uniform background intensity. In this paper, we study novel IRIS NUV observations of such loops in Mg II h and k lines and also show the behavior of hotter lines detected in the FUV channel. We obtained the spatial evolution of the velocities: near the loop top, the flow velocities are small and they are increasing toward the loop legs. Moreover, from slit-jaw image (SJI) movies, we observe some plasma upflows into the loops, which are also detectable in Mg II spectra. The brightness of the loops systematically decreases with increasing flow velocity, and we ascribe this to the effect of Doppler dimming, which works for Mg II lines. Emission profiles of Mg II were found to be extremely broad, and we explain this through the large unresolved non-thermal motions.

Structure and Dynamics of Cool Flare Loops Observed by the Interface Region Imaging Spectrograph

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mikuła, K.; Berlicki, A.; Heinzel, P.

Flare loops were well observed with the Interface Region Imaging Spectrograph ( IRIS ) during the gradual phase of two solar flares on 2014 March 29 and 2015 June 22. Cool flare loops are visible in various spectral lines formed at chromospheric and transition-region temperatures and exhibit large downflows which correspond to the standard scenario. The principal aim of this work is to analyze the structure and dynamics of cool flare loops observed in Mg ii lines. Synthetic profiles of the Mg ii h line are computed using the classical cloud model and assuming a uniform background intensity. In thismore » paper, we study novel IRIS NUV observations of such loops in Mg ii h and k lines and also show the behavior of hotter lines detected in the FUV channel. We obtained the spatial evolution of the velocities: near the loop top, the flow velocities are small and they are increasing toward the loop legs. Moreover, from slit-jaw image (SJI) movies, we observe some plasma upflows into the loops, which are also detectable in Mg ii spectra. The brightness of the loops systematically decreases with increasing flow velocity, and we ascribe this to the effect of Doppler dimming, which works for Mg ii lines. Emission profiles of Mg ii were found to be extremely broad, and we explain this through the large unresolved non-thermal motions.« less

Comparison of Multiple Molecular Dynamics Trajectories Calculated for the Drug-Resistant HIV-1 Integrase T66I/M154I Catalytic Domain

PubMed Central

Brigo, Alessandro; Lee, Keun Woo; Iurcu Mustata, Gabriela; Briggs, James M.

2005-01-01

HIV-1 integrase (IN) is an essential enzyme for the viral replication and an interesting target for the design of new pharmaceuticals for multidrug therapy of AIDS. Single and multiple mutations of IN at residues T66, S153, or M154 confer degrees of resistance to several inhibitors that prevent the enzyme from performing its normal strand transfer activity. Four different conformations of IN were chosen from a prior molecular dynamics (MD) simulation on the modeled IN T66I/M154I catalytic core domain as starting points for additional MD studies. The aim of this article is to understand the dynamic features that may play roles in the catalytic activity of the double mutant enzyme in the absence of any inhibitor. Moreover, we want to verify the influence of using different starting points on the MD trajectories and associated dynamical properties. By comparison of the trajectories obtained from these MD simulations we have demonstrated that the starting point does not affect the conformational space explored by this protein and that the time of the simulation is long enough to achieve convergence for this system. PMID:15764656

Genetic Algorithm Approaches to Prebiobiotic Chemistry Modeling

NASA Technical Reports Server (NTRS)

Lohn, Jason; Colombano, Silvano

1997-01-01

We model an artificial chemistry comprised of interacting polymers by specifying two initial conditions: a distribution of polymers and a fixed set of reversible catalytic reactions. A genetic algorithm is used to find a set of reactions that exhibit a desired dynamical behavior. Such a technique is useful because it allows an investigator to determine whether a specific pattern of dynamics can be produced, and if it can, the reaction network found can be then analyzed. We present our results in the context of studying simplified chemical dynamics in theorized protocells - hypothesized precursors of the first living organisms. Our results show that given a small sample of plausible protocell reaction dynamics, catalytic reaction sets can be found. We present cases where this is not possible and also analyze the evolved reaction sets.

Structural snapshots along the reaction pathway of Yersinia pestis RipA, a putative butyryl-CoA transferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Torres, Rodrigo; Lan, Benson; Latif, Yama

2014-04-01

The crystal structures of Y. pestis RipA mutants were determined to provide insights into the CoA transferase reaction pathway. Yersinia pestis, the causative agent of bubonic plague, is able to survive in both extracellular and intracellular environments within the human host, although its intracellular survival within macrophages is poorly understood. A novel Y. pestis three-gene rip (required for intracellular proliferation) operon, and in particular ripA, has been shown to be essential for survival and replication in interferon γ-induced macrophages. RipA was previously characterized as a putative butyryl-CoA transferase proposed to yield butyrate, a known anti-inflammatory shown to lower macrophage-produced NOmore » levels. RipA belongs to the family I CoA transferases, which share structural homology, a conserved catalytic glutamate which forms a covalent CoA-thioester intermediate and a flexible loop adjacent to the active site known as the G(V/I)G loop. Here, functional and structural analyses of several RipA mutants are presented in an effort to dissect the CoA transferase mechanism of RipA. In particular, E61V, M31G and F60M RipA mutants show increased butyryl-CoA transferase activities when compared with wild-type RipA. Furthermore, the X-ray crystal structures of E61V, M31G and F60M RipA mutants, when compared with the wild-type RipA structure, reveal important conformational changes orchestrated by a conserved acyl-group binding-pocket phenylalanine, Phe85, and the G(V/I)G loop. Binary structures of M31G RipA and F60M RipA with two distinct CoA substrate conformations are also presented. Taken together, these data provide CoA transferase reaction snapshots of an open apo RipA, a closed glutamyl-anhydride intermediate and an open CoA-thioester intermediate. Furthermore, biochemical analyses support essential roles for both the catalytic glutamate and the flexible G(V/I)G loop along the reaction pathway, although further research is required to fully understand the function of the acyl-group binding pocket in substrate specificity.« less

Nanostructures nucleation in carbon-metal gaseous phase: A molecular dynamics study

NASA Astrophysics Data System (ADS)

Galiullina, G. M.; Orekhov, N. D.; Stegailov, V. V.

2018-01-01

We perform nonequilibrium molecular dynamics simulation of carbon nanoclusters nucleation and early stages of growth from the gaseous phase. We analyze the catalytic effect of iron atoms on the nucleation kinetics and structure of the resultant nanoparticles. Reactive Force Field (ReaxFF) is used in the simulations for the description of bond formation and dissociation during the nucleation process at the nanoscale. The catalytic effect of iron reveals itself even on nanosecond simulation times: iron atoms accelerate the process of clustering but result in less graphitized carbon structures.

Geometric tuning of self-propulsion for Janus catalytic particles

NASA Astrophysics Data System (ADS)

Michelin, Sébastien; Lauga, Eric

2017-02-01

Catalytic swimmers have attracted much attention as alternatives to biological systems for examining collective microscopic dynamics and the response to physico-chemical signals. Yet, understanding and predicting even the most fundamental characteristics of their individual propulsion still raises important challenges. While chemical asymmetry is widely recognized as the cornerstone of catalytic propulsion, different experimental studies have reported that particles with identical chemical properties may propel in opposite directions. Here, we show that, beyond its chemical properties, the detailed shape of a catalytic swimmer plays an essential role in determining its direction of motion, demonstrating the compatibility of the classical theoretical framework with experimental observations.

Geometric tuning of self-propulsion for Janus catalytic particles

PubMed Central

Michelin, Sébastien; Lauga, Eric

2017-01-01

Catalytic swimmers have attracted much attention as alternatives to biological systems for examining collective microscopic dynamics and the response to physico-chemical signals. Yet, understanding and predicting even the most fundamental characteristics of their individual propulsion still raises important challenges. While chemical asymmetry is widely recognized as the cornerstone of catalytic propulsion, different experimental studies have reported that particles with identical chemical properties may propel in opposite directions. Here, we show that, beyond its chemical properties, the detailed shape of a catalytic swimmer plays an essential role in determining its direction of motion, demonstrating the compatibility of the classical theoretical framework with experimental observations. PMID:28205563

Geometric tuning of self-propulsion for Janus catalytic particles.

PubMed

Michelin, Sébastien; Lauga, Eric

2017-02-13

Catalytic swimmers have attracted much attention as alternatives to biological systems for examining collective microscopic dynamics and the response to physico-chemical signals. Yet, understanding and predicting even the most fundamental characteristics of their individual propulsion still raises important challenges. While chemical asymmetry is widely recognized as the cornerstone of catalytic propulsion, different experimental studies have reported that particles with identical chemical properties may propel in opposite directions. Here, we show that, beyond its chemical properties, the detailed shape of a catalytic swimmer plays an essential role in determining its direction of motion, demonstrating the compatibility of the classical theoretical framework with experimental observations.

All-digital phase-lock loops for noise-free signals

NASA Technical Reports Server (NTRS)

Anderson, T. O.

1973-01-01

Bit-synchronizers utilize all-digital phase-lock loops that are referenced to a high frequency digital clock. Phase-lock loop of first design acquires frequency within nominal range and tracks phase; second design is modified for random binary data by addition of simple transition detector; and third design acquires frequency over wide dynamic range.

Second International Workshop on Grid Simulator Testing of Wind Turbine

Science.gov Websites

, Clemson University, USA Update on the FSU-CAPS Megawatt Scale Power Hardware in the Loop Laboratory Loop Based Anti-Islanding Testing of PV Converters-Michael Steurer, Florida State University, USA Closed-Loop Control of Modern Test Benches Advanced Control Techniques for Dynamic Testing of Wind

Energy Systems Integration News | Energy Systems Integration Facility |

Science.gov Websites

-the-loop" (HIL) to connect physical devices to software models, EdgePower is drawing on NREL's are putting their controller into a synthetic environment that is called 'controller in-the-loop controller-in-the-loop platform allows us to observe the dynamics of these buildings as they implement the

Dislocation dynamics simulations of interactions between gliding dislocations and radiation induced prismatic loops in zirconium

NASA Astrophysics Data System (ADS)

Drouet, Julie; Dupuy, Laurent; Onimus, Fabien; Mompiou, Frédéric; Perusin, Simon; Ambard, Antoine

2014-06-01

The mechanical behavior of Pressurized Water Reactor fuel cladding tubes made of zirconium alloys is strongly affected by neutron irradiation due to the high density of radiation induced dislocation loops. In order to investigate the interaction mechanisms between gliding dislocations and loops in zirconium, a new nodal dislocation dynamics code, adapted to Hexagonal Close Packed metals, has been used. Various configurations have been systematically computed considering different glide planes, basal or prismatic, and different characters, edge or screw, for gliding dislocations with -type Burgers vectors. Simulations show various interaction mechanisms such as (i) absorption of a loop on an edge dislocation leading to the formation of a double super-jog, (ii) creation of a helical turn, on a screw dislocation, that acts as a strong pinning point or (iii) sweeping of a loop by a gliding dislocation. It is shown that the clearing of loops is more favorable when the dislocation glides in the basal plane than in the prismatic plane explaining the easy dislocation channeling in the basal plane observed after neutron irradiation by transmission electron microscopy.

Crystal structure of the bacterial luciferase/flavin complex provides insight into the function of the beta subunit.

PubMed

Campbell, Zachary T; Weichsel, Andrzej; Montfort, William R; Baldwin, Thomas O

2009-07-07

Bacterial luciferase from Vibrio harveyi is a heterodimer composed of a catalytic alpha subunit and a homologous but noncatalytic beta subunit. Despite decades of enzymological investigation, structural evidence defining the active center has been elusive. We report here the crystal structure of V. harveyi luciferase bound to flavin mononucleotide (FMN) at 2.3 A. The isoalloxazine ring is coordinated by an unusual cis-Ala-Ala peptide bond. The reactive sulfhydryl group of Cys106 projects toward position C-4a, the site of flavin oxygenation. This structure also provides the first data specifying the conformations of a mobile loop that is crystallographically disordered in both prior crystal structures [(1995) Biochemistry 34, 6581-6586; (1996) J. Biol. Chem. 271, 21956 21968]. This loop appears to be a boundary between solvent and the active center. Within this portion of the protein, a single contact was observed between Phe272 of the alpha subunit, not seen in the previous structures, and Tyr151 of the beta subunit. Substitutions at position 151 on the beta subunit caused reductions in activity and total quantum yield. Several of these mutants were found to have decreased affinity for reduced flavin mononucleotide (FMNH(2)). These findings partially address the long-standing question of how the beta subunit stabilizes the active conformation of the alpha subunit, thereby participating in the catalytic mechanism.

Deduced catalytic mechanism of d-amino acid amidase from Ochrobactrum anthropi SV3

PubMed Central

Okazaki, Seiji; Suzuki, Atsuo; Komeda, Hidenobu; Asano, Yasuhisa; Yamane, Takashi

2008-01-01

d-Amino acid amidase (DAA) from Ochrobactrum anthropi SV3 catalyzes d-stereospecific hydrolysis of amino acid amides. DAA has attracted attention as a catalyst for the stereospecific production of d-amino acids, although the mechanism that drives the reaction has not been clear. Previously, the structure of DAA was classified into two types, a substrate-bound state with an ordered Ω loop, and a ground state with a disordered Ω loop. Because the binding of the substrate facilitates ordering, this transition was regarded to be induced fit motion. The angles and distances of hydrogen bonds at Tyr149 Oη, Ser60 Oγ and Lys63 Nζ revealed that Tyr149 Oη donates an H atom to a water molecule in the substrate-bound state, and that Tyr149 Oη donates an H atom to Ser60 Oγ or Lys63 Nζ in the ground state. Taking into consideration the locations of the H atoms of Tyr149 Oη, Ser60 Oγ and Lys63 Nζ, a catalytic mechanism of DAA activity is presented, wherein a shift of an H atom at Tyr149 Oη in the substrate-bound versus the ground state plays a significant role in the reaction. This mechanism explains well why acylation proceeds and deacylation does not proceed in the substrate-bound state. PMID:18421151

Functional and structural characterization of the pentapeptide insertion of Theileria annulata lactate dehydrogenase by site-directed mutagenesis, comparative modeling and molecular dynamics simulations.

PubMed

Erdemir, Aysegul; Mutlu, Ozal

2017-06-01

Lactate dehydrogenase (LDH) is an important metabolic enzyme in glycolysis and it has been considered as the main energy source in many organisms including apicomplexan parasites. Differences at the active site loop of the host and parasite LDH's makes this enzyme an attractive target for drug inhibitors. In this study, five amino acid insertions in the active site pocket of Theileria annulata LDH (TaLDH) were deleted by PCR-based site-directed mutagenesis, expression and activity analysis of mutant and wild type TaLDH enzymes were performed. Removal of the insertion at the active site loop caused production of an inactive enzyme. Furthermore, structures of wild and mutant enzymes were predicted by comparative modeling and the importance of the insertions at the active site loop were also assigned by molecular docking and dynamics simulations in order to evaluate essential role of this loop for the enzymatic activity. Pentapeptide insertion removal resulted in loss of LDH activity due to deletion of Trp96 and conformational change of Arg98 because of loop instability. Analysis of wild type and mutant enzymes with comparative molecular dynamics simulations showed that the fluctuations of the loop residues increase in mutant enzyme. Together with in silico studies, in vitro results revealed that active site loop has a vital role in the enzyme activity and our findings promise hope for the further drug design studies against theileriosis and other apicomplexan parasite diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Dynamics of endoglucanase catalytic domains: implications towards thermostability

USDA-ARS?s Scientific Manuscript database

The function of proteins is controlled by their dynamics inherently determined by their structure. Exploring the protein structure-dynamics relationship is important to develop an understanding of protein function that allows tapping the potential of economically important proteins, such as endogluc...

Hysteresis losses and specific absorption rate measurements in magnetic nanoparticles for hyperthermia applications.

PubMed

Coïsson, Marco; Barrera, Gabriele; Celegato, Federica; Martino, Luca; Kane, Shashank N; Raghuvanshi, Saroj; Vinai, Franco; Tiberto, Paola

2017-06-01

Magnetic hysteresis loops areas and hyperthermia on magnetic nanoparticles have been studied with the aim of providing reliable and reproducible methods of measuring the specific absorption rate (SAR). The SAR of Fe 3 O 4 nanoparticles with two different mean sizes, and Ni 1-x Zn x Fe 2 O 4 ferrites with 0 ≤ x ≤ 0.8 has been measured with three approaches: static hysteresis loops areas, dynamic hysteresis loops areas and hyperthermia of a water solution. For dynamic loops and thermometric measurements, specific experimental setups have been developed, that operate at comparable frequencies (≈ 69kHz and ≈ 100kHz respectively) and rf magnetic field peak values (up to 100mT). The hyperthermia setup has been fully modelled to provide a direct measurement of the SAR of the magnetic nanoparticles by taking into account the heat exchange with the surrounding environment in non-adiabatic conditions and the parasitic heating of the water due to ionic currents. Dynamic hysteresis loops are shown to provide an accurate determination of the SAR except for superparamagnetic samples, where the boundary with a blocked regime could be crossed in dynamic conditions. Static hysteresis loops consistently underestimate the specific absorption rate but can be used to select the most promising samples. A means of reliably measure SAR of magnetic nanoparticles by different approaches for hyperthermia applications is presented and its validity discussed by comparing different methods. This work fits within the general subject of metrological traceability in medicine with a specific focus on magnetic hyperthermia. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader. Copyright © 2016 Elsevier B.V. All rights reserved.

Dynamics of nonlinear feedback control.

PubMed

Snippe, H P; van Hateren, J H

2007-05-01

Feedback control in neural systems is ubiquitous. Here we study the mathematics of nonlinear feedback control. We compare models in which the input is multiplied by a dynamic gain (multiplicative control) with models in which the input is divided by a dynamic attenuation (divisive control). The gain signal (resp. the attenuation signal) is obtained through a concatenation of an instantaneous nonlinearity and a linear low-pass filter operating on the output of the feedback loop. For input steps, the dynamics of gain and attenuation can be very different, depending on the mathematical form of the nonlinearity and the ordering of the nonlinearity and the filtering in the feedback loop. Further, the dynamics of feedback control can be strongly asymmetrical for increment versus decrement steps of the input. Nevertheless, for each of the models studied, the nonlinearity in the feedback loop can be chosen such that immediately after an input step, the dynamics of feedback control is symmetric with respect to increments versus decrements. Finally, we study the dynamics of the output of the control loops and find conditions under which overshoots and undershoots of the output relative to the steady-state output occur when the models are stimulated with low-pass filtered steps. For small steps at the input, overshoots and undershoots of the output do not occur when the filtering in the control path is faster than the low-pass filtering at the input. For large steps at the input, however, results depend on the model, and for some of the models, multiple overshoots and undershoots can occur even with a fast control path.

Alpha-canonical form representation of the open loop dynamics of the Space Shuttle main engine

NASA Technical Reports Server (NTRS)

Duyar, Almet; Eldem, Vasfi; Merrill, Walter C.; Guo, Ten-Huei

1991-01-01

A parameter and structure estimation technique for multivariable systems is used to obtain a state space representation of open loop dynamics of the space shuttle main engine in alpha-canonical form. The parameterization being used is both minimal and unique. The simplified linear model may be used for fault detection studies and control system design and development.

Non-active site mutations disturb the loop dynamics, dimerization, viral budding and egress of VP40 of the Ebola virus.

PubMed

Balmith, Marissa; Soliman, Mahmoud E S

2017-02-28

The first account of the dynamic features of the loop region of VP40 of the Ebola virus (EboV) using accelerated molecular dynamics (aMD) simulations is reported herein. Due to its major role in the Ebola life cycle, VP40 is considered a promising therapeutic target. The available experimental data on the N-terminal domain (NTD) loop indicates that mutations K127A, T129A and N130A demonstrate an unrecognized role for NTD-plasma membrane (PM) interaction for efficient VP40-PM localization, oligomerization, matrix assembly and egress. Despite experimental results, the molecular description of VP40 and the information it can provide still remain vague. Therefore, to gain further molecular insight into the effect of mutations on the loop region of VP40 and its effects on the overall protein conformation and VP40 dimerization, aMD simulations and post-dynamic analyses were employed for wildtype (WT) and mutant systems. The results showed significant variations in the presence of mutations as per RMSF, RMSD, R g , PCA and distance calculations in comparison to the WT. These results could provide researchers with insight with regards to the conformational aspects concerning VP40 and its close relation to the experimental data. We believe that the results presented in this study will ultimately provide a useful understanding of the structural landscape of the loop region of VP40, which would contribute towards the discovery of novel EboV inhibitors.

Structural dynamics of the lac repressor-DNA complex revealed by a multiscale simulation.

PubMed

Villa, Elizabeth; Balaeff, Alexander; Schulten, Klaus

2005-05-10

A multiscale simulation of a complex between the lac repressor protein (LacI) and a 107-bp-long DNA segment is reported. The complex between the repressor and two operator DNA segments is described by all-atom molecular dynamics; the size of the simulated system comprises either 226,000 or 314,000 atoms. The DNA loop connecting the operators is modeled as a continuous elastic ribbon, described mathematically by the nonlinear Kirchhoff differential equations with boundary conditions obtained from the coordinates of the terminal base pairs of each operator. The forces stemming from the looped DNA are included in the molecular dynamics simulations; the loop structure and the forces are continuously recomputed because the protein motions during the simulations shift the operators and the presumed termini of the loop. The simulations reveal the structural dynamics of the LacI-DNA complex in unprecedented detail. The multiple domains of LacI exhibit remarkable structural stability during the simulation, moving much like rigid bodies. LacI is shown to absorb the strain from the looped DNA mainly through its mobile DNA-binding head groups. Even with large fluctuating forces applied, the head groups tilt strongly and keep their grip on the operator DNA, while the remainder of the protein retains its V-shaped structure. A simulated opening of the cleft of LacI by 500-pN forces revealed the interactions responsible for locking LacI in the V-conformation.

Hydrodynamically induced oscillations and traffic dynamics in 1D microfludic networks

NASA Astrophysics Data System (ADS)

Bartolo, Denis; Jeanneret, Raphael

2011-03-01

We report on the traffic dynamics of particles driven through a minimal microfluidic network. Even in the minimal network consisting in a single loop, the traffic dynamics has proven to yield complex temporal patterns, including periodic, multi-periodic or chaotic sequences. This complex dynamics arises from the strongly nonlinear hydrodynamic interactions between the particles, that takes place at a junction. To better understand the consequences of this nontrivial coupling, we combined theoretical, numerical and experimental efforts and solved the 3-body problem in a 1D loop network. This apparently simple dynamical system revealed a rich and unexpected dynamics, including coherent spontaneous oscillations along closed orbits. Striking similarities between Hamiltonian systems and this driven dissipative system will be explained.

Watching Individual Enzymes at Work

NASA Astrophysics Data System (ADS)

Blank, Kerstin; Rocha, Susana; De Cremer, Gert; Roeffaers, Maarten B. J.; Uji-i, Hiroshi; Hofkens, Johan

Single-molecule fluorescence experiments are a powerful tool to analyze reaction mechanisms of enzymes. Because of their unique potential to detect heterogeneities in space and time, they have provided unprecedented insights into the nature and mechanisms of conformational changes related to the catalytic reaction. The most important finding from experiments with single enzymes is the generally observed phenomenon that the catalytic rate constants fluctuate over time (dynamic disorder). These fluctuations originate from conformational changes occurring on time scales, which are similar to or slower than that of the catalytic reaction. Here, we summarize experiments with enzymes that show dynamic disorder and introduce new experimental strategies showing how single-molecule fluorescence experiments can be applied to address other open questions in medical and industrial enzymology, such as enzyme inactivation processes, reactant transfer in cascade reactions, and the mechanisms of interfacial catalysis.

Periodic, Quasi-periodic and Chaotic Dynamics in Simple Gene Elements with Time Delays

PubMed Central

Suzuki, Yoko; Lu, Mingyang; Ben-Jacob, Eshel; Onuchic, José N.

2016-01-01

Regulatory gene circuit motifs play crucial roles in performing and maintaining vital cellular functions. Frequently, theoretical studies of gene circuits focus on steady-state behaviors and do not include time delays. In this study, the inclusion of time delays is shown to entirely change the time-dependent dynamics for even the simplest possible circuits with one and two gene elements with self and cross regulations. These elements can give rise to rich behaviors including periodic, quasi-periodic, weak chaotic, strong chaotic and intermittent dynamics. We introduce a special power-spectrum-based method to characterize and discriminate these dynamical modes quantitatively. Our simulation results suggest that, while a single negative feedback loop of either one- or two-gene element can only have periodic dynamics, the elements with two positive/negative feedback loops are the minimalist elements to have chaotic dynamics. These elements typically have one negative feedback loop that generates oscillations, and another unit that allows frequent switches among multiple steady states or between oscillatory and non-oscillatory dynamics. Possible dynamical features of several simple one- and two-gene elements are presented in details. Discussion is presented for possible roles of the chaotic behavior in the robustness of cellular functions and diseases, for example, in the context of cancer. PMID:26876008

Periodic, Quasi-periodic and Chaotic Dynamics in Simple Gene Elements with Time Delays

NASA Astrophysics Data System (ADS)

Suzuki, Yoko; Lu, Mingyang; Ben-Jacob, Eshel; Onuchic, José N.

2016-02-01

Regulatory gene circuit motifs play crucial roles in performing and maintaining vital cellular functions. Frequently, theoretical studies of gene circuits focus on steady-state behaviors and do not include time delays. In this study, the inclusion of time delays is shown to entirely change the time-dependent dynamics for even the simplest possible circuits with one and two gene elements with self and cross regulations. These elements can give rise to rich behaviors including periodic, quasi-periodic, weak chaotic, strong chaotic and intermittent dynamics. We introduce a special power-spectrum-based method to characterize and discriminate these dynamical modes quantitatively. Our simulation results suggest that, while a single negative feedback loop of either one- or two-gene element can only have periodic dynamics, the elements with two positive/negative feedback loops are the minimalist elements to have chaotic dynamics. These elements typically have one negative feedback loop that generates oscillations, and another unit that allows frequent switches among multiple steady states or between oscillatory and non-oscillatory dynamics. Possible dynamical features of several simple one- and two-gene elements are presented in details. Discussion is presented for possible roles of the chaotic behavior in the robustness of cellular functions and diseases, for example, in the context of cancer.

Activation of Latent Dihydroorotase from Aquifex aeolicus by Pressure*

PubMed Central

Hervé, Guy; Evans, Hedeel Guy; Fernado, Roshini; Patel, Chandni; Hachem, Fatme; Evans, David R.

2017-01-01

Elevated hydrostatic pressure was used to probe conformational changes of Aquifex aeolicus dihydroorotase (DHO), which catalyzes the third step in de novo pyrimidine biosynthesis. The isolated protein, a 45-kDa monomer, lacks catalytic activity but becomes active upon formation of a dodecameric complex with aspartate transcarbamoylase (ATC). X-ray crystallographic studies of the isolated DHO and of the complex showed that association induces several major conformational changes in the DHO structure. In the isolated DHO, a flexible loop occludes the active site blocking the access of substrates. The loop is mostly disordered but is tethered to the active site region by several electrostatic and hydrogen bonds. This loop becomes ordered and is displaced from the active site upon formation of DHO-ATC complex. The application of pressure to the complex causes its time-dependent dissociation and the loss of both DHO and ATC activities. Pressure induced irreversible dissociation of the obligate ATC trimer, and as a consequence the DHO is also inactivated. However, moderate hydrostatic pressure applied to the isolated DHO subunit mimics the complex formation and reversibly activates the isolated subunit in the absence of ATC, suggesting that the loop has been displaced from the active site. This effect of pressure is explained by the negative volume change associated with the disruption of ionic interactions and exposure of ionized amino acids to the solvent (electrostriction). The interpretation that the loop is relocated by pressure was validated by site-directed mutagenesis and by inhibition by small peptides that mimic the loop residues. PMID:27746403

Crystal structures of the class D beta-lactamase OXA-13 in the native form and in complex with meropenem.

PubMed

Pernot, L; Frénois, F; Rybkine, T; L'Hermite, G; Petrella, S; Delettré, J; Jarlier, V; Collatz, E; Sougakoff, W

2001-07-20

The therapeutic problems posed by class D beta-lactamases, a family of serine enzymes that hydrolyse beta-lactam antibiotics following an acylation-deacylation mechanism, are increased by the very low level of sensitivity of these enzymes to beta-lactamase inhibitors. To gain structural and mechanistic insights to aid the design of new inhibitors, we have determined the crystal structure of OXA-13 from Pseudomonas aeruginosa in the apo form and in complex with the carbapenem meropenem. The native form consisted of a dimer displaying an overall organisation similar to that found in the closely related enzyme OXA-10. In the acyl-enzyme complex, the positioning of the antibiotic appeared to be ensured mainly by (i) the covalent acyl bond and (ii) a strong salt-bridge involving the carboxylate moiety of the drug. Comparison of the structures of OXA-13 in the apo form and in complex with meropenem revealed an unsuspected flexibility in the region of the essential serine 115 residue, with possible consequences for the catalytic properties of the enzyme. In the apo form, the Ser115 side-chain is oriented outside the active site, whereas the general base Lys70 adopts a conformation that seems to be incompatible with the activation of the catalytic water molecule required for the deacylation step. In the OXA-13:meropenem complex, a 3.5 A movement of the backbone of the 114-116 loop towards the side-chain of Lys70 was observed, which seems to be driven by a displacement of the neighbouring 91-104 loop and which results in the repositioning of the side-chain hydroxyl group of Ser115 toward the catalytic centre. Concomitantly, the side-chain of Lys70 is forced to curve in the direction of the deacylating water molecule, which is then strongly bound and activated by this residue. However, a distance of ca 5 A separates the catalytic water molecule from the acyl carbonyl group of meropenem, a structural feature that accounts for the inhibition of OXA-13 by this drug. Finally, the low level of penicillinase activity revealed by the kinetic analysis of OXA-13 could be related to the specific presence in position 73 of a serine residue located close to the general base Lys70, which results in a decrease of the number of hydrogen-bonding interactions stabilising the catalytic water molecule. Copyright 2001 Academic Press.

Design and Implementation of an RTK-Based Vector Phase Locked Loop

PubMed Central

Shafaati, Ahmad; Lin, Tao; Broumandan, Ali; Lachapelle, Gérard

2018-01-01

This paper introduces a novel double-differential vector phase-locked loop (DD-VPLL) for Global Navigation Satellite Systems (GNSS) that leverages carrier phase position solutions as well as base station measurements in the estimation of rover tracking loop parameters. The use of double differencing alleviates the need for estimating receiver clock dynamics and atmospheric delays; therefore, the navigation filter consists of the baseline dynamic states only. It is shown that using vector processing for carrier phase tracking leads to a significant enhancement in the receiver sensitivity compared to using the conventional scalar-based tracking loop (STL) and vector frequency locked loop (VFLL). The sensitivity improvement of 8 to 10 dB compared to STL, and 7 to 8 dB compared to VFLL, is obtained based on the test cases reported in the paper. Also, an increased probability of ambiguity resolution in the proposed method results in better availability for real time kinematic (RTK) applications. PMID:29533994

Crystal structure of release factor RF3 trapped in the GTP state on a rotated conformation of the ribosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Jie; Lancaster, Laura; Trakhanov, Sergei

2012-03-26

The class II release factor RF3 is a GTPase related to elongation factor EF-G, which catalyzes release of class I release factors RF1 and RF2 from the ribosome after termination of protein synthesis. The 3.3 {angstrom} crystal structure of the RF3 {center_dot} GDPNP {center_dot} ribosome complex provides a high-resolution description of interactions and structural rearrangements that occur when binding of this translational GTPase induces large-scale rotational movements in the ribosome. RF3 induces a 7{sup o} rotation of the body and 14{sup o} rotation of the head of the 30S ribosomal subunit, and itself undergoes inter- and intradomain conformational rearrangements. Wemore » suggest that ordering of critical elements of switch loop I and the P loop, which help to form the GTPase catalytic site, are caused by interactions between the G domain of RF3 and the sarcin-ricin loop of 23S rRNA. The rotational movements in the ribosome induced by RF3, and its distinctly different binding orientation to the sarcin-ricin loop of 23S rRNA, raise interesting implications for the mechanism of action of EF-G in translocation.« less

The flexibility of a distant loop modulates active site motion and product release in ribonuclease A.

PubMed

Doucet, Nicolas; Watt, Eric D; Loria, J Patrick

2009-08-04

The role of the flexible loop 1 in protein conformational motion and in the dissociation of enzymatic product from ribonuclease A (RNase A) was investigated by creation of a chimeric enzyme in which a 6-residue loop 1 from the RNase A homologue, eosinophil cationic protein (ECP), replaced the 12-residue loop 1 in RNase A. The chimera (RNase A(ECP)) experiences only local perturbations in NMR backbone chemical shifts compared to WT RNase A. Many of the flexible residues that were previously identified in WT as involved in an important conformational change now experience no NMR-detected millisecond motions in the chimera. Likewise, binding of the product analogue, 3'-CMP, to RNase A(ECP) results in only minor chemical shift changes in the enzyme similar to what is observed for the H48A mutant of RNase A and in contrast to WT enzyme. For both RNase A(ECP) and H48A there is a 10-fold decrease in the product release rate constant, k(off), compared to WT, in agreement with previous studies indicating the importance of flexibility in RNase A in the overall rate-limiting product release step. Together, these NMR and biochemical experiments provide additional insight into the mechanism of millisecond motions in the RNase A catalytic cycle.

Concerted action of the MutLβ heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion

PubMed Central

Duroc, Yann; Kumar, Rajeev; Ranjha, Lepakshi; Adam, Céline; Guérois, Raphaël; Md Muntaz, Khan; Marsolier-Kergoat, Marie-Claude; Dingli, Florent; Laureau, Raphaëlle; Loew, Damarys; Llorente, Bertrand; Charbonnier, Jean-Baptiste; Cejka, Petr; Borde, Valérie

2017-01-01

Gene conversions resulting from meiotic recombination are critical in shaping genome diversification and evolution. How the extent of gene conversions is regulated is unknown. Here we show that the budding yeast mismatch repair related MutLβ complex, Mlh1-Mlh2, specifically interacts with the conserved meiotic Mer3 helicase, which recruits it to recombination hotspots, independently of mismatch recognition. This recruitment is essential to limit gene conversion tract lengths genome-wide, without affecting crossover formation. Contrary to expectations, Mer3 helicase activity, proposed to extend the displacement loop (D-loop) recombination intermediate, does not influence the length of gene conversion events, revealing non-catalytical roles of Mer3. In addition, both purified Mer3 and MutLβ preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutLβ is an integral part of a new regulatory step of meiotic recombination, which has implications to prevent rapid allele fixation and hotspot erosion in populations. DOI: http://dx.doi.org/10.7554/eLife.21900.001 PMID:28051769

Dynamics and unfolding pathway of chimeric azurin variants: insights from molecular dynamics simulation.

PubMed

Evoli, Stefania; Guzzi, Rita; Rizzuti, Bruno

2013-10-01

The spectroscopic, thermal, and functional properties of blue copper proteins can be modulated by mutations in the metal binding loop. Molecular dynamics simulation was used to compare the conformational properties of azurin and two chimeric variants, which were obtained by inserting into the azurin scaffold the copper binding loop of amicyanin and plastocyanin, respectively. Simulations at room temperature show that the proteins retain their overall structure and exhibit concerted motions among specific inner regions, as revealed by principal component analysis. Molecular dynamics at high temperature indicates that the first events in the unfolding pathway are structurally similar in the three proteins and unfolding starts from the region of the α-helix that is far from the metal binding loop. The results provide details of the denaturation process that are consistent with experimental data and in close agreement with other computational approaches, suggesting a distinct mechanism of unfolding of azurin and its chimeric variants. Moreover, differences observed in the dynamics of specific regions in the three proteins correlate with their thermal behavior, contributing to the determination of the basic factors that influence the stability.

Conformational and dynamics changes induced by bile acids binding to chicken liver bile acid binding protein.

PubMed

Eberini, Ivano; Guerini Rocco, Alessandro; Ientile, Anna Rita; Baptista, António M; Gianazza, Elisabetta; Tomaselli, Simona; Molinari, Henriette; Ragona, Laura

2008-06-01

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family. (c) 2008 Wiley-Liss, Inc.

Dynamics of the active site architecture in plant-type ferredoxin-NADP(+) reductases catalytic complexes.

PubMed

Sánchez-Azqueta, Ana; Catalano-Dupuy, Daniela L; López-Rivero, Arleth; Tondo, María Laura; Orellano, Elena G; Ceccarelli, Eduardo A; Medina, Milagros

2014-10-01

Kinetic isotope effects in reactions involving hydride transfer and their temperature dependence are powerful tools to explore dynamics of enzyme catalytic sites. In plant-type ferredoxin-NADP(+) reductases the FAD cofactor exchanges a hydride with the NADP(H) coenzyme. Rates for these processes are considerably faster for the plastidic members (FNR) of the family than for those belonging to the bacterial class (FPR). Hydride transfer (HT) and deuteride transfer (DT) rates for the NADP(+) coenzyme reduction of four plant-type FNRs (two representatives of the plastidic type FNRs and the other two from the bacterial class), and their temperature dependences are here examined applying a full tunnelling model with coupled environmental fluctuations. Parameters for the two plastidic FNRs confirm a tunnelling reaction with active dynamics contributions, but isotope effects on Arrhenius factors indicate a larger contribution for donor-acceptor distance (DAD) dynamics in the Pisum sativum FNR reaction than in the Anabaena FNR reaction. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT coordinate and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and rigid than those of FNRs. These differences must be due to adaptation of the active sites and catalytic mechanisms to fulfil their particular metabolic roles, establishing a compromise between protein flexibility and functional optimisation. Analysis of site-directed mutants in plastidic enzymes additionally indicates the requirement of a minimal optimal architecture in the catalytic complex to provide a favourable gating contribution. Copyright © 2014 Elsevier B.V. All rights reserved.

Dynamics of knotted flexible loops settling under a constant force in a viscous fluid

NASA Astrophysics Data System (ADS)

Gruziel, Magdalena; Thyagarajan, Krishnan; Dietler, Giovanni; Szymczak, Piotr; Ekiel-Jezewska, Maria

2017-11-01

Sedimenting chains of metal beads knotted to a topology of a torus knot tend to stabilize in the form of extended, flat, tightly interwound loops. In this configuration they perform an oscillatory motion of the loops swirling periodically around each other. Stokesian dynamics simulations of elastic fibers confirm the long-lasting character of the traveling wave-like swirling motion and show also the accompanying rotation of the system. Moreover, the periodic motion shows striking resemblance to the stable solutions for the evolution of vortices of torus knot topology. Using the results of the simulations we study the dependence of the frequencies and sedimentation velocities on the length of the fiber. We also notice the dependence of the knot dynamics on the bending stiffness of the fiber and the knot rank. NCN-2015/19/D/ST8/03199.

Structure of the kinase domain of Gilgamesh from Drosophila melanogaster

PubMed Central

Han, Ni; Chen, CuiCui; Shi, Zhubing; Cheng, Dianlin

2014-01-01

The CK1 family kinases regulate multiple cellular aspects and play important roles in Wnt/Wingless and Hedgehog signalling. The kinase domain of Drosophila Gilgamesh isoform I (Gilgamesh-I), a homologue of human CK1-γ, was purified and crystallized. Crystals of methylated Gilgamesh-I kinase domain with a D210A mutation diffracted to 2.85 Å resolution and belonged to space group P43212, with unit-cell parameters a = b = 52.025, c = 291.727 Å. The structure of Gilgamesh-I kinase domain, which was determined by molecular replacement, has conserved catalytic elements and an active conformation. Structural comparison indicates that an extended loop between the α1 helix and the β4 strand exists in the Gilgamesh-I kinase domain. This extended loop may regulate the activity and function of Gilgamesh-I. PMID:24699734

Study of a Simulation Tool to Determine Achievable Control Dynamics and Control Power Requirements with Perfect Tracking

NASA Technical Reports Server (NTRS)

Ostroff, Aaron J.

1998-01-01

This paper contains a study of two methods for use in a generic nonlinear simulation tool that could be used to determine achievable control dynamics and control power requirements while performing perfect tracking maneuvers over the entire flight envelope. The two methods are NDI (nonlinear dynamic inversion) and the SOFFT(Stochastic Optimal Feedforward and Feedback Technology) feedforward control structure. Equivalent discrete and continuous SOFFT feedforward controllers have been developed. These equivalent forms clearly show that the closed-loop plant model loop is a plant inversion and is the same as the NDI formulation. The main difference is that the NDI formulation has a closed-loop controller structure whereas SOFFT uses an open-loop command model. Continuous, discrete, and hybrid controller structures have been developed and integrated into the formulation. Linear simulation results show that seven different configurations all give essentially the same response, with the NDI hybrid being slightly different. The SOFFT controller gave better tracking performance compared to the NDI controller when a nonlinear saturation element was added. Future plans include evaluation using a nonlinear simulation.

Catalytic reaction processes revealed by scanning probe microscopy. [corrected].

PubMed

Jiang, Peng; Bao, Xinhe; Salmeron, Miquel

2015-05-19

Heterogeneous catalysis is of great importance for modern society. About 80% of the chemicals are produced by catalytic reactions. Green energy production and utilization as well as environmental protection also need efficient catalysts. Understanding the reaction mechanisms is crucial to improve the existing catalysts and develop new ones with better activity, selectivity, and stability. Three components are involved in one catalytic reaction: reactant, product, and catalyst. The catalytic reaction process consists of a series of elementary steps: adsorption, diffusion, reaction, and desorption. During reaction, the catalyst surface can change at the atomic level, with roughening, sintering, and segregation processes occurring dynamically in response to the reaction conditions. Therefore, it is imperative to obtain atomic-scale information for understanding catalytic reactions. Scanning probe microscopy (SPM) is a very appropriate tool for catalytic research at the atomic scale because of its unique atomic-resolution capability. A distinguishing feature of SPM, compared to other surface characterization techniques, such as X-ray photoelectron spectroscopy, is that there is no intrinsic limitation for SPM to work under realistic reaction conditions (usually high temperature and high pressure). Therefore, since it was introduced in 1981, scanning tunneling microscopy (STM) has been widely used to investigate the adsorption, diffusion, reaction, and desorption processes on solid catalyst surfaces at the atomic level. STM can also monitor dynamic changes of catalyst surfaces during reactions. These invaluable microscopic insights have not only deepened the understanding of catalytic processes, but also provided important guidance for the development of new catalysts. This Account will focus on elementary reaction processes revealed by SPM. First, we will demonstrate the power of SPM to investigate the adsorption and diffusion process of reactants on catalyst surfaces at the atomic level. Then the dynamic processes, including surface reconstruction, roughening, sintering, and phase separation, studied by SPM will be discussed. Furthermore, SPM provides valuable insights toward identifying the active sites and understanding the reaction mechanisms. We also illustrate here how both ultrahigh vacuum STM and high pressure STM provide valuable information, expanding the understanding provided by traditional surface science. We conclude with highlighting remarkable recent progress in noncontact atomic force microscopy (NC-AFM) and inelastic electron tunneling spectroscopy (IETS), and their impact on single-chemical-bond level characterization for catalytic reaction processes in the future.

Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme.

PubMed

McCord, Lauren A; Liang, Wenguang G; Dowdell, Evan; Kalas, Vasilios; Hoey, Robert J; Koide, Akiko; Koide, Shohei; Tang, Wei-Jen

2013-08-20

Insulin-degrading enzyme (IDE) selectively degrades the monomer of amyloidogenic peptides and contributes to clearance of amyloid β (Aβ). Thus, IDE retards the progression of Alzheimer's disease. IDE possesses an enclosed catalytic chamber that engulfs and degrades its peptide substrates; however, the molecular mechanism of IDE function, including substrate access to the chamber and recognition, remains elusive. Here, we captured a unique IDE conformation by using a synthetic antibody fragment as a crystallization chaperone. An unexpected displacement of a door subdomain creates an ~18-Å opening to the chamber. This swinging-door mechanism permits the entry of short peptides into the catalytic chamber and disrupts the catalytic site within IDE door subdomain. Given the propensity of amyloidogenic peptides to convert into β-strands for their polymerization into amyloid fibrils, they also use such β-strands to stabilize the disrupted catalytic site resided at IDE door subdomain for their degradation by IDE. Thus, action of the swinging door allows IDE to recognize amyloidogenicity by substrate-induced stabilization of the IDE catalytic cleft. Small angle X-ray scattering (SAXS) analysis revealed that IDE exists as a mixture of closed and open states. These open states, which are distinct from the swinging door state, permit entry of larger substrates (e.g., Aβ, insulin) to the chamber and are preferred in solution. Mutational studies confirmed the critical roles of the door subdomain and hinge loop joining the N- and C-terminal halves of IDE for catalysis. Together, our data provide insights into the conformational changes of IDE that govern the selective destruction of amyloidogenic peptides.

Spatiotemporal Dynamics of a Network of Coupled Time-Delay Digital Tanlock Loops

NASA Astrophysics Data System (ADS)

Paul, Bishwajit; Banerjee, Tanmoy; Sarkar, B. C.

The time-delay digital tanlock loop (TDTLs) is an important class of phase-locked loop that is widely used in electronic communication systems. Although nonlinear dynamics of an isolated TDTL has been studied in the past but the collective behavior of TDTLs in a network is an important topic of research and deserves special attention as in practical communication systems separate entities are rarely isolated. In this paper, we carry out the detailed analysis and numerical simulations to explore the spatiotemporal dynamics of a network of a one-dimensional ring of coupled TDTLs with nearest neighbor coupling. The equation representing the network is derived and we carry out analytical calculations using the circulant matrix formalism to obtain the stability criteria. An extensive numerical simulation reveals that with the variation of gain parameter and coupling strength the network shows a variety of spatiotemporal dynamics such as frozen random pattern, pattern selection, spatiotemporal intermittency and fully developed spatiotemporal chaos. We map the distinct dynamical regions of the system in two-parameter space. Finally, we quantify the spatiotemporal dynamics by using quantitative measures like Lyapunov exponent and the average quadratic deviation of the full network.

Sampling-based real-time motion planning under state uncertainty for autonomous micro-aerial vehicles in GPS-denied environments.

PubMed

Li, Dachuan; Li, Qing; Cheng, Nong; Song, Jingyan

2014-11-18

This paper presents a real-time motion planning approach for autonomous vehicles with complex dynamics and state uncertainty. The approach is motivated by the motion planning problem for autonomous vehicles navigating in GPS-denied dynamic environments, which involves non-linear and/or non-holonomic vehicle dynamics, incomplete state estimates, and constraints imposed by uncertain and cluttered environments. To address the above motion planning problem, we propose an extension of the closed-loop rapid belief trees, the closed-loop random belief trees (CL-RBT), which incorporates predictions of the position estimation uncertainty, using a factored form of the covariance provided by the Kalman filter-based estimator. The proposed motion planner operates by incrementally constructing a tree of dynamically feasible trajectories using the closed-loop prediction, while selecting candidate paths with low uncertainty using efficient covariance update and propagation. The algorithm can operate in real-time, continuously providing the controller with feasible paths for execution, enabling the vehicle to account for dynamic and uncertain environments. Simulation results demonstrate that the proposed approach can generate feasible trajectories that reduce the state estimation uncertainty, while handling complex vehicle dynamics and environment constraints.

Sampling-Based Real-Time Motion Planning under State Uncertainty for Autonomous Micro-Aerial Vehicles in GPS-Denied Environments

PubMed Central

Li, Dachuan; Li, Qing; Cheng, Nong; Song, Jingyan

2014-01-01

This paper presents a real-time motion planning approach for autonomous vehicles with complex dynamics and state uncertainty. The approach is motivated by the motion planning problem for autonomous vehicles navigating in GPS-denied dynamic environments, which involves non-linear and/or non-holonomic vehicle dynamics, incomplete state estimates, and constraints imposed by uncertain and cluttered environments. To address the above motion planning problem, we propose an extension of the closed-loop rapid belief trees, the closed-loop random belief trees (CL-RBT), which incorporates predictions of the position estimation uncertainty, using a factored form of the covariance provided by the Kalman filter-based estimator. The proposed motion planner operates by incrementally constructing a tree of dynamically feasible trajectories using the closed-loop prediction, while selecting candidate paths with low uncertainty using efficient covariance update and propagation. The algorithm can operate in real-time, continuously providing the controller with feasible paths for execution, enabling the vehicle to account for dynamic and uncertain environments. Simulation results demonstrate that the proposed approach can generate feasible trajectories that reduce the state estimation uncertainty, while handling complex vehicle dynamics and environment constraints. PMID:25412217

Application of the Tool for Turbine Engine Closed-loop Transient Analysis (TTECTrA) for Dynamic Systems Analysis

NASA Technical Reports Server (NTRS)

Csank, Jeffrey; Zinnecker, Alicia

2014-01-01

Systems analysis involves steady-state simulations of combined components to evaluate the steady-state performance, weight, and cost of a system; dynamic considerations are not included until later in the design process. The Dynamic Systems Analysis task, under NASAs Fixed Wing project, is developing the capability for assessing dynamic issues at earlier stages during systems analysis. To provide this capability the Tool for Turbine Engine Closed-loop Transient Analysis (TTECTrA) has been developed to design a single flight condition controller (defined as altitude and Mach number) and, ultimately, provide an estimate of the closed-loop performance of the engine model. This tool has been integrated with the Commercial Modular Aero-Propulsion System Simulation 40,000(CMAPSS40k) engine model to demonstrate the additional information TTECTrA makes available for dynamic systems analysis. This dynamic data can be used to evaluate the trade-off between performance and safety, which could not be done with steady-state systems analysis data. TTECTrA has been designed to integrate with any turbine engine model that is compatible with the MATLABSimulink (The MathWorks, Inc.) environment.

Application of the Tool for Turbine Engine Closed-loop Transient Analysis (TTECTrA) for Dynamic Systems Analysis

NASA Technical Reports Server (NTRS)

Csank, Jeffrey Thomas; Zinnecker, Alicia Mae

2014-01-01

Systems analysis involves steady-state simulations of combined components to evaluate the steady-state performance, weight, and cost of a system; dynamic considerations are not included until later in the design process. The Dynamic Systems Analysis task, under NASAs Fixed Wing project, is developing the capability for assessing dynamic issues at earlier stages during systems analysis. To provide this capability the Tool for Turbine Engine Closed-loop Transient Analysis (TTECTrA) has been developed to design a single flight condition controller (defined as altitude and Mach number) and, ultimately, provide an estimate of the closed-loop performance of the engine model. This tool has been integrated with the Commercial Modular Aero-Propulsion System Simulation 40,000 (CMAPSS 40k) engine model to demonstrate the additional information TTECTrA makes available for dynamic systems analysis. This dynamic data can be used to evaluate the trade-off between performance and safety, which could not be done with steady-state systems analysis data. TTECTrA has been designed to integrate with any turbine engine model that is compatible with the MATLAB Simulink (The MathWorks, Inc.) environment.

X-38 Experimental Controls Laws

NASA Technical Reports Server (NTRS)

Munday, Steve; Estes, Jay; Bordano, Aldo J.

2000-01-01

X-38 Experimental Control Laws X-38 is a NASA JSC/DFRC experimental flight test program developing a series of prototypes for an International Space Station (ISS) Crew Return Vehicle, often called an ISS "lifeboat." X- 38 Vehicle 132 Free Flight 3, currently scheduled for the end of this month, will be the first flight test of a modem FCS architecture called Multi-Application Control-Honeywell (MACH), originally developed by the Honeywell Technology Center. MACH wraps classical P&I outer attitude loops around a modem dynamic inversion attitude rate loop. The dynamic inversion process requires that the flight computer have an onboard aircraft model of expected vehicle dynamics based upon the aerodynamic database. Dynamic inversion is computationally intensive, so some timing modifications were made to implement MACH on the slower flight computers of the subsonic test vehicles. In addition to linear stability margin analyses and high fidelity 6-DOF simulation, hardware-in-the-loop testing is used to verify the implementation of MACH and its robustness to aerodynamic and environmental uncertainties and disturbances.

Improvements in flight table dynamic transparency for hardware-in-the-loop facilities

NASA Astrophysics Data System (ADS)

DeMore, Louis A.; Mackin, Rob; Swamp, Michael; Rusterholtz, Roger

2000-07-01

Flight tables are a 'necessary evil' in the Hardware-In-The- Loop (HWIL) simulation. Adding the actual or prototypic flight hardware to the loop, in order to increase the realism of the simulation, forces us to add motion simulation to the process. Flight table motion bases bring unwanted dynamics, non- linearities, transport delays, etc to an already difficult problem sometimes requiring the simulation engineer to compromise the results. We desire that the flight tables be 'dynamically transparent' to the simulation scenario. This paper presents a State Variable Feedback (SVF) control system architecture with feed-forward techniques that improves the flight table's dynamic transparency by significantly reducing the table's low frequency phase lag. We offer some actual results with existing flight tables that demonstrate the improved transparency. These results come from a demonstration conducted on a flight table in the KHILS laboratory at Eglin AFB and during a refurbishment of a flight table for the Boeing Company of St. Charles, Missouri.

Metalloprotein structures at ambient conditions and in real-time: biological crystallography and spectroscopy using X-ray free electron lasers

DOE PAGES

Kern, Jan; Yachandra, Vittal K.; Yano, Junko

2015-09-02

We have studied the structure of enzymes and the chemistry at the catalytic sites, intensively and have acquired an understanding of the atomic-scale chemistry which requires a new approach beyond steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure of metallo-enzymes at ambient conditions, while overcoming the severe X-ray-induced changes to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by the intense and ultra-short femtosecond (fs) X-ray pulses from an X-ray free electron laser (XFEL) by acquiring a signal before the samplemore » is destroyed. Our review describes the recent and pioneering uses of XFELs to study the protein structure and dynamics of metallo-enzymes using crystallography and scattering, as well as the chemical structure and dynamics of the catalytic complexes (charge, spin, and covalency) using spectroscopy during the reaction to understand the electron-transfer processes and elucidate the mechanism.« less

Metalloprotein structures at ambient conditions and in real-time: biological crystallography and spectroscopy using X-ray free electron lasers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kern, Jan; Yachandra, Vittal K.; Yano, Junko

We have studied the structure of enzymes and the chemistry at the catalytic sites, intensively and have acquired an understanding of the atomic-scale chemistry which requires a new approach beyond steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure of metallo-enzymes at ambient conditions, while overcoming the severe X-ray-induced changes to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by the intense and ultra-short femtosecond (fs) X-ray pulses from an X-ray free electron laser (XFEL) by acquiring a signal before the samplemore » is destroyed. Our review describes the recent and pioneering uses of XFELs to study the protein structure and dynamics of metallo-enzymes using crystallography and scattering, as well as the chemical structure and dynamics of the catalytic complexes (charge, spin, and covalency) using spectroscopy during the reaction to understand the electron-transfer processes and elucidate the mechanism.« less

A loop-based neural architecture for structured behavior encoding and decoding.

PubMed

Gisiger, Thomas; Boukadoum, Mounir

2018-02-01

We present a new type of artificial neural network that generalizes on anatomical and dynamical aspects of the mammal brain. Its main novelty lies in its topological structure which is built as an array of interacting elementary motifs shaped like loops. These loops come in various types and can implement functions such as gating, inhibitory or executive control, or encoding of task elements to name a few. Each loop features two sets of neurons and a control region, linked together by non-recurrent projections. The two neural sets do the bulk of the loop's computations while the control unit specifies the timing and the conditions under which the computations implemented by the loop are to be performed. By functionally linking many such loops together, a neural network is obtained that may perform complex cognitive computations. To demonstrate the potential offered by such a system, we present two neural network simulations. The first illustrates the structure and dynamics of a single loop implementing a simple gating mechanism. The second simulation shows how connecting four loops in series can produce neural activity patterns that are sufficient to pass a simplified delayed-response task. We also show that this network reproduces electrophysiological measurements gathered in various regions of the brain of monkeys performing similar tasks. We also demonstrate connections between this type of neural network and recurrent or long short-term memory network models, and suggest ways to generalize them for future artificial intelligence research. Copyright © 2017 Elsevier Ltd. All rights reserved.

Movement Forms: A Graph-Dynamic Perspective

PubMed Central

Saltzman, Elliot; Holt, Ken

2014-01-01

The focus of this paper is on characterizing the physical movement forms (e.g., walk, crawl, roll, etc.) that can be used to actualize abstract, functionally-specified behavioral goals (e.g., locomotion). Emphasis is placed on how such forms are distinguished from one another, in part, by the set of topological patterns of physical contact between agent and environment (i.e., the set of physical graphs associated with each form) and the transitions among these patterns displayed over the course of performance (i.e., the form’s physical graph dynamics). Crucial in this regard is the creation and dissolution of loops in these graphs, which can be related to the distinction between open and closed kinematic chains. Formal similarities are described within the theoretical framework of task-dynamics between physically-closed kinematic chains (physical loops) that are created during various movement forms and functionally-closed kinematic chains (functional loops) that are associated with task-space control of end-effectors; it is argued that both types of loop must be flexibly incorporated into the coordinative structures that govern skilled action. Final speculation is focused on the role of graphs and their dynamics, not only in processes of coordination and control for individual agents, but also in processes of inter-agent coordination and the coupling of agents with (non-sentient) environmental objects. PMID:24910507

Movement Forms: A Graph-Dynamic Perspective.

PubMed

Saltzman, Elliot; Holt, Ken

2014-01-01

The focus of this paper is on characterizing the physical movement forms (e.g., walk, crawl, roll, etc.) that can be used to actualize abstract, functionally-specified behavioral goals (e.g., locomotion). Emphasis is placed on how such forms are distinguished from one another, in part, by the set of topological patterns of physical contact between agent and environment (i.e., the set of physical graphs associated with each form) and the transitions among these patterns displayed over the course of performance (i.e., the form's physical graph dynamics ). Crucial in this regard is the creation and dissolution of loops in these graphs, which can be related to the distinction between open and closed kinematic chains. Formal similarities are described within the theoretical framework of task-dynamics between physically-closed kinematic chains (physical loops) that are created during various movement forms and functionally-closed kinematic chains (functional loops) that are associated with task-space control of end-effectors; it is argued that both types of loop must be flexibly incorporated into the coordinative structures that govern skilled action. Final speculation is focused on the role of graphs and their dynamics, not only in processes of coordination and control for individual agents, but also in processes of inter-agent coordination and the coupling of agents with (non-sentient) environmental objects.

Crystal Structure of Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26 at 0.95 Å Resolution: Dynamics of Catalytic Residues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oakley, Aaron J.; Klvana, Martin; Otyepka, Michal

We present the structure of LinB, a 33-kDa haloalkane dehalogenase from Sphingomonas paucimobilis UT26, at 0.95 {angstrom} resolution. The data have allowed us to directly observe the anisotropic motions of the catalytic residues. In particular, the side-chain of the catalytic nucleophile, Asp108, displays a high degree of disorder. It has been modeled in two conformations, one similar to that observed previously (conformation A) and one strained (conformation B) that approached the catalytic base (His272). The strain in conformation B was mainly in the C{sub {alpha}}-C{sub {beta}}-C{sub {gamma}} angle (126{sup o}) that deviated by 13.4{sup o} from the 'ideal' bond anglemore » of 112.6{sup o}. On the basis of these observations, we propose a role for the charge state of the catalytic histidine in determining the geometry of the catalytic residues. We hypothesized that double-protonation of the catalytic base (His272) reduces the distance between the side-chain of this residue and that of the Asp108. The results of molecular dynamics simulations were consistent with the structural data showing that protonation of the His272 side-chain nitrogen atoms does indeed reduce the distance between the side-chains of the residues in question, although the simulations failed to demonstrate the same degree of strain in the Asp108 C{sub {alpha}}-C{sub {beta}}-C{sub {gamma}} angle. Instead, the changes in the molecular dynamics structures were distributed over several bond and dihedral angles. Quantum mechanics calculations on LinB with 1-chloro-2,2-dimethylpropane as a substrate were performed to determine which active site conformations and protonation states were most likely to result in catalysis. It was shown that His272 singly protonated at N{sub {delta}1} and Asp108 in conformation A gave the most exothermic reaction ({Delta}H = -22 kcal/mol). With His272 doubly protonated at N{sub {delta}1} and N{sub {epsilon}2}, the reactions were only slightly exothermic or were endothermic. In all calculations starting with Asp108 in conformation B, the Asp108 C{sub {alpha}}-C{sub {beta}}-C{sub {gamma}} angle changed during the reaction and the Asp108 moved to conformation A. The results presented here indicate that the positions of the catalytic residues and charge state of the catalytic base are important for determining reaction energetics in LinB.« less

Dynamic coupling between the LID and NMP domain motions in the catalytic conversion of ATP and AMP to ADP by adenylate kinase

NASA Astrophysics Data System (ADS)

Jana, Biman; Adkar, Bharat V.; Biswas, Rajib; Bagchi, Biman

2011-01-01

The catalytic conversion of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP) by adenylate kinase (ADK) involves large amplitude, ligand induced domain motions, involving the opening and the closing of ATP binding domain (LID) and AMP binding domain (NMP) domains, during the repeated catalytic cycle. We discover and analyze an interesting dynamical coupling between the motion of the two domains during the opening, using large scale atomistic molecular dynamics trajectory analysis, covariance analysis, and multidimensional free energy calculations with explicit water. Initially, the LID domain must open by a certain amount before the NMP domain can begin to open. Dynamical correlation map shows interesting cross-peak between LID and NMP domain which suggests the presence of correlated motion between them. This is also reflected in our calculated two-dimensional free energy surface contour diagram which has an interesting elliptic shape, revealing a strong correlation between the opening of the LID domain and that of the NMP domain. Our free energy surface of the LID domain motion is rugged due to interaction with water and the signature of ruggedness is evident in the observed root mean square deviation variation and its fluctuation time correlation functions. We develop a correlated dynamical disorder-type theoretical model to explain the observed dynamic coupling between the motion of the two domains in ADK. Our model correctly reproduces several features of the cross-correlation observed in simulations.

3D-Stereoscopic Analysis of Solar Active Region Loops: I: SoHo/EIT Observations at Temperatures of 1.0-1.5 MK

NASA Technical Reports Server (NTRS)

Aschwanden, Markus J.; Newmark, Jeff; Delaboudiniere, Jean-Pierre; Neupert, Werner M.; Portier-Fozzani, Fabrice; Gary, G. Allen; Zucker, Arik

1998-01-01

The three-dimensional (3D) structure of solar active region NOAA 7986 observed on 1996 August 30 with the Extrem-ultraviolet Imaging Telescope (EIT) onboard the Solar and Heliospheric Observatory (SoHO) is analyzed. We develop a new method of Dynamic Stereoscopy to reconstruct the 3D geometry of dynamically changing loops, which allows us to determine the orientation of the loop plane with respect to the line-of-sight, a prerequisite to correct properly for projection effects in 3D loop models. With this method and the filter-ratio technique applied to EIT 171 A and 195 A images we determine the 3D coordinates (x(s), y(s), z(s)), the loop width) w(s), the electron density n(sub e)(s), and the electron temperature T(sub e)(s) as function of the loop length s for 30 loop segments. Fitting the loop densities with an exponential density model n(sub e)(h) we find that the so inferred scale height temperatures, T(sub e)(sup lambda) = 1.22 +/- 0.23 MK, match closely the EIT filter-ratio temperatures, T(sub e)(sup FIT) = 1.21 +/- 0.06 MK. We conclude that these rather large-scale loops (with heights of h approx. equals 50 - 200 Mm) that dominate EIT 171 A images are close to thermal equilibrium. Most of the loops show no significant thickness variation w(s), but many exhibit a trend of increasing temperature (dT/ds greater than 0) above the footpoint.

Radiation Enhanced Absorption of Frank Loops by Nanovoids in Cu

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Youxing; Zhang, Xinghang; Wang, Jian

Neutron and heavy ion irradiation generally induces voids in metallic materials, and continuous radiations typically result in void swelling and mechanical failure of the irradiated materials. Recent experiments showed that nanovoids in nanotwinned copper could act as sinks for radiation-induced Frank loops, significantly mitigating radiation damage [Y. Chen et al., Nat. Commun. 6:7036 (2015)]. In this paper, we report on structural evolution of Frank loops under cascades and address the role of nanovoids in absorbing Frank loops in detail by using molecular dynamics simulations. Results show that a stand-alone Frank loop is stable under cascades. When Frank loops are adjacentmore » to nanovoids, the diffusion of a group of atoms from the loop into nanovoids is accomplished via the formation and propagation of dislocation loops. The loop-nanovoid interactions result in the shrinkage of the nanovoids and the Frank loops.« less

Radiation Enhanced Absorption of Frank Loops by Nanovoids in Cu

DOE PAGES

Chen, Youxing; Zhang, Xinghang; Wang, Jian

2016-11-01

Neutron and heavy ion irradiation generally induces voids in metallic materials, and continuous radiations typically result in void swelling and mechanical failure of the irradiated materials. Recent experiments showed that nanovoids in nanotwinned copper could act as sinks for radiation-induced Frank loops, significantly mitigating radiation damage [Y. Chen et al., Nat. Commun. 6:7036 (2015)]. In this paper, we report on structural evolution of Frank loops under cascades and address the role of nanovoids in absorbing Frank loops in detail by using molecular dynamics simulations. Results show that a stand-alone Frank loop is stable under cascades. When Frank loops are adjacentmore » to nanovoids, the diffusion of a group of atoms from the loop into nanovoids is accomplished via the formation and propagation of dislocation loops. The loop-nanovoid interactions result in the shrinkage of the nanovoids and the Frank loops.« less

Asymmetric Preorganization of Inverted Pair Residues in the Sodium-Calcium Exchanger

PubMed Central

Giladi, Moshe; Almagor, Lior; van Dijk, Liat; Hiller, Reuben; Man, Petr; Forest, Eric; Khananshvili, Daniel

2016-01-01

In analogy with many other proteins, Na+/Ca2+ exchangers (NCX) adapt an inverted twofold symmetry of repeated structural elements, while exhibiting a functional asymmetry by stabilizing an outward-facing conformation. Here, structure-based mutant analyses of the Methanococcus jannaschii Na+/Ca2+ exchanger (NCX_Mj) were performed in conjunction with HDX-MS (hydrogen/deuterium exchange mass spectrometry) to identify the structure-dynamic determinants of functional asymmetry. HDX-MS identified hallmark differences in backbone dynamics at ion-coordinating residues of apo-NCX_Mj, whereas Na+or Ca2+ binding to the respective sites induced relatively small, but specific, changes in backbone dynamics. Mutant analysis identified ion-coordinating residues affecting the catalytic capacity (kcat/Km), but not the stability of the outward-facing conformation. In contrast, distinct “noncatalytic” residues (adjacent to the ion-coordinating residues) control the stability of the outward-facing conformation, but not the catalytic capacity. The helix-breaking signature sequences (GTSLPE) on the α1 and α2 repeats (at the ion-binding core) differ in their folding/unfolding dynamics, while providing asymmetric contributions to transport activities. The present data strongly support the idea that asymmetric preorganization of the ligand-free ion-pocket predefines catalytic reorganization of ion-bound residues, where secondary interactions with adjacent residues couple the alternating access. These findings provide a structure-dynamic basis for ion-coupled alternating access in NCX and similar proteins. PMID:26876271

Probing Ultrafast Electron Dynamics at Surfaces Using Soft X-Ray Transient Reflectivity Spectroscopy

NASA Astrophysics Data System (ADS)

Baker, L. Robert; Husek, Jakub; Biswas, Somnath; Cirri, Anthony

The ability to probe electron dynamics with surface sensitivity on the ultrafast time scale is critical for understanding processes such as charge separation, injection, and surface trapping that mediate efficiency in catalytic and energy conversion materials. Toward this goal, we have developed a high harmonic generation (HHG) light source for femtosecond soft x-ray reflectivity. Using this light source we investigated the ultrafast carrier dynamics at the surface of single crystalline α-Fe2O3, polycrystalline α-Fe2O3, and the mixed metal oxide, CuFeO2. We have recently demonstrated that CuFeO2 in particular is a selective catalyst for photo-electrochemical CO2 reduction to acetate; however, the role of electronic structure and charge carrier dynamics in mediating catalytic selectivity has not been well understood. Soft x-ray reflectivity measurements probe the M2,3, edges of the 3d transition metals, which provide oxidation and spin state resolution with element specificity. In addition to chemical state specificity, these measurements are also surface sensitive, and by independently simulating the contributions of the real and imaginary components of the complex refractive index, we can differentiate between surface and sub-surface contributions to the excited state spectrum. Accordingly, this work demonstrates the ability to probe ultrafast carrier dynamics in catalytic materials with element and chemical state specificity and with surface sensitivity.

Dynamics of safety performance and culture: a group model building approach.

PubMed

Goh, Yang Miang; Love, Peter E D; Stagbouer, Greg; Annesley, Chris

2012-09-01

The management of occupational health and safety (OHS) including safety culture interventions is comprised of complex problems that are often hard to scope and define. Due to the dynamic nature and complexity of OHS management, the concept of system dynamics (SD) is used to analyze accident prevention. In this paper, a system dynamics group model building (GMB) approach is used to create a causal loop diagram of the underlying factors influencing the OHS performance of a major drilling and mining contractor in Australia. While the organization has invested considerable resources into OHS their disabling injury frequency rate (DIFR) has not been decreasing. With this in mind, rich individualistic knowledge about the dynamics influencing the DIFR was acquired from experienced employees with operations, health and safety and training background using a GMB workshop. Findings derived from the workshop were used to develop a series of causal loop diagrams that includes a wide range of dynamics that can assist in better understanding the causal influences OHS performance. The causal loop diagram provides a tool for organizations to hypothesize the dynamics influencing effectiveness of OHS management, particularly the impact on DIFR. In addition the paper demonstrates that the SD GMB approach has significant potential in understanding and improving OHS management. Copyright © 2011 Elsevier Ltd. All rights reserved.

Many-body dynamics of chemically propelled nanomotors

NASA Astrophysics Data System (ADS)

Colberg, Peter H.; Kapral, Raymond

2017-08-01

The collective behavior of chemically propelled sphere-dimer motors made from linked catalytic and noncatalytic spheres in a quasi-two-dimensional confined geometry is studied using a coarse-grained microscopic dynamical model. Chemical reactions at the catalytic spheres that convert fuel to product generate forces that couple to solvent degrees of freedom as a consequence of momentum conservation in the microscopic dynamics. The collective behavior of the many-body system is influenced by direct intermolecular interactions among the motors, chemotactic effects due to chemical gradients, hydrodynamic coupling, and thermal noise. Segregation into high and low density phases and globally homogeneous states with strong fluctuations are investigated as functions of the motor characteristics. Factors contributing to this behavior are discussed in the context of active Brownian models.

Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

DOE PAGES

Manthei, Kelly A.; Hill, Morgan C.; Burke, Jordan E.; ...

2015-03-23

RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. In this paper, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ~90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATPmore » hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. Finally, this bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.« less

Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases.

PubMed

Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X Edward; West, Graham M; Kovach, Amanda; Tan, M H Eileen; Suino-Powell, Kelly M; He, Yuanzheng; Xu, Yong; Chalmers, Michael J; Brunzelle, Joseph S; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R; Melcher, Karsten; Xu, H Eric

2012-01-06

Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

Dynamic Modification of Pore Opening of SAPO-34 by Adsorbed Surface Methoxy Species during Induction of Catalytic Methanol-to-Olefins Reactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lo, Benedict T W.; Ye, Lin; Change, G.G. Z.

Here, we report that the pore opening of SAPO-34 can be significantly modified by an adsorbed surface methoxy species during induction of the catalytic methanol-to-olefins process, which offers molecular sieving properties due to physical obstacle of the methoxy group and its adsorption modification to other hydrocarbons. X-ray powder diffraction and Rietveld refinement clearly reveal that the adsorbed single carbon atom as the methoxy group is dynamically created from methanol dehydration on a Brønsted acid site in close proximity to the pore windows. As a result, industrial desirable smaller olefins such as ethylene and propylene can be favourably made at themore » expenses of higher olefins. The structures and fundamental understanding in alteration in the olefins selectivity during induction may allow rational optimisation in catalytic performance under the complex fluidisation conditions.« less

Conserved water-mediated H-bonding dynamics of catalytic Asn 175 in plant thiol protease.

PubMed

Nandi, Tapas K; Bairagya, Hridoy R; Mukhopadhyay, Bishnu P; Sekar, K; Sukul, Dipankar; Bera, Asim K

2009-03-01

The role of invariant water molecules in the activity of plant cysteine protease is ubiquitous in nature. On analysing the 11 different Protein DataBank (PDB) structures of plant thiol proteases, the two invariant water molecules W1 and W2 (W220 and W222 in the template 1PPN structure) were observed to form H-bonds with the O b atom of Asn 175. Extensive energy minimization and molecular dynamics simulation studies up to 2 ns on all the PDB and solvated structures clearly revealed the involvement of the H-bonding association of the two water molecules in fixing the orientation of the asparagine residue of the catalytic triad. From this study,it is suggested that H-bonding of the water molecule at the W1 invariant site better stabilizes the Asn residue at the active site of the catalytic triad.

Protein Topology Determines Cysteine Oxidation Fate: The Case of Sulfenyl Amide Formation among Protein Families

PubMed Central

Defelipe, Lucas A.; Lanzarotti, Esteban; Gauto, Diego; Marti, Marcelo A.; Turjanski, Adrián G.

2015-01-01

Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function. PMID:25741692

Active site remodeling during the catalytic cycle in metal-dependent fructose-1,6-bisphosphate aldolases.

PubMed

Jacques, Benoit; Coinçon, Mathieu; Sygusch, Jurgen

2018-03-28

Crystal structures of two bacterial metal (Zn) dependent D-fructose 1,6-bisphosphate (FBP) aldolases in complex with substrate, analogues, and triose-P reaction products were determined to 1.5-2.0 Å resolution. The ligand complexes cryotrapped in native or mutant H. pylori aldolase crystals enabled a novel mechanistic description of FBP C 3 -C 4 bond cleavage. The reaction mechanism uses active site remodelling during the catalytic cycle implicating relocation of the Zn cofactor that is mediated by conformational changes of active site loops. Substrate binding initiates conformational changes, triggered upon P 1 -phosphate binding, which liberates the Zn chelating His180, allowing it to act as a general base for the proton abstraction at the FBP C 4 -hydroxyl group. A second zinc chelating His83 hydrogen bonds the substrate C 4 - hydroxyl group and assists cleavage by stabilizing the developing negative charge during proton abstraction. Cleavage is concerted with relocation of the metal cofactor from an interior to a surface exposed site, thereby stabilizing the nascent enediolate form. Conserved residue Glu142 is essential for protonation of the enediolate form, prior to product release. A D-tagatose 1,6-bisphosphate enzymatic complex reveals how His180 mediated proton abstraction controls stereospecificity of the cleavage reaction. Recognition and discrimination of the reaction products, dihydroxyacetone-P and D-glyceraldehyde-3-P, occurs via charged hydrogen bonds between hydroxyl groups of the triose-Ps and conserved residues, Asp82 and Asp255, respectively, and are crucial aspects of the enzyme's role in gluconeogenesis. Conformational changes in mobile loops β5-α7 and β6-α8 (containing catalytic residues Glu142 and His180, respectively) drive active site remodelling enabling the relocation of the metal cofactor. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

A class III chitinase without disulfide bonds from the fern, Pteris ryukyuensis: crystal structure and ligand-binding studies.

PubMed

Kitaoku, Yoshihito; Umemoto, Naoyuki; Ohnuma, Takayuki; Numata, Tomoyuki; Taira, Toki; Sakuda, Shohei; Fukamizo, Tamo

2015-10-01

We first solved the crystal structure of class III catalytic domain of a chitinase from fern (PrChiA-cat), and found a structural difference between PrChiA-cat and hevamine. PrChiA-cat was found to have reduced affinities to chitin oligosaccharides and allosamidin. Plant class III chitinases are subdivided into enzymes with three disulfide bonds and those without disulfide bonds. We here referred to the former enzymes as class IIIa chitinases and the latter as class IIIb chitinases. In this study, we solved the crystal structure of the class IIIb catalytic domain of a chitinase from the fern Pteris ryukyuensis (PrChiA-cat), and compared it with that of hevamine, a class IIIa chitinase from Hevea brasiliensis. PrChiA-cat was found to adopt an (α/β)8 fold typical of GH18 chitinases in a similar manner to that of hevamine. However, PrChiA-cat also had two large loops that extruded from the catalytic site, and the corresponding loops in hevamine were markedly smaller than those of PrChiA-cat. An HPLC analysis of the enzymatic products revealed that the mode of action of PrChiA-cat toward chitin oligosaccharides, (GlcNAc) n (n = 4-6), differed from those of hevamine and the other class IIIa chitinases. The binding affinities of (GlcNAc)3 and (GlcNAc)4 toward the inactive mutant of PrChiA-cat were determined by isothermal titration calorimetry, and were markedly lower than those toward other members of the GH18 family. The affinity and the inhibitory activity of allosamidin toward PrChiA-cat were also lower than those toward the GH18 chitinases investigated to date. Several hydrogen bonds found in the crystal structure of hevamine-allosamidin complex were missing in the modeled structure of PrChiA-cat-allosamidin complex. The structural findings for PrChiA-cat successfully interpreted the functional data presented.

Tackling Critical Catalytic Residues in Helicobacter pylori l-Asparaginase

PubMed Central

Maggi, Maristella; Chiarelli, Laurent R; Valentini, Giovanna; Scotti, Claudia

2015-01-01

Bacterial asparaginases (amidohydrolases, EC 3.5.1.1) are important enzymes in cancer therapy, especially for Acute Lymphoblastic Leukemia. They are tetrameric enzymes able to catalyze the deamination of l-ASN and, to a variable extent, of l-GLN, on which leukemia cells are dependent for survival. In contrast to other known l-asparaginases, Helicobacter pylori CCUG 17874 type II enzyme (HpASNase) is cooperative and has a low affinity towards l-GLN. In this study, some critical amino acids forming the active site of HpASNase (T16, T95 and E289) have been tackled by rational engineering in the attempt to better define their role in catalysis and to achieve a deeper understanding of the peculiar cooperative behavior of this enzyme. Mutations T16E, T95D and T95H led to a complete loss of enzymatic activity. Mutation E289A dramatically reduced the catalytic activity of the enzyme, but increased its thermostability. Interestingly, E289 belongs to a loop that is very variable in l-asparaginases from the structure, sequence and length point of view, and which could be a main determinant of their different catalytic features. PMID:25826146

Tackling Critical Catalytic Residues in Helicobacter pylori L-Asparaginase.

PubMed

Maggi, Maristella; Chiarelli, Laurent R; Valentini, Giovanna; Scotti, Claudia

2015-03-27

Bacterial asparaginases (amidohydrolases, EC 3.5.1.1) are important enzymes in cancer therapy, especially for Acute Lymphoblastic Leukemia. They are tetrameric enzymes able to catalyze the deamination of L-ASN and, to a variable extent, of L-GLN, on which leukemia cells are dependent for survival. In contrast to other known L-asparaginases, Helicobacter pylori CCUG 17874 type II enzyme (HpASNase) is cooperative and has a low affinity towards L-GLN. In this study, some critical amino acids forming the active site of HpASNase (T16, T95 and E289) have been tackled by rational engineering in the attempt to better define their role in catalysis and to achieve a deeper understanding of the peculiar cooperative behavior of this enzyme. Mutations T16E, T95D and T95H led to a complete loss of enzymatic activity. Mutation E289A dramatically reduced the catalytic activity of the enzyme, but increased its thermostability. Interestingly, E289 belongs to a loop that is very variable in L-asparaginases from the structure, sequence and length point of view, and which could be a main determinant of their different catalytic features.

Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase.

PubMed

Asgeirsson, Bjarni; Adalbjörnsson, Björn Vidar; Gylfason, Gudjón Andri

2007-06-01

Alkaline phosphatase is an extracellular enzyme that is membrane-bound in eukaryotes but resides in the periplasmic space of bacteria. It normally carries four cysteine residues that form two disulfide bonds, for instance in the APs of Escherichia coli and vertebrates. An AP variant from a Vibrio sp. has only one cysteine residue. This cysteine is second next to the nucleophilic serine in the active site. We have individually modified seven residues to cysteine that are on two loops predicted to be within a 5 A radius. Four of them formed a disulfide bond to the endogenous cysteine. Thermal stability was monitored by circular dichroism and activity measurements. Global stability was similar to the wild-type enzyme. However, a significant increase in heat-stability was observed for the disulfide-containing variants using activity as a measure, together with a large reduction in catalytic rates (k(cat)) and a general decrease in Km values. The results suggest that a high degree of mobility near the active site and in the helix carrying the endogenous cysteine is essential for full catalytic efficiency in the cold-adapted AP.

Initial Binding of Ions to the Interhelical Loops of Divalent Ion Transporter CorA: Replica Exchange Molecular Dynamics Simulation Study

PubMed Central

Zhang, Tong; Mu, Yuguang

2012-01-01

Crystal structures of Thermotoga maritima magnesium transporter CorA, reported in 2006, revealed its homo-pentameric constructions. However, the structure of the highly conserved extracellular interhelical loops remains unsolved, due to its high flexibility. We have explored the configurations of the loops through extensive replica exchange molecular dynamics simulations in explicit solvent model with the presence of either Co(III) Hexamine ions or Mg2+ ions. We found that there are multiple binding sites available on the interhelical loops in which the negatively charged residues, E316 and E320, are located notably close to the positively charged ions during the simulations. Our simulations resolved the distinct binding patterns of the two kinds of ions: Co(III) Hexamine ions were found to bind stronger with the loop than Mg2+ ions with binding free energy −7.3 kJ/mol lower, which is nicely consistent with the previous data. Our study provides an atomic basis description of the initial binding process of Mg2+ ions on the extracellular interhelical loops of CorA and the detailed inhibition mechanism of Co(III) Hexamine ions on CorA ions transportation. PMID:22952795

Closed-Loop HIRF Experiments Performed on a Fault Tolerant Flight Control Computer

NASA Technical Reports Server (NTRS)

Belcastro, Celeste M.

1997-01-01

ABSTRACT Closed-loop HIRF experiments were performed on a fault tolerant flight control computer (FCC) at the NASA Langley Research Center. The FCC used in the experiments was a quad-redundant flight control computer executing B737 Autoland control laws. The FCC was placed in one of the mode-stirred reverberation chambers in the HIRF Laboratory and interfaced to a computer simulation of the B737 flight dynamics, engines, sensors, actuators, and atmosphere in the Closed-Loop Systems Laboratory. Disturbances to the aircraft associated with wind gusts and turbulence were simulated during tests. Electrical isolation between the FCC under test and the simulation computer was achieved via a fiber optic interface for the analog and discrete signals. Closed-loop operation of the FCC enabled flight dynamics and atmospheric disturbances affecting the aircraft to be represented during tests. Upset was induced in the FCC as a result of exposure to HIRF, and the effect of upset on the simulated flight of the aircraft was observed and recorded. This paper presents a description of these closed- loop HIRF experiments, upset data obtained from the FCC during these experiments, and closed-loop effects on the simulated flight of the aircraft.

Implementation of Push Recovery Strategy Using Triple Linear Inverted Pendulum Model in “T-FloW” Humanoid Robot

NASA Astrophysics Data System (ADS)

Dimas Pristovani, R.; Raden Sanggar, D.; Dadet, Pramadihanto.

2018-04-01

Push recovery is one of humanbehaviorwhich is a strategy to defend the body from anexternal force in any environment. This paper describes push recovery strategy which usesMIMO decoupled control system method. The dynamics system uses aquasi-dynamic system based on triple linear inverted pendulum model (TLIPM). The analysis of TLIPMuses zero moment point (ZMP) calculation from ZMP simplification in last research. By using this simplification of dynamics system, the control design can be simplified into 3 serial SISOwith known and uncertain disturbance models in each inverted pendulum. Each pendulum has different plan to damp the external force effect. In this experiment, PID controller (closed- loop)is used to arrange the damp characteristic.The experiment result shows thatwhen using push recovery control strategy (closed-loop control) is about 85.71% whilewithout using push recovery control strategy (open-loop control) it is about 28.57%.

Hybrid automata models of cardiac ventricular electrophysiology for real-time computational applications.

PubMed

Andalam, Sidharta; Ramanna, Harshavardhan; Malik, Avinash; Roop, Parthasarathi; Patel, Nitish; Trew, Mark L

2016-08-01

Virtual heart models have been proposed for closed loop validation of safety-critical embedded medical devices, such as pacemakers. These models must react in real-time to off-the-shelf medical devices. Real-time performance can be obtained by implementing models in computer hardware, and methods of compiling classes of Hybrid Automata (HA) onto FPGA have been developed. Models of ventricular cardiac cell electrophysiology have been described using HA which capture the complex nonlinear behavior of biological systems. However, many models that have been used for closed-loop validation of pacemakers are highly abstract and do not capture important characteristics of the dynamic rate response. We developed a new HA model of cardiac cells which captures dynamic behavior and we implemented the model in hardware. This potentially enables modeling the heart with over 1 million dynamic cells, making the approach ideal for closed loop testing of medical devices.

Dynamics for a 2-vertex quantum gravity model

NASA Astrophysics Data System (ADS)

Borja, Enrique F.; Díaz-Polo, Jacobo; Garay, Iñaki; Livine, Etera R.

2010-12-01

We use the recently introduced U(N) framework for loop quantum gravity to study the dynamics of spin network states on the simplest class of graphs: two vertices linked with an arbitrary number N of edges. Such graphs represent two regions, in and out, separated by a boundary surface. We study the algebraic structure of the Hilbert space of spin networks from the U(N) perspective. In particular, we describe the algebra of operators acting on that space and discuss their relation to the standard holonomy operator of loop quantum gravity. Furthermore, we show that it is possible to make the restriction to the isotropic/homogeneous sector of the model by imposing the invariance under a global U(N) symmetry. We then propose a U(N)-invariant Hamiltonian operator and study the induced dynamics. Finally, we explore the analogies between this model and loop quantum cosmology and sketch some possible generalizations of it.

System identification of dynamic closed-loop control of total peripheral resistance by arterial and cardiopulmonary baroreceptors

NASA Astrophysics Data System (ADS)

Nikolai Aljuri, A.; Bursac, Nenad; Marini, Robert; Cohen, Richard J.

2001-08-01

Prolonged exposure to microgravity in space flight missions (days) impairs the mechanisms responsible for defense of arterial blood pressure (ABP) and cardiac output (CO) against orthostatic stress in the post-flight period. The mechanisms responsible for the observed orthostatic intolerance are not yet completely understood. Additionally, effective counter measures to attenuate this pathophysiological response are not available. The aim of this study was to investigate the ability of our proposed system identification method to predict closed-loop dynamic changes in TPR induced by changes in mean arterial pressure (MAP) and right atrial pressure (RAP). For this purpose we designed and employed a novel experimental animal model for the examination of arterial and cardiopulmonary baroreceptors in the dynamic closed-loop control of total peripheral resistance (TPR), and applied system identification to the analysis of beat-to-beat fluctuations in the measured signals.

Structural Studies of a Rationally Selected Multi-Drug Resistant HIV-1 Protease Reveal Synergistic Effect of Distal Mutations on Flap Dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agniswamy, Johnson; Louis, John M.; Roche, Julien

We report structural analysis of HIV protease variant PRS17 which was rationally selected by machine learning to represent wide classes of highly drug-resistant variants. Crystal structures were solved of PRS17 in the inhibitor-free form and in complex with antiviral inhibitor, darunavir. Despite its 17 mutations, PRS17 has only one mutation (V82S) in the inhibitor/substrate binding cavity, yet exhibits high resistance to all clinical inhibitors. PRS17 has none of the major mutations (I47V, I50V, I54ML, L76V and I84V) associated with darunavir resistance, but has 10,000-fold weaker binding affinity relative to the wild type PR. Comparable binding affinity of 8000-fold weaker thanmore » PR is seen for drug resistant mutant PR20, which bears 3 mutations associated with major resistance to darunavir (I47V, I54L and I84V). Inhibitor-free PRS17 shows an open flap conformation with a curled tip correlating with G48V flap mutation. NMR studies on inactive PRS17 D25N unambiguously confirm that the flaps adopt mainly an open conformation in solution very similar to that in the inhibitor-free crystal structure. In PRS17, the hinge loop cluster of mutations, E35D, M36I and S37D, contributes to the altered flap dynamics by a mechanism similar to that of PR20. An additional K20R mutation anchors an altered conformation of the hinge loop. Flap mutations M46L and G48V in PRS17/DRV complex alter the Phe53 conformation by steric hindrance between the side chains. Unlike the L10F mutation in PR20, L10I in PRS17 does not break the inter-subunit ion pair or diminish the dimer stability, consistent with a very low dimer dissociation constant comparable to that of wild type PR. Distal mutations A71V, L90M and I93L propagate alterations to the catalytic site of PRS17. PRS17 exhibits a molecular mechanism whereby mutations act synergistically to alter the flap dynamics resulting in significantly weaker binding yet maintaining active site contacts with darunavir.« less

Dynamically limiting energy consumed by cooling apparatus

DOEpatents

Chainer, Timothy J.; David, Milnes P.; Iyengar, Madhusudan K.; Parida, Pritish R.; Schmidt, Roger R.; Schultz, Mark D.

2015-05-26

Cooling apparatuses and methods are provided which include one or more coolant-cooled structures associated with an electronics rack, a coolant loop coupled in fluid communication with one or more passages of the coolant-cooled structure(s), one or more heat exchange units coupled to facilitate heat transfer from coolant within the coolant loop, and N controllable components associated with the coolant loop or the heat exchange unit(s), wherein N.gtoreq.1. The N controllable components facilitate circulation of coolant through the coolant loop or transfer of heat from the coolant via the heat exchange unit(s). A controller is coupled to the N controllable components, and dynamically adjusts operation of the N controllable components, based on Z input parameters and one or more specified constraints, to provide a specified cooling to the coolant-cooled structure(s), while limiting energy consumed by the N controllable components, wherein Z.gtoreq.1.

Dynamically limiting energy consumed by cooling apparatus

DOEpatents

Chainer, Timothy J.; David, Milnes P.; Iyengar, Madhusudan K.; Parida, Pritish R.; Schmidt, Roger R.; Schultz, Mark D.

2015-06-09

Cooling methods are provided which include providing: one or more coolant-cooled structures associated with an electronics rack, a coolant loop coupled in fluid communication with one or more passages of the coolant-cooled structure(s), one or more heat exchange units coupled to facilitate heat transfer from coolant within the coolant loop, and N controllable components associated with the coolant loop or the heat exchange unit(s), wherein N.gtoreq.1. The N controllable components facilitate circulation of coolant through the coolant loop or transfer of heat from the coolant via the heat exchange unit(s). A controller is also provided to dynamically adjust operation of the N controllable components, based on Z input parameters and one or more specified constraints, and provide a specified cooling to the coolant-cooled structure(s), while limiting energy consumed by the N controllable components, wherein Z.gtoreq.1.

The Solar Dynamic radiator with a historical perspective

NASA Technical Reports Server (NTRS)

Mclallin, K. L.; Fleming, M. L.; Hoehn, F. W.; Howerton, R.

1988-01-01

A historical perspective on pumped loop space radiators provides a basis for the design of the Space Station Solar Dynamic (SD) power module radiator. SD power modules, capable of generating 25 kWe each, are planned for growth Station power requirements. The Brayton (cycle) SD module configuration incorporates a pumped loop radiator that must reject up to 99 kW. The thermal/hydraulic design conditions in combination with required radiator orientation and packaging envelope form a unique set of constraints as compared to previous pumped loop radiator systems. Nevertheless, past program successes have demonstrated a technology base which can be applied to the SD radiator development program to ensure a low risk, low cost system.

Growth rate effects on the formation of dislocation loops around deep helium bubbles in Tungsten

DOE PAGES

Sandoval, Luis; Perez, Danny; Uberuaga, Blas P.; ...

2016-11-15

Here, the growth process of spherical helium bubbles located 6 nm below a (100) surface is studied using molecular dynamics and parallel replica dynamics simulations, over growth rates from 10 6 to 10 12 helium atoms per second. Slower growth rates lead to a release of pressure and lower helium content as compared with fast growth cases. In addition, at slower growth rates, helium bubbles are not decorated by multiple dislocation loops, as these tend to merge or emit given sufficient time. At faster rates, dislocation loops nucleate faster than they can emit, leading to a more complicated dislocation structuremore » around the bubble.« less

Silicon photonic dynamic optical channel leveler with external feedback loop.

PubMed

Doylend, J K; Jessop, P E; Knights, A P

2010-06-21

We demonstrate a dynamic optical channel leveler composed of a variable optical attenuator (VOA) integrated monolithically with a defect-mediated photodiode in a silicon photonic waveguide device. An external feedback loop mimics an analog circuit such that the photodiode directly controls the VOA to provide blind channel leveling within +/-1 dB across a 7-10 dB dynamic range for wavelengths from 1530 nm to 1570 nm. The device consumes approximately 50 mW electrical power and occupies a 6 mm x 0.1 mm footprint per channel. Dynamic leveling is accomplished without tapping optical power from the output path to the photodiode and thus the loss penalty is minimized.

Supercomputer optimizations for stochastic optimal control applications

NASA Technical Reports Server (NTRS)

Chung, Siu-Leung; Hanson, Floyd B.; Xu, Huihuang

1991-01-01

Supercomputer optimizations for a computational method of solving stochastic, multibody, dynamic programming problems are presented. The computational method is valid for a general class of optimal control problems that are nonlinear, multibody dynamical systems, perturbed by general Markov noise in continuous time, i.e., nonsmooth Gaussian as well as jump Poisson random white noise. Optimization techniques for vector multiprocessors or vectorizing supercomputers include advanced data structures, loop restructuring, loop collapsing, blocking, and compiler directives. These advanced computing techniques and superconducting hardware help alleviate Bellman's curse of dimensionality in dynamic programming computations, by permitting the solution of large multibody problems. Possible applications include lumped flight dynamics models for uncertain environments, such as large scale and background random aerospace fluctuations.

Dynamic magnetic hysteresis properties of two-dimensional ferrimagnetic structures containing high-spin (S = 5/2) and low-spin (S = 1/2)

NASA Astrophysics Data System (ADS)

Batı, Mehmet; Ertaş, Mehmet

2017-09-01

The dynamic hysteresis behaviors of a containing high spin-5/2 and low spin-1/2 Ising ferrimagnetic system on a square lattice are studied by using the dynamic mean-field approximation. The influences of the temperature, the single-ion anisotropy and the frequency on dynamic hysteresis behaviors are investigated in detail. Somewhat characteristic behaviors are found, such as the presence of triple hysteresis loop for appropriate values of the crystal field or temperature. Besides, we observed that, hysteresis loop area and phase transition points are very sensitive to changes in frequency and thus have profound importance in device application.

Analysis of in vitro evolution reveals the underlying distribution of catalytic activity among random sequences.

PubMed

Pressman, Abe; Moretti, Janina E; Campbell, Gregory W; Müller, Ulrich F; Chen, Irene A

2017-08-21

The emergence of catalytic RNA is believed to have been a key event during the origin of life. Understanding how catalytic activity is distributed across random sequences is fundamental to estimating the probability that catalytic sequences would emerge. Here, we analyze the in vitro evolution of triphosphorylating ribozymes and translate their fitnesses into absolute estimates of catalytic activity for hundreds of ribozyme families. The analysis efficiently identified highly active ribozymes and estimated catalytic activity with good accuracy. The evolutionary dynamics follow Fisher's Fundamental Theorem of Natural Selection and a corollary, permitting retrospective inference of the distribution of fitness and activity in the random sequence pool for the first time. The frequency distribution of rate constants appears to be log-normal, with a surprisingly steep dropoff at higher activity, consistent with a mechanism for the emergence of activity as the product of many independent contributions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Depression as a systemic syndrome: mapping the feedback loops of major depressive disorder.

PubMed

Wittenborn, A K; Rahmandad, H; Rick, J; Hosseinichimeh, N

2016-02-01

Depression is a complex public health problem with considerable variation in treatment response. The systemic complexity of depression, or the feedback processes among diverse drivers of the disorder, contribute to the persistence of depression. This paper extends prior attempts to understand the complex causal feedback mechanisms that underlie depression by presenting the first broad boundary causal loop diagram of depression dynamics. We applied qualitative system dynamics methods to map the broad feedback mechanisms of depression. We used a structured approach to identify candidate causal mechanisms of depression in the literature. We assessed the strength of empirical support for each mechanism and prioritized those with support from validation studies. Through an iterative process, we synthesized the empirical literature and created a conceptual model of major depressive disorder. The literature review and synthesis resulted in the development of the first causal loop diagram of reinforcing feedback processes of depression. It proposes candidate drivers of illness, or inertial factors, and their temporal functioning, as well as the interactions among drivers of depression. The final causal loop diagram defines 13 key reinforcing feedback loops that involve nine candidate drivers of depression. Future research is needed to expand upon this initial model of depression dynamics. Quantitative extensions may result in a better understanding of the systemic syndrome of depression and contribute to personalized methods of evaluation, prevention and intervention.

Depression as a systemic syndrome: mapping the feedback loops of major depressive disorder

PubMed Central

Wittenborn, A. K.; Rahmandad, H.; Rick, J.; Hosseinichimeh, N.

2016-01-01

Background Depression is a complex public health problem with considerable variation in treatment response. The systemic complexity of depression, or the feedback processes among diverse drivers of the disorder, contribute to the persistence of depression. This paper extends prior attempts to understand the complex causal feedback mechanisms that underlie depression by presenting the first broad boundary causal loop diagram of depression dynamics. Method We applied qualitative system dynamics methods to map the broad feedback mechanisms of depression. We used a structured approach to identify candidate causal mechanisms of depression in the literature. We assessed the strength of empirical support for each mechanism and prioritized those with support from validation studies. Through an iterative process, we synthesized the empirical literature and created a conceptual model of major depressive disorder. Results The literature review and synthesis resulted in the development of the first causal loop diagram of reinforcing feedback processes of depression. It proposes candidate drivers of illness, or inertial factors, and their temporal functioning, as well as the interactions among drivers of depression. The final causal loop diagram defines 13 key reinforcing feedback loops that involve nine candidate drivers of depression. Conclusions Future research is needed to expand upon this initial model of depression dynamics. Quantitative extensions may result in a better understanding of the systemic syndrome of depression and contribute to personalized methods of evaluation, prevention and intervention. PMID:26621339

Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase.

PubMed

Zhai, Xiang; Amyes, Tina L; Richard, John P

2015-12-09

The side chains of Y208 and S211 from loop 7 of triosephosphate isomerase (TIM) form hydrogen bonds to backbone amides and carbonyls from loop 6 to stabilize the caged enzyme-substrate complex. The effect of seven mutations [Y208T, Y208S, Y208A, Y208F, S211G, S211A, Y208T/S211G] on the kinetic parameters for TIM catalyzed reactions of the whole substrates dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate [(k(cat)/K(m))(GAP) and (k(cat)/K(m))DHAP] and of the substrate pieces glycolaldehyde and phosphite dianion (k(cat)/K(HPi)K(GA)) are reported. The linear logarithmic correlation between these kinetic parameters, with slope of 1.04 ± 0.03, shows that most mutations of TIM result in an identical change in the activation barriers for the catalyzed reactions of whole substrate and substrate pieces, so that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The second linear logarithmic correlation [slope = 0.53 ± 0.16] between k(cat) for isomerization of GAP and K(d)(⧧) for phosphite dianion binding to the transition state for wildtype and many mutant TIM-catalyzed reactions of substrate pieces shows that ca. 50% of the wildtype TIM dianion binding energy, eliminated by these mutations, is expressed at the wildtype Michaelis complex, and ca. 50% is only expressed at the wildtype transition state. Negative deviations from this correlation are observed when the mutation results in a decrease in enzyme reactivity at the catalytic site. The main effect of Y208T, Y208S, and Y208A mutations is to cause a reduction in the total intrinsic dianion binding energy, but the effect of Y208F extends to the catalytic site.

EVIDENCE FOR COLLAPSING FIELDS IN THE CORONA AND PHOTOSPHERE DURING THE 2011 FEBRUARY 15 X2.2 FLARE: SDO/AIA AND HMI OBSERVATIONS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gosain, S., E-mail: sgosain@nso.edu; Udaipur Solar Observatory, P.O. Box 198, Dewali, Udaipur, Rajasthan 313001

2012-04-10

We use high-resolution Solar Dynamics Observatory (SDO)/Atmospheric Imaging Assembly observations to study the evolution of the coronal loops in a flaring solar active region, NOAA 11158. We identify three distinct phases of the coronal loop dynamics during this event: (1) slow-rise phase: slow rising motion of the loop-tops prior to the flare in response to the slow rise of the underlying flux rope; (2) collapse phase: sudden contraction of the loop-tops, with the lower loops collapsing earlier than the higher loops; and (3) oscillation phase: the loops exhibit global kink oscillations after the collapse phase at different periods, with themore » period decreasing with the decreasing height of the loops. The period of these loop oscillations is used to estimate the field strength in the coronal loops. Furthermore, we also use SDO/Helioseismic and Magnetic Imager (HMI) observations to study the photospheric changes close to the polarity inversion line (PIL). The longitudinal magnetograms show a stepwise permanent decrease in the magnetic flux after the flare over a coherent patch along the PIL. Furthermore, we examine the HMI Stokes I, Q, U, V profiles over this patch and find that the Stokes-V signal systematically decreases while the Stokes-Q and U signals increase after the flare. These observations suggest that close to the PIL the field configuration became more horizontal after the flare. We also use HMI vector magnetic field observations to quantify the changes in the field inclination angle and find an inward collapse of the field lines toward the PIL by {approx}10 Degree-Sign . These observations are consistent with the 'coronal implosion' scenario and its predictions about flare-related photospheric field changes.« less

All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

PubMed Central

Xu, Wu; Amire-Brahimi, Benjamin; Xie, Xiao-Jun; Huang, Liying; Ji, Jun-Yuan

2014-01-01

The Mediator, a conserved multisubunit protein complex in eukaryotic organisms, regulates gene expression by bridging sequence-specific DNA-binding transcription factors to the general RNA polymerase II machinery. In yeast, Mediator complex is organized in three core modules (head, middle and tail) and a separable ‘CDK8 submodule’ consisting of four subunits including Cyclin-dependent kinase CDK8 (CDK8), Cyclin C (CycC), MED12, and MED13. The 3-D structure of human CDK8-CycC complex has been recently experimentally determined. To take advantage of this structure and the improved theoretical calculation methods, we have performed molecular dynamic simulations to study dynamics of CDK8 and two CDK8 point mutations (D173A and D189N), which have been identified in human cancers, with and without full length of the A-loop as well as the binding between CDK8 and CycC. We found that CDK8 structure gradually loses two helical structures during the 50-ns molecular dynamic simulation, likely due to the presence of the full-length A-loop. In addition, our studies showed the hydrogen bond occupation of the CDK8 A-loop increases during the first 20-ns MD simulation and stays stable during the later 30-ns MD simulation. Four residues in the A-loop of CDK8 have high hydrogen bond occupation, while the rest residues have low or no hydrogen bond occupation. The hydrogen bond dynamic study of the A-loop residues exhibits three types of changes: increasing, decreasing, and stable. Furthermore, the 3-D structures of CDK8 point mutations D173A, D189N, T196A and T196D have been built by molecular modeling and further investigated by 50-ns molecular dynamic simulations. D173A has the highest average potential energy, while T196D has the lowest average potential energy, indicating that T196D is the most stable structure. Finally, we calculated theoretical binding energy of CDK8 and CycC by MM/PBSA and MM/GBSA methods, and the negative values obtained from both methods demonstrate stability of CDK8-CycC complex. Taken together, these analyses will improve our understanding of the exact functions of CDK8 and the interaction with its partner CycC. PMID:24754906

Coronal rain in magnetic bipolar weak fields

NASA Astrophysics Data System (ADS)

Xia, C.; Keppens, R.; Fang, X.

2017-07-01

Aims: We intend to investigate the underlying physics for the coronal rain phenomenon in a representative bipolar magnetic field, including the formation and the dynamics of coronal rain blobs. Methods: With the MPI-AMRVAC code, we performed three dimensional radiative magnetohydrodynamic (MHD) simulation with strong heating localized on footpoints of magnetic loops after a relaxation to quiet solar atmosphere. Results: Progressive cooling and in-situ condensation starts at the loop top due to radiative thermal instability. The first large-scale condensation on the loop top suffers Rayleigh-Taylor instability and becomes fragmented into smaller blobs. The blobs fall vertically dragging magnetic loops until they reach low-β regions and start to fall along the loops from loop top to loop footpoints. A statistic study of the coronal rain blobs finds that small blobs with masses of less than 1010 g dominate the population. When blobs fall to lower regions along the magnetic loops, they are stretched and develop a non-uniform velocity pattern with an anti-parallel shearing pattern seen to develop along the central axis of the blobs. Synthetic images of simulated coronal rain with Solar Dynamics Observatory Atmospheric Imaging Assembly well resemble real observations presenting dark falling clumps in hot channels and bright rain blobs in a cool channel. We also find density inhomogeneities during a coronal rain "shower", which reflects the observed multi-stranded nature of coronal rain. Movies associated to Figs. 3 and 7 are available at http://www.aanda.org

Dynamical behaviour in coronal loops

NASA Technical Reports Server (NTRS)

Haisch, Bernhard M.

1986-01-01

Rapid variability has been found in two active region coronal loops observed by the X-ray Polychromator (XRP) and the Hard X-ray Imaging Spectrometer (HXIS) onboard the Solar Maximum Mission (SMM). There appear to be surprisingly few observations of the short-time scale behavior of hot loops, and the evidence presented herein lends support to the hypothesis that coronal heating may be impulsive and driven by flaring.

Dynamical behaviour in coronal loops

NASA Astrophysics Data System (ADS)

Haisch, Bernhard M.

Rapid variability has been found in two active region coronal loops observed by the X-ray Polychromator (XRP) and the Hard X-ray Imaging Spectrometer (HXIS) onboard the Solar Maximum Mission (SMM). There appear to be surprisingly few observations of the short-time scale behavior of hot loops, and the evidence presented herein lends support to the hypothesis that coronal heating may be impulsive and driven by flaring.

Structure-function similarities between a plant receptor-like kinase and the human interleukin-1 receptor-associated kinase-4.

PubMed

Klaus-Heisen, Dörte; Nurisso, Alessandra; Pietraszewska-Bogiel, Anna; Mbengue, Malick; Camut, Sylvie; Timmers, Ton; Pichereaux, Carole; Rossignol, Michel; Gadella, Theodorus W J; Imberty, Anne; Lefebvre, Benoit; Cullimore, Julie V

2011-04-01

Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension α-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general.

Instant Integrated Ultradeep Quantitative-structural Membrane Proteomics Discovered Post-translational Modification Signatures for Human Cys-loop Receptor Subunit Bias.

PubMed

Zhang, Xi

2016-12-01

Neurotransmitter ligand-gated ion channels (LGICs) are widespread and pivotal in brain functions. Unveiling their structure-function mechanisms is crucial to drive drug discovery, and demands robust proteomic quantitation of expression, post-translational modifications (PTMs) and dynamic structures. Yet unbiased digestion of these modified transmembrane proteins-at high efficiency and peptide reproducibility-poses the obstacle. Targeting both enzyme-substrate contacts and PTMs for peptide formation and detection, we devised flow-and-detergent-facilitated protease and de-PTM digestions for deep sequencing (FDD) method that combined omni-compatible detergent, tandem immobilized protease/PNGase columns, and Cys-selective reduction/alkylation, to achieve streamlined ultradeep peptide preparation within minutes not days, at high peptide reproducibility and low abundance-bias. FDD transformed enzyme-protein contacts into equal catalytic travel paths through enzyme-excessive columns regardless of protein abundance, removed products instantly preventing inhibition, tackled intricate structures via sequential multiple micro-digestions along the flow, and precisely controlled peptide formation by flow rate. Peptide-stage reactions reduced steric bias; low contamination deepened MS/MS scan; distinguishing disulfide from M oxidation and avoiding gain/loss artifacts unmasked protein-endogenous oxidation states. Using a recent interactome of 285-kDa human GABA type A receptor, this pilot study validated FDD platform's applicability to deep sequencing (up to 99% coverage), H/D-exchange and TMT-based structural mapping. FDD discovered novel subunit-specific PTM signatures, including unusual nontop-surface N-glycosylations, that may drive subunit biases in human Cys-loop LGIC assembly and pharmacology, by redefining subunit/ligand interfaces and connecting function domains. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Disproportionating Enzyme 1 (DPE1)*

PubMed Central

O'Neill, Ellis C.; Stevenson, Clare E. M.; Tantanarat, Krit; Latousakis, Dimitrios; Donaldson, Matthew I.; Rejzek, Martin; Nepogodiev, Sergey A.; Limpaseni, Tipaporn; Field, Robert A.; Lawson, David M.

2015-01-01

The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The “gate” is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms. PMID:26504082

Analysis of the function of E. coli 23S rRNA helix-loop 69 by mutagenesis

PubMed Central

Liiv, Aivar; Karitkina, Diana; Maiväli, Ülo; Remme, Jaanus

2005-01-01

Background The ribosome is a two-subunit enzyme known to exhibit structural dynamism during protein synthesis. The intersubunit bridges have been proposed to play important roles in decoding, translocation, and the peptidyl transferase reaction; yet the physical nature of their contributions is ill understood. An intriguing intersubunit bridge, B2a, which contains 23S rRNA helix 69 as a major component, has been implicated by proximity in a number of catalytically important regions. In addition to contacting the small ribosomal subunit, helix 69 contacts both the A and P site tRNAs and several translation factors. Results We scanned the loop of helix 69 by mutagenesis and analyzed the mutant ribosomes using a plasmid-borne IPTG-inducible expression system. We assayed the effects of 23S rRNA mutations on cell growth, contribution of mutant ribosomes to cellular polysome pools and the ability of mutant ribosomes to function in cell-free translation. Mutations A1912G, and A1919G have very strong growth phenotypes, are inactive during in vitro protein synthesis, and under-represented in the polysomes. Mutation Ψ1917C has a very strong growth phenotype and leads to a general depletion of the cellular polysome pool. Mutation A1916G, having a modest growth phenotype, is apparently defective in the assembly of the 70S ribosome. Conclusion Mutations A1912G, A1919G, and Ψ1917C of 23S rRNA strongly inhibit translation. Mutation A1916G causes a defect in the 50S subunit or 70S formation. Mutations Ψ1911C, A1913G, C1914A, Ψ1915C, and A1918G lack clear phenotypes. PMID:16053518

A consensus-guided approach yields a heat-stable alkane-producing enzyme and identifies residues promoting thermostability.

PubMed

Shakeel, Tabinda; Gupta, Mayank; Fatma, Zia; Kumar, Rakesh; Kumar, Raubins; Singh, Rahul; Sharma, Medha; Jade, Dhananjay; Gupta, Dinesh; Fatma, Tasneem; Yazdani, Syed Shams

2018-06-15

Aldehyde-deformylating oxygenase (ADO) is an essential enzyme for production of long-chain alkanes as drop-in biofuels, which are compatible with existing fuel systems. The most active ADOs are present in mesophilic cyanobacteria, especially Nostoc punctiforme Given the potential applications of thermostable enzymes in biorefineries, here we generated a thermostable (Cts)-ADO based on a consensus of ADO sequences from several thermophilic cyanobacterial strains. Using an in silico design pipeline and a metagenome library containing 41 hot-spring microbial communities, we created Cts-ADO. Cts-ADO displayed a 3.8-fold increase in pentadecane production on raising the temperature from 30 to 42 °C, whereas ADO from N. punctiforme (Np-ADO) exhibited a 1.7-fold decline. 3D structure modeling and molecular dynamics simulations of Cts- and Np-ADO at different temperatures revealed differences between the two enzymes in residues clustered on exposed loops of these variants, which affected the conformation of helices involved in forming the ADO catalytic core. In Cts-ADO, this conformational change promoted ligand binding to its preferred iron, Fe2, in the di-iron cluster at higher temperature, but the reverse was observed in Np-ADO. Detailed mapping of residues conferring Cts-ADO thermostability identified four amino acids, which we substituted individually and together in Np-ADO. Among these substitution variants, A161E was remarkably similar to Cts-ADO in terms of activity optima, kinetic parameters, and structure at higher temperature. A161E was located in loop L6, which connects helices H5 and H6, and supported ligand binding to Fe2 at higher temperatures, thereby promoting optimal activity at these temperatures and explaining the increased thermostability of Cts-ADO. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

An Allosteric Cross-Talk Between the Activation Loop and the ATP Binding Site Regulates the Activation of Src Kinase

NASA Astrophysics Data System (ADS)

Pucheta-Martínez, Encarna; Saladino, Giorgio; Morando, Maria Agnese; Martinez-Torrecuadrada, Jorge; Lelli, Moreno; Sutto, Ludovico; D'Amelio, Nicola; Gervasio, Francesco Luigi

2016-04-01

Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe.

The Role of Protein Loops and Linkers in Conformational Dynamics and Allostery.

PubMed

Papaleo, Elena; Saladino, Giorgio; Lambrughi, Matteo; Lindorff-Larsen, Kresten; Gervasio, Francesco Luigi; Nussinov, Ruth

2016-06-08

Proteins are dynamic entities that undergo a plethora of conformational changes that may take place on a wide range of time scales. These changes can be as small as the rotation of one or a few side-chain dihedral angles or involve concerted motions in larger portions of the three-dimensional structure; both kinds of motions can be important for biological function and allostery. It is becoming increasingly evident that "connector regions" are important components of the dynamic personality of protein structures. These regions may be either disordered loops, i.e., poorly structured regions connecting secondary structural elements, or linkers that connect entire protein domains. Experimental and computational studies have, however, revealed that these regions are not mere connectors, and their role in allostery and conformational changes has been emerging in the last few decades. Here we provide a detailed overview of the structural properties and classification of loops and linkers, as well as a discussion of the main computational methods employed to investigate their function and dynamical properties. We also describe their importance for protein dynamics and allostery using as examples key proteins in cellular biology and human diseases such as kinases, ubiquitinating enzymes, and transcription factors.

Catalytic Steam and Partial Oxidation Reforming of Liquid Fuels for Application in Improving the Efficiency of Internal Combustion Engines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brookshear, Daniel William; Pihl, Josh A.; Szybist, James P.

Here, this study investigated the potential for catalytically reforming liquid fuels in a simulated exhaust gas recirculation (EGR) mixture loop for the purpose of generating reformate that could be used to increase stoichiometric combustion engine efficiency. The experiments were performed on a simulated exhaust flow reactor using a Rh/Al 2O 3 reformer catalyst, and the fuels evaluated included iso-octane, ethanol, and gasoline. Both steam reforming and partial oxidation reforming were examined as routes for the production of reformate. Steam reforming was determined to be an ineffective option for reforming in an EGR loop, because of the high exhaust temperatures (inmore » excess of 700 °C) required to produce adequate concentrations of reformate, regardless of fuel. However, partial oxidation reforming is capable of producing hydrogen concentrations as high as 10%–16%, depending on fuel and operating conditions in the simulated EGR gas mixture. Meanwhile, measurements of total fuel enthalpy retention were shown to have favorable energetics under a range of conditions, although a tradeoff between fuel enthalpy retention and reformate production was observed. Of the three fuels evaluated, iso-octane exhibited the best overall performance, followed by ethanol and then gasoline. Overall, it was found that partial oxidation reforming of liquid fuels in a simulated EGR mixture over the Rh/Al 2O 3 catalyst demonstrated sufficiently high reformate yields and favorable energetics to warrant further evaluation in the EGR system of a stoichiometric combustion engine.« less

Catalytic Steam and Partial Oxidation Reforming of Liquid Fuels for Application in Improving the Efficiency of Internal Combustion Engines

DOE PAGES

Brookshear, Daniel William; Pihl, Josh A.; Szybist, James P.

2018-02-07

Here, this study investigated the potential for catalytically reforming liquid fuels in a simulated exhaust gas recirculation (EGR) mixture loop for the purpose of generating reformate that could be used to increase stoichiometric combustion engine efficiency. The experiments were performed on a simulated exhaust flow reactor using a Rh/Al 2O 3 reformer catalyst, and the fuels evaluated included iso-octane, ethanol, and gasoline. Both steam reforming and partial oxidation reforming were examined as routes for the production of reformate. Steam reforming was determined to be an ineffective option for reforming in an EGR loop, because of the high exhaust temperatures (inmore » excess of 700 °C) required to produce adequate concentrations of reformate, regardless of fuel. However, partial oxidation reforming is capable of producing hydrogen concentrations as high as 10%–16%, depending on fuel and operating conditions in the simulated EGR gas mixture. Meanwhile, measurements of total fuel enthalpy retention were shown to have favorable energetics under a range of conditions, although a tradeoff between fuel enthalpy retention and reformate production was observed. Of the three fuels evaluated, iso-octane exhibited the best overall performance, followed by ethanol and then gasoline. Overall, it was found that partial oxidation reforming of liquid fuels in a simulated EGR mixture over the Rh/Al 2O 3 catalyst demonstrated sufficiently high reformate yields and favorable energetics to warrant further evaluation in the EGR system of a stoichiometric combustion engine.« less

A study of digital gyro compensation loops. [data conversion routines and breadboard models

NASA Technical Reports Server (NTRS)

1975-01-01

The feasibility is discussed of replacing existing state-of-the-art analog gyro compensation loops with digital computations. This was accomplished by designing appropriate compensation loops for the dry turned TDF gyro, selecting appropriate data conversion and processing techniques and algorithms, and breadboarding the design for laboratory evaluation. A breadboard design was established in which one axis of a Teledyne turned-gimbal TDF gyro was caged digitally while the other was caged using conventional analog electronics. The digital loop was designed analytically to closely resemble the analog loop in performance. The breadboard was subjected to various static and dynamic tests in order to establish the relative stability characteristics and frequency responses of the digital and analog loops. Several variations of the digital loop configuration were evaluated. The results were favorable.

Coronal Loop Evolution Observed with AIA and Hi-C

NASA Technical Reports Server (NTRS)

Mulu-Moore, Fana; Winebarger, A.; Cirtain, J.; Kobayashi, K.; Korreck, K.; Golub, L.; Kuzin. S.; Walsh, R.; DeForest, C.; DePontieu, B.;

It's positive to be negative: Achilles tendon work loops during human locomotion.

PubMed

Zelik, Karl E; Franz, Jason R

2017-01-01

Ultrasound imaging is increasingly used with motion and force data to quantify tendon dynamics during human movement. Frequently, tendon dynamics are estimated indirectly from muscle fascicle kinematics (by subtracting muscle from muscle-tendon unit length), but there is mounting evidence that this Indirect approach yields implausible tendon work loops. Since tendons are passive viscoelastic structures, when they undergo a loading-unloading cycle they must exhibit a negative work loop (i.e., perform net negative work). However, prior studies using this Indirect approach report large positive work loops, often estimating that tendons return 2-5 J of elastic energy for every 1 J of energy stored. More direct ultrasound estimates of tendon kinematics have emerged that quantify tendon elongations by tracking either the muscle-tendon junction or localized tendon tissue. However, it is unclear if these yield more plausible estimates of tendon dynamics. Our objective was to compute tendon work loops and hysteresis losses using these two Direct tendon kinematics estimates during human walking. We found that Direct estimates generally resulted in negative work loops, with average tendon hysteresis losses of 2-11% at 1.25 m/s and 33-49% at 0.75 m/s (N = 8), alluding to 0.51-0.98 J of tendon energy returned for every 1 J stored. We interpret this finding to suggest that Direct approaches provide more plausible estimates than the Indirect approach, and may be preferable for understanding tendon energy storage and return. However, the Direct approaches did exhibit speed-dependent trends that are not consistent with isolated, in vitro tendon hysteresis losses of about 5-10%. These trends suggest that Direct estimates also contain some level of error, albeit much smaller than Indirect estimates. Overall, this study serves to highlight the complexity and difficulty of estimating tendon dynamics non-invasively, and the care that must be taken to interpret biological function from current ultrasound-based estimates.

Catalytic dimer nanomotors: continuum theory and microscopic dynamics.

PubMed

Reigh, Shang Yik; Kapral, Raymond

2015-04-28

Synthetic chemically-powered motors with various geometries have potentially new applications involving dynamics on very small scales. Self-generated concentration and fluid flow fields, which depend on geometry, play essential roles in motor dynamics. Sphere-dimer motors, comprising linked catalytic and noncatalytic spheres, display more complex versions of such fields, compared to the often-studied spherical Janus motors. By making use of analytical continuum theory and particle-based simulations we determine the concentration fields, and both the complex structure of the near-field and point-force dipole nature of the far-field behavior of the solvent velocity field that are important for studies of collective motor motion. We derive the dependence of motor velocity on geometric factors such as sphere size and dimer bond length and, thus, show how to construct motors with specific characteristics.

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

PubMed Central

Itoh, Takafumi; Hibi, Takao; Suzuki, Fumiko; Sugimoto, Ikumi; Fujiwara, Akihiro; Inaka, Koji; Tanaka, Hiroaki; Ohta, Kazunori; Fujii, Yutaka; Taketo, Akira; Kimoto, Hisashi

2016-01-01

The Gram-positive bacterium Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 Å resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed β-helix domain (carbohydrate-binding module family 54, CBM-54), a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st β/α-barrel catalytic domain (glycoside hydrolase family 18, GH-18), 2nd Ig-like fold domain and 2nd β/α-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, β-1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation. PMID:27907169

Structural and Thermodynamic Comparison of the Catalytic Domain of AMSH and AMSH-LP: Nearly Identical Fold but Different Stability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davies, Christopher W.; Paul, Lake N.; Kim, Myung-Il

2012-02-07

AMSH plays a critical role in the ESCRT (endosomal sorting complexes required for transport) machinery, which facilitates the down-regulation and degradation of cell-surface receptors. It displays a high level of specificity toward cleavage of Lys63-linked polyubiquitin chains, the structural basis of which has been understood recently through the crystal structure of a highly related, but ESCRT-independent, protein AMSH-LP (AMSH-like protein). We have determined the X-ray structure of two constructs representing the catalytic domain of AMSH: AMSH244, the JAMM (JAB1/MPN/MOV34)-domain-containing polypeptide segment from residues 244 to 424, and AMSH219{sup E280A}, an active-site mutant, Glu280 to Ala, of the segment from 219more » to 424. In addition to confirming the expected zinc coordination in the protein, the structures reveal that the catalytic domains of AMSH and AMSH-LP are nearly identical; however, guanidine-hydrochloride-induced unfolding studies show that the catalytic domain of AMSH is thermodynamically less stable than that of AMSH-LP, indicating that the former is perhaps structurally more plastic. Much to our surprise, in the AMSH219{sup E280A} structure, the catalytic zinc was still held in place, by the compensatory effect of an aspartate from a nearby loop moving into a position where it could coordinate with the zinc, once again suggesting the plasticity of AMSH. Additionally, a model of AMSH244 bound to Lys63-linked diubiquitin reveals a type of interface for the distal ubiquitin significantly different from that seen in AMSH-LP. Altogether, we believe that our data provide important insight into the structural difference between the two proteins that may translate into the difference in their biological function.« less

Mining protein loops using a structural alphabet and statistical exceptionality

PubMed Central

2010-01-01

Background Protein loops encompass 50% of protein residues in available three-dimensional structures. These regions are often involved in protein functions, e.g. binding site, catalytic pocket... However, the description of protein loops with conventional tools is an uneasy task. Regular secondary structures, helices and strands, have been widely studied whereas loops, because they are highly variable in terms of sequence and structure, are difficult to analyze. Due to data sparsity, long loops have rarely been systematically studied. Results We developed a simple and accurate method that allows the description and analysis of the structures of short and long loops using structural motifs without restriction on loop length. This method is based on the structural alphabet HMM-SA. HMM-SA allows the simplification of a three-dimensional protein structure into a one-dimensional string of states, where each state is a four-residue prototype fragment, called structural letter. The difficult task of the structural grouping of huge data sets is thus easily accomplished by handling structural letter strings as in conventional protein sequence analysis. We systematically extracted all seven-residue fragments in a bank of 93000 protein loops and grouped them according to the structural-letter sequence, named structural word. This approach permits a systematic analysis of loops of all sizes since we consider the structural motifs of seven residues rather than complete loops. We focused the analysis on highly recurrent words of loops (observed more than 30 times). Our study reveals that 73% of loop-lengths are covered by only 3310 highly recurrent structural words out of 28274 observed words). These structural words have low structural variability (mean RMSd of 0.85 Å). As expected, half of these motifs display a flanking-region preference but interestingly, two thirds are shared by short (less than 12 residues) and long loops. Moreover, half of recurrent motifs exhibit a significant level of amino-acid conservation with at least four significant positions and 87% of long loops contain at least one such word. We complement our analysis with the detection of statistically over-represented patterns of structural letters as in conventional DNA sequence analysis. About 30% (930) of structural words are over-represented, and cover about 40% of loop lengths. Interestingly, these words exhibit lower structural variability and higher sequential specificity, suggesting structural or functional constraints. Conclusions We developed a method to systematically decompose and study protein loops using recurrent structural motifs. This method is based on the structural alphabet HMM-SA and not on structural alignment and geometrical parameters. We extracted meaningful structural motifs that are found in both short and long loops. To our knowledge, it is the first time that pattern mining helps to increase the signal-to-noise ratio in protein loops. This finding helps to better describe protein loops and might permit to decrease the complexity of long-loop analysis. Detailed results are available at http://www.mti.univ-paris-diderot.fr/publication/supplementary/2009/ACCLoop/. PMID:20132552

Mining protein loops using a structural alphabet and statistical exceptionality.

PubMed

Regad, Leslie; Martin, Juliette; Nuel, Gregory; Camproux, Anne-Claude

2010-02-04

Protein loops encompass 50% of protein residues in available three-dimensional structures. These regions are often involved in protein functions, e.g. binding site, catalytic pocket... However, the description of protein loops with conventional tools is an uneasy task. Regular secondary structures, helices and strands, have been widely studied whereas loops, because they are highly variable in terms of sequence and structure, are difficult to analyze. Due to data sparsity, long loops have rarely been systematically studied. We developed a simple and accurate method that allows the description and analysis of the structures of short and long loops using structural motifs without restriction on loop length. This method is based on the structural alphabet HMM-SA. HMM-SA allows the simplification of a three-dimensional protein structure into a one-dimensional string of states, where each state is a four-residue prototype fragment, called structural letter. The difficult task of the structural grouping of huge data sets is thus easily accomplished by handling structural letter strings as in conventional protein sequence analysis. We systematically extracted all seven-residue fragments in a bank of 93000 protein loops and grouped them according to the structural-letter sequence, named structural word. This approach permits a systematic analysis of loops of all sizes since we consider the structural motifs of seven residues rather than complete loops. We focused the analysis on highly recurrent words of loops (observed more than 30 times). Our study reveals that 73% of loop-lengths are covered by only 3310 highly recurrent structural words out of 28274 observed words). These structural words have low structural variability (mean RMSd of 0.85 A). As expected, half of these motifs display a flanking-region preference but interestingly, two thirds are shared by short (less than 12 residues) and long loops. Moreover, half of recurrent motifs exhibit a significant level of amino-acid conservation with at least four significant positions and 87% of long loops contain at least one such word. We complement our analysis with the detection of statistically over-represented patterns of structural letters as in conventional DNA sequence analysis. About 30% (930) of structural words are over-represented, and cover about 40% of loop lengths. Interestingly, these words exhibit lower structural variability and higher sequential specificity, suggesting structural or functional constraints. We developed a method to systematically decompose and study protein loops using recurrent structural motifs. This method is based on the structural alphabet HMM-SA and not on structural alignment and geometrical parameters. We extracted meaningful structural motifs that are found in both short and long loops. To our knowledge, it is the first time that pattern mining helps to increase the signal-to-noise ratio in protein loops. This finding helps to better describe protein loops and might permit to decrease the complexity of long-loop analysis. Detailed results are available at http://www.mti.univ-paris-diderot.fr/publication/supplementary/2009/ACCLoop/.

Flexibility Correlation between Active Site Regions Is Conserved across Four AmpC β-Lactamase Enzymes.

PubMed

Brown, Jenna R; Livesay, Dennis R

2015-01-01

β-lactamases are bacterial enzymes that confer resistance to β-lactam antibiotics, such as penicillins and cephalosporins. There are four classes of β-lactamase enzymes, each with characteristic sequence and structure properties. Enzymes from class A are the most common and have been well characterized across the family; however, less is known about how physicochemical properties vary across the C and D families. In this report, we compare the dynamical properties of four AmpC (class C) β-lactamases using our distance constraint model (DCM). The DCM reliably predicts thermodynamic and mechanical properties in an integrated way. As a consequence, quantitative stability/flexibility relationships (QSFR) can be determined and compared across the whole family. The DCM calculates a large number of QSFR metrics. Perhaps the most useful is the flexibility index (FI), which quantifies flexibility along the enzyme backbone. As typically observed in other systems, FI is well conserved across the four AmpC enzymes. Cooperativity correlation (CC), which quantifies intramolecular couplings within structure, is rarely conserved across protein families; however, it is in AmpC. In particular, the bulk of each structure is composed of a large rigid cluster, punctuated by three flexibly correlated regions located at the active site. These regions include several catalytic residues and the Ω-loop. This evolutionary conservation combined with active their site location strongly suggests that these coupled dynamical modes are important for proper functioning of the enzyme.

Flexibility Correlation between Active Site Regions Is Conserved across Four AmpC β-Lactamase Enzymes

PubMed Central

Brown, Jenna R.; Livesay, Dennis R.

2015-01-01

β-lactamases are bacterial enzymes that confer resistance to β-lactam antibiotics, such as penicillins and cephalosporins. There are four classes of β-lactamase enzymes, each with characteristic sequence and structure properties. Enzymes from class A are the most common and have been well characterized across the family; however, less is known about how physicochemical properties vary across the C and D families. In this report, we compare the dynamical properties of four AmpC (class C) β-lactamases using our distance constraint model (DCM). The DCM reliably predicts thermodynamic and mechanical properties in an integrated way. As a consequence, quantitative stability/flexibility relationships (QSFR) can be determined and compared across the whole family. The DCM calculates a large number of QSFR metrics. Perhaps the most useful is the flexibility index (FI), which quantifies flexibility along the enzyme backbone. As typically observed in other systems, FI is well conserved across the four AmpC enzymes. Cooperativity correlation (CC), which quantifies intramolecular couplings within structure, is rarely conserved across protein families; however, it is in AmpC. In particular, the bulk of each structure is composed of a large rigid cluster, punctuated by three flexibly correlated regions located at the active site. These regions include several catalytic residues and the Ω-loop. This evolutionary conservation combined with active their site location strongly suggests that these coupled dynamical modes are important for proper functioning of the enzyme. PMID:26018804

Identification of the hot spot residues for pyridine derivative inhibitor CCT251455 and ATP substrate binding on monopolar spindle 1 (MPS1) kinase by molecular dynamic simulation.

PubMed

Chen, Kai; Duan, Wenxiu; Han, Qianqian; Sun, Xuan; Li, Wenqian; Hu, Shuangyun; Wan, Jiajia; Wu, Jiang; Ge, Yushu; Liu, Dan

2018-03-08

Protein kinase monopolar spindle 1 plays an important role in spindle assembly checkpoint at the onset of mitosis. Over expression of MPS1 correlated with a wide range of human tumors makes it an attractive target for finding an effective and specific inhibitor. In this work, we performed molecular dynamics simulations of protein MPS1 itself as well as protein bound systems with the inhibitor and natural substrate based on crystal structures. The reported orally bioavailable 1 h-pyrrolo [3,2-c] pyridine inhibitors of MPS1 maintained stable binding in the catalytic site, while natural substrate ATP could not stay. Comparative study of stability and flexibility of three systems reveals position shifting of β-sheet region within the catalytic site, which indicates inhibition mechanism was through stabilizing the β-sheet region. Binding free energies calculated with MM-GB/PBSA method shows different binding affinity for inhibitor and ATP. Finally, interactions between protein and inhibitor during molecular dynamic simulations were measured and counted. Residue Gly605 and Leu654 were suggested as important hot spots for stable binding of inhibitor by molecular dynamic simulation. Our results reveal an important position shifting within catalytic site for non-inhibited proteins. Together with hot spots found by molecular dynamic simulation, the results provide important information of inhibition mechanism and will be referenced for designing novel inhibitors.

Scanning mass spectrometry with integrated constant distance positioning

NASA Astrophysics Data System (ADS)

Li, Nan; Eckhard, Kathrin; Aßmann, Jens; Hagen, Volker; Otto, Horst; Chen, Xingxing; Schuhmann, Wolfgang; Muhler, Martin

2006-08-01

Scanning mass spectrometry is of growing importance for the characterization of catalytically active surfaces. The instrument presented here is capable of measuring catalytic activity spatially resolved by means of two concentric capillaries. The outer one is used for cofeeding reactants such as ethene and hydrogen to the sample surface, whereas the inner one is pumping off the product mixture as inlet to a quadrupole mass spectrometer. Three-dimensional measurements under stagnant-point flow conditions become possible based on a home-built capillary positioning unit. Step-motor driven positioning stages exhibiting a minimum step width of 2.5μm̸half step are used for the x, y positioning, and the step motor in z direction has a resolution of 1μm̸half step. The system is additionally equipped with a feedback loop for following the topography of the sample throughout scanning. Hence, the obtained catalytic data are unimpaired by signal changes caused by the morphology of the investigated structure. For distance control the argon ion current is used originating from externally fed argon diffusing into the confined space between the accurately positioned capillaries and the sample surface. A well-defined microchannel flow field with 400μm wide channels and 200μm wide mounds was chosen to evaluate the developed method. The catalytic activity of a Pt catalyst deposited on glassy carbon was successfully visualized in constant probe to sample distance. Simultaneously, the topography of the sample was recorded derived from the z positioning of the capillaries.

Modulation of dynamic modes by interplay between positive and negative feedback loops in gene regulatory networks

NASA Astrophysics Data System (ADS)

Wang, Liu-Suo; Li, Ning-Xi; Chen, Jing-Jia; Zhang, Xiao-Peng; Liu, Feng; Wang, Wei

2018-04-01

A positive and a negative feedback loop can induce bistability and oscillation, respectively, in biological networks. Nevertheless, they are frequently interlinked to perform more elaborate functions in many gene regulatory networks. Coupled positive and negative feedback loops may exhibit either oscillation or bistability depending on the intensity of the stimulus in some particular networks. It is less understood how the transition between the two dynamic modes is modulated by the positive and negative feedback loops. We developed an abstract model of such systems, largely based on the core p53 pathway, to explore the mechanism for the transformation of dynamic behaviors. Our results show that enhancing the positive feedback may promote or suppress oscillations depending on the strength of both feedback loops. We found that the system oscillates with low amplitudes in response to a moderate stimulus and switches to the on state upon a strong stimulus. When the positive feedback is activated much later than the negative one in response to a strong stimulus, the system exhibits long-term oscillations before switching to the on state. We explain this intriguing phenomenon using quasistatic approximation. Moreover, early switching to the on state may occur when the system starts from a steady state in the absence of stimuli. The interplay between the positive and negative feedback plays a key role in the transitions between oscillation and bistability. Of note, our conclusions should be applicable only to some specific gene regulatory networks, especially the p53 network, in which both oscillation and bistability exist in response to a certain type of stimulus. Our work also underscores the significance of transient dynamics in determining cellular outcome.

Formation of prismatic loops from C15 Laves phase interstitial clusters in body-centered cubic iron

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yongfeng; Bai, Xian-Ming; Tonks, Michael R.

2015-03-01

This Letter reports the transition of C15 phase self-interstitial clusters to loops in body-centered-cubic Iron. Molecular dynamics simulations are performed to evaluate the relative stabilities of difference interstitial cluster configurations including C15 phase structure and <100> and <111>/2 loops. Within a certain size range, C15 cluster are found more stable than loops, and the relative stabilities are reversed beyond that range. In accordance to the crossover in relative stabilities, C15 clusters may grow by absorbing individual interstitials at small sizes and transitions into loops eventually. The transition takes place by nucleation and reaction of <111>/2 loop segments. These observations explainmore » the absence of C15 phase interstitial clusters predicted by density-functional-theory calculations in previous experimental observations. More importantly, the current results provide a new formation mechanism of <100> loops which requires no interaction of loops.« less

An alternative for presenting interactive dynamic data sets in electronic presentations: a scrollable flash movie loop.

PubMed

Yam, Chun-Shan

2007-11-01

The purpose of this article is to describe an alternative for creating scrollable movie loops for electronic presentations including PowerPoint. The alternative provided in this article enables academic radiologists to present scrollable movie loops in PowerPoint. The scrolling capability is created using Flash ActionScript. A Flash template with the required ActionScript code is provided. Users can simply download the template and follow the step-by-step demonstration to create scrollable movie loops. No previous ActionScript programming knowledge is necessary.

Stability of disclination loop in pure twist nematic liquid crystals

NASA Astrophysics Data System (ADS)

Kadivar, Erfan

2018-04-01

In this work, the annihilations dynamics and stability of disclination loop in a bulk pure twist nematic liquid crystal are investigated. This work is based on the Frank free energy and the nematodynamics equations. The energy dissipation is calculated by using two methods. In the first method, the energy dissipation is obtained from the Frank free energy. In the second method, it is calculated by using the nematodynamics equations. Finally, we derive a critical radius of disclination loop that above this radius, loop creation is energetically forbidden.

NMR Studies of the Dynamics of Nitrophorin 2 Bound to Nitric Oxide†

PubMed Central

Muthu, Dhanasekaran; Berry, Robert E.; Zhang, Hongjun; Walker, F. Ann

2013-01-01

The Rhodnius nitrophorins are β-barrel proteins of the lipocalin fold with a heme protruding from the open end of the barrel. They are found in the saliva of the blood-sucking insect Rhodnius prolixus, which synthesizes and stores nitric oxide (NO) in the salivary glands, where NO is bound to iron. NO is released by dilution and pH rise when the insect spits its saliva into the tissues of a victim, to aid in obtaining a blood meal. In the adult insect there are four nitrophorins, NP1, NP2, NP3 and NP4. At pH 7.3, NP4 releases NO 17 times faster than does NP2, as measured by stopped-flow kinetics. A number of crystal structures of the least abundant protein, NP4, are available. These structures have been used to propose that two loops between adjacent β-strands at the front opening of the protein, the A-B and G-H loops, determine the rate of NO release. In order to learn how the protein loops contribute to release of NO for each of the nitrophorins, the dynamics of these proteins are being studied in our laboratory. In this work, the NP2-NO complex has been investigated by NMR relaxation measurements to probe the pico- to nanosecond and micro- to millisecond time scale motions at three pH values, 5.0, 6.5, and 7.3. It is found that at pH 5.0 and 6.5, NP2-NO is rigid and only a few residues in the loop regions show dynamics, while at pH 7.3 somewhat more dynamics, particularly of the A-B loop, are observed. Comparison to other lipocalins shows that all are relatively rigid, and that the dynamics of lipocalins in general are much more subtle than those of mainly α-helical proteins. PMID:24116947

The solar dynamic radiator with a historical perspective

NASA Technical Reports Server (NTRS)

Mclallin, K. L.; Fleming, M. L.; Hoehn, F. W.; Howerton, R. L.

1988-01-01

A historical perspective on pumped-fluid loop space radiators provides a basis for the design of the Space Station Solar Dynamic (SD) power module radiator. SD power modules, capable of generating 25 kW (electrical) each, are planned for growth in Station power requirements. The Brayton cycle SD module configuration incorporates a pumped-fluid loop radiator that must reject up to 99 kW (thermal). The thermal/hydraulic design conditions in combination with required radiator orientation and packaging envelope form a unique set of constraints as compared to previous pumped-fluid loop radiator systems. Nevertheless, past program successes have demonstrated a technology base that can be applied to the SD radiator development program to ensure a low risk, low cost system.

Prominence condensation and magnetic levitation in a coronal loop

NASA Technical Reports Server (NTRS)

Van Hoven, G.; Mok, Y.; Drake, J. F.

1992-01-01

The results of a model dynamic simulation of the formation and support of a narrow prominence at the apex of a coronal magnetic loop or arcade are described. The condensation process proceeds via an initial radiative cooling and pressure drop, and a secondary siphon flow from the dense chromospheric ends. The antibuoyancy effect as the prominence forms causes a bending of the confining magnetic field, which propagates toward the semirigid ends of the magnetic loop. Thus, a wide magnetic 'hammock' or well (of the normal-polarity Kippenhahn-Schlueter-type) is formed, which supports the prominence at or near the field apex. The simplicity of this 1.5-dimensional model, with its accompanying diagnostics, elucidates the various contributions to the nonlinear dynamics of prominence condensation and levitation.

Modeling the protonation states of β-secretase binding pocket by molecular dynamics simulations and docking studies.

PubMed

Sabbah, Dima A; Zhong, Haizhen A

2016-07-01

β-secretase (BACE1) is an aspartyl protease that processes the β-amyloid peptide in the human brain in patients with Alzheimer's disease. There are two catalytic aspartates (ASP32 and ASP228) in the active domain of BACE1. Although it is believed that the net charge of the Asp dyad is -1, the exact protonation state still remains a matter of debate. We carried out molecular dynamic (MD) simulations for the four protonation states of BACE1 proteins. We applied Glide docking studies to 21 BACE1 inhibitors against the MD extracted conformations. The dynamic results infer that the protein/ligand complex remains stable during the entire simulation course for HD32D228 model. The results show that the hydrogen bonds between the inhibitor and the Asp dyad are maintained in the 10,000th ps snapshot of HD32D228 model. Our results also reveal the significant loop residues in maintaining the active binding conformation in the HD32D228 model. Molecular docking results show that the HD32D228 model provided the best enrichment factor score, suggesting that this model was able to recognize the most active compounds. Our observations provide an evidence for the preference of the anionic state (HD32D228) in BACE1 binding site and are in accord with reported computational data. The protonation state study would provide significant information to assign the correct protonation state for structure-based drug design and docking studies targeting the BACE1 proteins as a tactic to develop potential AD inhibitors. Copyright © 2016 Elsevier Inc. All rights reserved.

Monomerization alters the dynamics of the lid region in Campylobacter jejuni CstII: an MD simulation study.

PubMed

Prabhakar, Pradeep Kumar; Srivastava, Alka; Rao, K Krishnamurthy; Balaji, Petety V

2016-01-01

CstII, a bifunctional (α2,3/8) sialyltransferase from Campylobacter jejuni, is a homotetramer. It has been reported that mutation of the interface residues Phe121 (F121D) or Tyr125 (Y125Q) leads to monomerization and partial loss of enzyme activity, without any change in the secondary or tertiary structures. MD simulations of both tetramer and monomer, with and without bound donor substrate, were performed for the two mutants and WT to understand the reasons for partial loss of activity due to monomerization since the active site is located within each monomer. RMSF values were found to correlate with the crystallographic B-factor values indicating that the simulations are able to capture the flexibility of the molecule effectively. There were no gross changes in either the secondary or tertiary structure of the proteins during MD simulations. However, interface is destabilized by the mutations, and more importantly the flexibility of the lid region (Gly152-Lys190) is affected. The lid region accesses three major conformations named as open, intermediate, and closed conformations. In both Y121Q and F121D mutants, the closed conformation is accessed predominantly. In this conformation, the catalytic base His188 is also displaced. Normal mode analysis also revealed differences in the lid movement in tetramer and monomer. This provides a possible explanation for the partial loss of enzyme activity in both interface mutants. The lid region controls the traffic of substrates and products in and out of the active site, and the dynamics of this region is regulated by tetramerization. Thus, this study provides valuable insights into the role of loop dynamics in enzyme activity of CstII.

Understanding the role of dynamics in the iron sulfur cluster molecular machine.

PubMed

di Maio, Danilo; Chandramouli, Balasubramanian; Yan, Robert; Brancato, Giuseppe; Pastore, Annalisa

2017-01-01

The bacterial proteins IscS, IscU and CyaY, the bacterial orthologue of frataxin, play an essential role in the biological machine that assembles the prosthetic FeS cluster groups on proteins. They form functionally binary and ternary complexes both in vivo and in vitro. Yet, the mechanism by which they work remains unclear. We carried out extensive molecular dynamics simulations to understand the nature of their interactions and the role of dynamics starting from the crystal structure of a IscS-IscU complex and the experimentally-based model of a ternary IscS-IscU-CyaY complex and used nuclear magnetic resonance to experimentally test the interface. We show that, while being firmly anchored to IscS, IscU has a pivotal motion around the interface. Our results also describe how the catalytic loop of IscS can flip conformation to allow FeS cluster assembly. This motion is hampered in the ternary complex explaining its inhibitory properties in cluster formation. We conclude that the observed 'fluid' IscS-IscU interface provides the binary complex with a functional adaptability exploited in partner recognition and unravels the molecular determinants of the reported inhibitory action of CyaY in the IscS-IscU-CyaY complex explained in terms of the hampering effect on specific IscU-IscS movements. Our study provides the first mechanistic basis to explain how the IscS-IscU complex selects its binding partners and supports the inhibitory role of CyaY in the ternary complex. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Investigating the Structure and Dynamics of the PIK3CA Wild-Type and H1047R Oncogenic Mutant

PubMed Central

Pavlaki, Maria; Lazani, Vasiliki; Christoforidis, Savvas; Agianian, Bogos; Cournia, Zoe

2014-01-01

The PIK3CA gene is one of the most frequently mutated oncogenes in human cancers. It encodes p110α, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kα), which activates signaling cascades leading to cell proliferation, survival, and cell growth. The most frequent mutation in PIK3CA is H1047R, which results in enzymatic overactivation. Understanding how the H1047R mutation causes the enhanced activity of the protein in atomic detail is central to developing mutant-specific therapeutics for cancer. To this end, Surface Plasmon Resonance (SPR) experiments and Molecular Dynamics (MD) simulations were carried out for both wild-type (WT) and H1047R mutant proteins. An expanded positive charge distribution on the membrane binding regions of the mutant with respect to the WT protein is observed through MD simulations, which justifies the increased ability of the mutated protein variant to bind to membranes rich in anionic lipids in our SPR experiments. Our results further support an auto-inhibitory role of the C-terminal tail in the WT protein, which is abolished in the mutant protein due to loss of crucial intermolecular interactions. Moreover, Functional Mode Analysis reveals that the H1047R mutation alters the twisting motion of the N-lobe of the kinase domain with respect to the C-lobe and shifts the position of the conserved P-loop residues in the vicinity of the active site. These findings demonstrate the dynamical and structural differences of the two proteins in atomic detail and propose a mechanism of overactivation for the mutant protein. The results may be further utilized for the design of mutant-specific PI3Kα inhibitors that exploit the altered mutant conformation. PMID:25340423

Three flavor Nambu-Jona-Lasinio model with Polyakov loop and competition with nuclear matter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ciminale, M.; Ippolito, N. D.; Nardulli, G.

2008-03-01

We study the phase diagram of the three flavor Polyakov-Nambu-Jona-Lasinio (PNJL) model and, in particular, the interplay between chiral symmetry restoration and deconfinement crossover. We compute chiral condensates, quark densities, and the Polyakov loop at several values of temperature and chemical potential. Moreover we investigate on the role of the Polyakov loop dynamics in the transition from nuclear matter to quark matter.

Plasma dynamics above solar flare soft x-ray loop tops

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doschek, G. A.; Warren, H. P.; McKenzie, D. E.

2014-06-10

We measure non-thermal motions in flare loop tops and above the loop tops using profiles of highly ionized spectral lines of Fe XXIV and Fe XXIII formed at multimillion-degree temperatures. Non-thermal motions that may be due to turbulence or multiple flow regions along the line of sight are extracted from the line profiles. The non-thermal motions are measured for four flares seen at or close to the solar limb. The profile data are obtained using the Extreme-ultraviolet Imaging Spectrometer on the Hinode spacecraft. The multimillion-degree non-thermal motions are between 20 and 60 km s{sup –1} and appear to increase withmore » height above the loop tops. Motions determined from coronal lines (i.e., lines formed at about 1.5 MK) tend to be smaller. The multimillion-degree temperatures in the loop tops and above range from about 11 MK to 15 MK and also tend to increase with height above the bright X-ray-emitting loop tops. The non-thermal motions measured along the line of sight, as well as their apparent increase with height, are supported by Solar Dynamics Observatory Atmospheric Imaging Assembly measurements of turbulent velocities in the plane of the sky.« less

A time domain inverse dynamic method for the end point tracking control of a flexible manipulator

NASA Technical Reports Server (NTRS)

Kwon, Dong-Soo; Book, Wayne J.

1991-01-01

The inverse dynamic equation of a flexible manipulator was solved in the time domain. By dividing the inverse system equation into the causal part and the anticausal part, we calculated the torque and the trajectories of all state variables for a given end point trajectory. The interpretation of this method in the frequency domain was explained in detail using the two-sided Laplace transform and the convolution integral. The open loop control of the inverse dynamic method shows an excellent result in simulation. For real applications, a practical control strategy is proposed by adding a feedback tracking control loop to the inverse dynamic feedforward control, and its good experimental performance is presented.

Dual allosteric activation mechanisms in monomeric human glucokinase

PubMed Central

Whittington, A. Carl; Larion, Mioara; Bowler, Joseph M.; Ramsey, Kristen M.; Brüschweiler, Rafael; Miller, Brian G.

2015-01-01

Cooperativity in human glucokinase (GCK), the body’s primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme’s small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the 1H-13C heteronuclear multiple quantum coherence (HMQC) spectrum of 13C-Ile–labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the 1H-13C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems. PMID:26283387

Dual allosteric activation mechanisms in monomeric human glucokinase.

PubMed

Whittington, A Carl; Larion, Mioara; Bowler, Joseph M; Ramsey, Kristen M; Brüschweiler, Rafael; Miller, Brian G

2015-09-15

Cooperativity in human glucokinase (GCK), the body's primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme's small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) spectrum of (13)C-Ile-labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the (1)H-(13)C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems.

Mass gap in the weak coupling limit of (2 +1 )-dimensional SU(2) lattice gauge theory

NASA Astrophysics Data System (ADS)

Anishetty, Ramesh; Sreeraj, T. P.

2018-04-01

We develop the dual description of (2 +1 )-dimensional SU(2) lattice gauge theory as interacting "Abelian-like" electric loops by using Schwinger bosons. "Point splitting" of the lattice enables us to construct explicit Hilbert space for the gauge invariant theory which in turn makes dynamics more transparent. Using path integral representation in phase space, the interacting closed loop dynamics is analyzed in the weak coupling limit to get the mass gap.

Dynamic stability and handling qualities tests on a highly augmented, statically unstable airplane

NASA Technical Reports Server (NTRS)

Gera, Joseph; Bosworth, John T.

1987-01-01

This paper describes some novel flight tests and analysis techniques in the flight dynamics and handling qualities area. These techniques were utilized during the initial flight envelope clearance of the X-29A aircraft and were largely responsible for the completion of the flight controls clearance program without any incidents or significant delays. The resulting open-loop and closed-loop frequency responses and the time history comparison using flight and linear simulation data are discussed.

Thermoelectric power generator with intermediate loop

DOEpatents

Bell, Lon E; Crane, Douglas Todd

2013-05-21

A thermoelectric power generator is disclosed for use to generate electrical power from heat, typically waste heat. An intermediate heat transfer loop forms a part of the system to permit added control and adjustability in the system. This allows the thermoelectric power generator to more effectively and efficiently generate power in the face of dynamically varying temperatures and heat flux conditions, such as where the heat source is the exhaust of an automobile, or any other heat source with dynamic temperature and heat flux conditions.

Thermoelectric power generator with intermediate loop

DOEpatents

Bel,; Lon, E [Altadena, CA; Crane, Douglas Todd [Pasadena, CA

2009-10-27

A thermoelectric power generator is disclosed for use to generate electrical power from heat, typically waste heat. An intermediate heat transfer loop forms a part of the system to permit added control and adjustability in the system. This allows the thermoelectric power generator to more effectively and efficiently generate power in the face of dynamically varying temperatures and heat flux conditions, such as where the heat source is the exhaust of an automobile, or any other heat source with dynamic temperature and heat flux conditions.

Paraxial Stage of the Spatiotemporal Dynamics of Loop Solitons

NASA Astrophysics Data System (ADS)

Sazonov, Sergey V.

2018-03-01

Using the averaged Lagrangian method, an analytic study of the spatiotemporal dynamics of extremely short loop solitons of the generalized Vakhnenko-Schäfer-Wayne equation is carried out. The conditions under which soliton propagation occurs in the modes of defocusing and self-focusing are determined. It is shown that transverse defocusing is accompanied by a temporal compression of the soliton and a decrease in its amplitude. At the same time, with transverse self-focusing, its temporal broadening and peak amplification occur.

Toward a blueprint for UDP-glucose pyrophosphorylase structure/function properties: homology-modeling analyses.

PubMed

Geisler, Matt; Wilczynska, Malgorzata; Karpinski, Stanislaw; Kleczkowski, Leszek A

2004-11-01

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of synthesis of sucrose, cellulose, and several other polysaccharides in all plants. The protein is evolutionarily conserved among eukaryotes, but has little relation, aside from its catalytic reaction, to UGPases of prokaryotic origin. Using protein homology modeling strategy, 3D structures for barley, poplar, and Arabidopsis UGPases have been derived, based on recently published crystal structure of human UDP-N-acetylglucosamine pyrophosphorylase. The derived 3D structures correspond to a bowl-shaped protein with the active site at a central groove, and a C-terminal domain that includes a loop (I-loop) possibly involved in dimerization. Data on a plethora of earlier described UGPase mutants from a variety of eukaryotic organisms have been revisited, and we have, in most cases, verified the role of each mutation in enzyme catalysis/regulation/structural integrity. We have also found that one of two alternatively spliced forms of poplar UGPase has a very short I-loop, suggesting differences in oligomerization ability of the two isozymes. The derivation of the structural model for plant UGPase should serve as a useful blueprint for further function/structure studies on this protein.

Structural characterization of the novel aminoglycoside phosphotransferase AphVIII from Streptomyces rimosus with enzymatic activity modulated by phosphorylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boyko, Konstantin M., E-mail: kmb@inbi.ras.ru; National Research Center “Kurchatov Institute”, Kurchatov Complex of NBICS-technologies, Akad. Kurchatova sqr., 1, Moscow, 123182; Gorbacheva, Marina A.

2016-09-02

Aminoglycoside phosphotransferases represent a broad class of enzymes that promote bacterial resistance to aminoglycoside antibiotics via the phosphorylation of hydroxyl groups in the latter. Here we report the spatial structure of the 3′-aminoglycoside phosphotransferase of novel VIII class (AphVIII) solved by X-ray diffraction method with a resolution of 2.15 Å. Deep analysis of APHVIII structure and its comparison with known structures of aminoglycoside phosphotransferases of various types reveals that AphVIII has a typical two-domain fold and, however, possesses some unique characteristics that distinguish the enzyme from its known homologues. The most important difference is the presence of the activation loop withmore » unique Ser146 residue. We demonstrate that in the apo-state of the enzyme the activation loop does not interact with other parts of the enzyme and seems to adopt catalytically competent state only after substrate binding. - Highlights: • 3D structure of the novel aminoglycoside phosphotransferase AphVIII was obtained. • AphVIII activation loop is clearly identified in the electron density. • AphVIII has some unique structural features in its substrate C-ring binding pocket.« less

Mobile application MDDCS for modeling the expansion dynamics of a dislocation loop in FCC metals

NASA Astrophysics Data System (ADS)

Kirilyuk, Vasiliy; Petelin, Alexander; Eliseev, Andrey

2017-11-01

A mobile version of the software package Dynamic Dislocation of Crystallographic Slip (MDDCS) designed for modeling the expansion dynamics of dislocation loops and formation of a crystallographic slip zone in FCC-metals is examined. The paper describes the possibilities for using MDDCS, the application interface, and the database scheme. The software has a simple and intuitive interface and does not require special training. The user can set the initial parameters of the experiment, carry out computational experiments, export parameters and results of the experiment into separate text files, and display the experiment results on the device screen.

Linearizing feedforward/feedback attitude control

NASA Technical Reports Server (NTRS)

Paielli, Russell A.; Bach, Ralph E.

1991-01-01

An approach to attitude control theory is introduced in which a linear form is postulated for the closed-loop rotation error dynamics, then the exact control law required to realize it is derived. The nonminimal (four-component) quaternion form is used to attitude because it is globally nonsingular, but the minimal (three-component) quaternion form is used for attitude error because it has no nonlinear constraints to prevent the rotational error dynamics from being linearized, and the definition of the attitude error is based on quaternion algebra. This approach produces an attitude control law that linearizes the closed-loop rotational error dynamics exactly, without any attitude singularities, even if the control errors become large.

Replacing Arginine 33 for Alanine in the Hemophore HasA from Pseudomonas aeruginosa Causes Closure of the H32 Loop in the Apo-Protein

PubMed Central

Kumar, Ritesh; Qi, Yifei; Matsumura, Hirotoshi; Lovell, Scott; Yao, Huili; Battaile, Kevin P.; Im, Wonpil; Moënne-Loccoz, Pierre; Rivera, Mario

2017-01-01

Previous characterization of hemophores from Serratia marcescens (HasAs), Pseudomonas aeruginosa (HasAp) and Yersinia pestis (HasAyp) showed that hemin binds between two loops, where it is axially coordinated by H32 and Y75. The Y75 loop is structurally conserved in all three hemophores and harbors conserved ligand Y75. The other loop contains H32 in HasAs and HasAp, but a noncoordinating Q32 in HasAyp. The H32 loop in apo-HasAs and apo-HasAp is in an open conformation, which places H32 about 30 Å from the hemin-binding site. Hence, hemin binding onto the Y75 loop of HasAs or HasAp triggers a large relocation of the H32 loop from an open- to a closed-loop conformation and enables coordination of the hemin-iron by H32. In comparison, the Q32 loop in apo-HasAyp is in the closed conformation and hemin binding occurs with minimal reorganization and without coordinative interactions with the Q32 loop. Studies in crystallo and in solution have established that the open H32 loop in apo-HasAp and apo-HasAs is well structured and minimally affected by conformational dynamics. In this study we address the intriguing issue of the stability of the H32 loop in apo-HasAp and how hemin binding triggers its relocation. We address this question with a combination of NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations and find that R33 is critical to the stability of the open H32 loop. Replacing R33 with A causes the H32 loop in R33A apo-HasAp to adopt a conformation similar to that of holo-HasAp. Finally, stopped-flow absorption and resonance Raman analyses of hemin binding to apo-R33A HasAp indicates that the closed H32 loop slows down the insertion of the heme inside the binding pocket, presumably as it obstructs access to the hydrophobic platform on the Y75 loop, but accelerate the completion of the heme iron coordination. PMID:27074415

Hippocampal closed-loop modeling and implications for seizure stimulation design

NASA Astrophysics Data System (ADS)

Sandler, Roman A.; Song, Dong; Hampson, Robert E.; Deadwyler, Sam A.; Berger, Theodore W.; Marmarelis, Vasilis Z.

2015-10-01

Objective. Traditional hippocampal modeling has focused on the series of feedforward synapses known as the trisynaptic pathway. However, feedback connections from CA1 back to the hippocampus through the entorhinal cortex (EC) actually make the hippocampus a closed-loop system. By constructing a functional closed-loop model of the hippocampus, one may learn how both physiological and epileptic oscillations emerge and design efficient neurostimulation patterns to abate such oscillations. Approach. Point process input-output models where estimated from recorded rodent hippocampal data to describe the nonlinear dynamical transformation from CA3 → CA1, via the schaffer-collateral synapse, and CA1 → CA3 via the EC. Each Volterra-like subsystem was composed of linear dynamics (principal dynamic modes) followed by static nonlinearities. The two subsystems were then wired together to produce the full closed-loop model of the hippocampus. Main results. Closed-loop connectivity was found to be necessary for the emergence of theta resonances as seen in recorded data, thus validating the model. The model was then used to identify frequency parameters for the design of neurostimulation patterns to abate seizures. Significance. Deep-brain stimulation (DBS) is a new and promising therapy for intractable seizures. Currently, there is no efficient way to determine optimal frequency parameters for DBS, or even whether periodic or broadband stimuli are optimal. Data-based computational models have the potential to be used as a testbed for designing optimal DBS patterns for individual patients. However, in order for these models to be successful they must incorporate the complex closed-loop structure of the seizure focus. This study serves as a proof-of-concept of using such models to design efficient personalized DBS patterns for epilepsy.

Hippocampal closed-loop modeling and implications for seizure stimulation design.

PubMed

Sandler, Roman A; Song, Dong; Hampson, Robert E; Deadwyler, Sam A; Berger, Theodore W; Marmarelis, Vasilis Z

2015-10-01

Traditional hippocampal modeling has focused on the series of feedforward synapses known as the trisynaptic pathway. However, feedback connections from CA1 back to the hippocampus through the entorhinal cortex (EC) actually make the hippocampus a closed-loop system. By constructing a functional closed-loop model of the hippocampus, one may learn how both physiological and epileptic oscillations emerge and design efficient neurostimulation patterns to abate such oscillations. Point process input-output models where estimated from recorded rodent hippocampal data to describe the nonlinear dynamical transformation from CA3 → CA1, via the schaffer-collateral synapse, and CA1 → CA3 via the EC. Each Volterra-like subsystem was composed of linear dynamics (principal dynamic modes) followed by static nonlinearities. The two subsystems were then wired together to produce the full closed-loop model of the hippocampus. Closed-loop connectivity was found to be necessary for the emergence of theta resonances as seen in recorded data, thus validating the model. The model was then used to identify frequency parameters for the design of neurostimulation patterns to abate seizures. Deep-brain stimulation (DBS) is a new and promising therapy for intractable seizures. Currently, there is no efficient way to determine optimal frequency parameters for DBS, or even whether periodic or broadband stimuli are optimal. Data-based computational models have the potential to be used as a testbed for designing optimal DBS patterns for individual patients. However, in order for these models to be successful they must incorporate the complex closed-loop structure of the seizure focus. This study serves as a proof-of-concept of using such models to design efficient personalized DBS patterns for epilepsy.

Hippocampal Closed-Loop Modeling and Implications for Seizure Stimulation Design

PubMed Central

Sandler, Roman A.; Song, Dong; Hampson, Robert E.; Deadwyler, Sam A.; Berger, Theodore W.; Marmarelis, Vasilis Z.

2016-01-01

Objective Traditional hippocampal modeling has focused on the series of feedforward synapses known as the trisynaptic pathway. However, feedback connections from CA1 back to the hippocampus through the Entorhinal Cortex (EC) actually make the hippocampus a closed-loop system. By constructing a functional closed-loop model of the hippocampus, one may learn how both physiological and epileptic oscillations emerge and design efficient neurostimulation patterns to abate such oscillations. Approach Point process input-output models where estimated from recorded rodent hippocampal data to describe the nonlinear dynamical transformation from CA3→CA1, via the Schaffer-Collateral synapse, and CA1→CA3 via the EC. Each Volterra-like subsystem was composed of linear dynamics (Principal Dynamic Modes) followed by static nonlinearities. The two subsystems were then wired together to produce the full closed-loop model of the hippocampus. Main Results Closed-loop connectivity was found to be necessary for the emergence of theta resonances as seen in recorded data, thus validating the model. The model was then used to identify frequency parameters for the design of neurostimulation patterns to abate seizures. Significance DBS is a new and promising therapy for intractable seizures. Currently, there is no efficient way to determine optimal frequency parameters for DBS, or even whether periodic or broadband stimuli are optimal. Data-based computational models have the potential to be used as a testbed for designing optimal DBS patterns for individual patients. However, in order for these models to be successful they must incorporate the complex closed-loop structure of the seizure focus. This study serves as a proof-of-concept of using such models to design efficient personalized DBS patterns for epilepsy. PMID:26355815

Actin Hydrophobic Loop (262-274) and Filament Nucleation and Elongation

PubMed Central

Shvetsov, Alexander; Galkin, Vitold E.; Orlova, Albina; Phillips, Martin; Bergeron, Sarah E.; Rubenstein, Peter A.; Egelman, Edward H.; Reisler, Emil

2014-01-01

Summary The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently using a yeast actin mutant, L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked G-actin does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin - to assist with actin nucleation - and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not alone, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin the helical twist of F-actin changes by ~ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin, and a change of twist by ~ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or a competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics to both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors. PMID:18037437

Control-structure interaction in precision pointing servo loops

NASA Technical Reports Server (NTRS)

Spanos, John T.

1989-01-01

The control-structure interaction problem is addressed via stability analysis of a generic linear servo loop model. With the plant described by the rigid body mode and a single elastic mode, structural flexibility is categorized into one of three types: (1) appendage, (2) in-the-loop minimum phase, and (3) in-the-loop nonminimum phase. Closing the loop with proportional-derivative (PD) control action and introducing sensor roll-off dynamics in the feedback path, stability conditions are obtained. Trade studies are conducted with modal frequency, modal participation, modal damping, loop bandwidth, and sensor bandwidth treated as free parameters. Results indicate that appendage modes are most likely to produce instability if they are near the sensor rolloff, whereas in-the-loop modes are most dangerous near the loop bandwidth. The main goal of this paper is to provide a fundamental understanding of the control-structure interaction problem so that it may benefit the design of complex spacecraft and pointing system servo loops. In this framework, the JPL Pathfinder gimbal pointer is considered as an example.

Towards the use of Structural Loop Analysis to Study System Behaviour of Socio-Ecological Systems.

NASA Astrophysics Data System (ADS)

Abram, Joseph; Dyke, James

2016-04-01

Maintaining socio-ecological systems in desirable states is key to developing a growing economy, alleviating poverty and achieving a sustainable future. While the driving forces of an environmental system are often well known, the dynamics impacting these drivers can be hidden within a tangled structure of causal chains and feedback loops. A lack of understanding of a system's dynamic structure and its influence on a system's behaviour can cause unforeseen side-effects during model scenario testing and policy implementation. Structural Loop analysis of socio-ecological system models identifies dominant feedback structures during times of behavioural shift, allowing the user to monitor key influential drivers during model simulation. This work carries out Loop Eigenvalue Elasticity Analysis (LEEA) on three system dynamic models, exploring tipping points in lake systems undergoing eutrophication. The purpose is to explore the potential benefits and limitations of the technique in the field of socio-ecology. The LEEA technique shows promise for socio-ecological systems which undergo regime shifts or express oscillatory trends, but shows limited usefulness with large models. The results of this work highlight changes in feedback loop dominance, years prior to eutrophic tipping events in lake systems. LEEA could be used as an early warning signal to impending system changes, complementary to other known early warning signals. This approach could improve our understanding during critical times of a system's behaviour, changing how we approach model analysis and the way scenario testing and policy implementation are addressed in socio-ecological system models.

The insertion of human dynamics models in the flight control loops of V/STOL research aircraft. Appendix 2: The optimal control model of a pilot in V/STOL aircraft control loops

NASA Technical Reports Server (NTRS)

Zipf, Mark E.

1989-01-01

An overview is presented of research work focussed on the design and insertion of classical models of human pilot dynamics within the flight control loops of V/STOL aircraft. The pilots were designed and configured for use in integrated control system research and design. The models of human behavior that were considered are: McRuer-Krendel (a single variable transfer function model); and Optimal Control Model (a multi-variable approach based on optimal control and stochastic estimation theory). These models attempt to predict human control response characteristics when confronted with compensatory tracking and state regulation tasks. An overview, mathematical description, and discussion of predictive limitations of the pilot models is presented. Design strategies and closed loop insertion configurations are introduced and considered for various flight control scenarios. Models of aircraft dynamics (both transfer function and state space based) are developed and discussed for their use in pilot design and application. Pilot design and insertion are illustrated for various flight control objectives. Results of pilot insertion within the control loops of two V/STOL research aricraft (Sikorski Black Hawk UH-60A, McDonnell Douglas Harrier II AV-8B) are presented and compared against actual pilot flight data. Conclusions are reached on the ability of the pilot models to adequately predict human behavior when confronted with similar control objectives.

Structural consequences of cutting a binding loop: two circularly permuted variants of streptavidin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Le Trong, Isolde; University of Washington, Box 357742, Seattle, WA 98195-7742; Chu, Vano

2013-06-01

The crystal structures of two circularly permuted streptavidins probe the role of a flexible loop in the tight binding of biotin. Molecular-dynamics calculations for one of the mutants suggests that increased fluctuations in a hydrogen bond between the protein and biotin are associated with cleavage of the binding loop. Circular permutation of streptavidin was carried out in order to investigate the role of a main-chain amide in stabilizing the high-affinity complex of the protein and biotin. Mutant proteins CP49/48 and CP50/49 were constructed to place new N-termini at residues 49 and 50 in a flexible loop involved in stabilizing themore » biotin complex. Crystal structures of the two mutants show that half of each loop closes over the binding site, as observed in wild-type streptavidin, while the other half adopts the open conformation found in the unliganded state. The structures are consistent with kinetic and thermodynamic data and indicate that the loop plays a role in enthalpic stabilization of the bound state via the Asn49 amide–biotin hydrogen bond. In wild-type streptavidin, the entropic penalties of immobilizing a flexible portion of the protein to enhance binding are kept to a manageable level by using a contiguous loop of medium length (six residues) which is already constrained by its anchorage to strands of the β-barrel protein. A molecular-dynamics simulation for CP50/49 shows that cleavage of the binding loop results in increased structural fluctuations for Ser45 and that these fluctuations destabilize the streptavidin–biotin complex.« less

Kalman Orbit Optimized Loop Tracking

NASA Technical Reports Server (NTRS)

Young, Lawrence E.; Meehan, Thomas K.

2011-01-01

Under certain conditions of low signal power and/or high noise, there is insufficient signal to noise ratio (SNR) to close tracking loops with individual signals on orbiting Global Navigation Satellite System (GNSS) receivers. In addition, the processing power available from flight computers is not great enough to implement a conventional ultra-tight coupling tracking loop. This work provides a method to track GNSS signals at very low SNR without the penalty of requiring very high processor throughput to calculate the loop parameters. The Kalman Orbit-Optimized Loop (KOOL) tracking approach constitutes a filter with a dynamic model and using the aggregate of information from all tracked GNSS signals to close the tracking loop for each signal. For applications where there is not a good dynamic model, such as very low orbits where atmospheric drag models may not be adequate to achieve the required accuracy, aiding from an IMU (inertial measurement unit) or other sensor will be added. The KOOL approach is based on research JPL has done to allow signal recovery from weak and scintillating signals observed during the use of GPS signals for limb sounding of the Earth s atmosphere. That approach uses the onboard PVT (position, velocity, time) solution to generate predictions for the range, range rate, and acceleration of the low-SNR signal. The low- SNR signal data are captured by a directed open loop. KOOL builds on the previous open loop tracking by including feedback and observable generation from the weak-signal channels so that the MSR receiver will continue to track and provide PVT, range, and Doppler data, even when all channels have low SNR.

OBSERVATIONAL SIGNATURES OF CORONAL LOOP HEATING AND COOLING DRIVEN BY FOOTPOINT SHUFFLING

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dahlburg, R. B.; Taylor, B. D.; Einaudi, G.

The evolution of a coronal loop is studied by means of numerical simulations of the fully compressible three-dimensional magnetohydrodynamic equations using the HYPERION code. The footpoints of the loop magnetic field are advected by random motions. As a consequence, the magnetic field in the loop is energized and develops turbulent nonlinear dynamics characterized by the continuous formation and dissipation of field-aligned current sheets: energy is deposited at small scales where heating occurs. Dissipation is nonuniformly distributed so that only a fraction of the coronal mass and volume gets heated at any time. Temperature and density are highly structured at scalesmore » that, in the solar corona, remain observationally unresolved: the plasma of our simulated loop is multithermal, where highly dynamical hotter and cooler plasma strands are scattered throughout the loop at sub-observational scales. Numerical simulations of coronal loops of 50,000 km length and axial magnetic field intensities ranging from 0.01 to 0.04 T are presented. To connect these simulations to observations, we use the computed number densities and temperatures to synthesize the intensities expected in emission lines typically observed with the Extreme Ultraviolet Imaging Spectrometer on Hinode. These intensities are used to compute differential emission measure distributions using the Monte Carlo Markov Chain code, which are very similar to those derived from observations of solar active regions. We conclude that coronal heating is found to be strongly intermittent in space and time, with only small portions of the coronal loop being heated: in fact, at any given time, most of the corona is cooling down.« less

The Metabolic Core and Catalytic Switches Are Fundamental Elements in the Self-Regulation of the Systemic Metabolic Structure of Cells

PubMed Central

De la Fuente, Ildefonso M.; Cortes, Jesus M.; Perez-Pinilla, Martin B.; Ruiz-Rodriguez, Vicente; Veguillas, Juan

2011-01-01

Background Experimental observations and numerical studies with dissipative metabolic networks have shown that cellular enzymatic activity self-organizes spontaneously leading to the emergence of a metabolic core formed by a set of enzymatic reactions which are always active under all environmental conditions, while the rest of catalytic processes are only intermittently active. The reactions of the metabolic core are essential for biomass formation and to assure optimal metabolic performance. The on-off catalytic reactions and the metabolic core are essential elements of a Systemic Metabolic Structure which seems to be a key feature common to all cellular organisms. Methodology/Principal Findings In order to investigate the functional importance of the metabolic core we have studied different catalytic patterns of a dissipative metabolic network under different external conditions. The emerging biochemical data have been analysed using information-based dynamic tools, such as Pearson's correlation and Transfer Entropy (which measures effective functionality). Our results show that a functional structure of effective connectivity emerges which is dynamical and characterized by significant variations of bio-molecular information flows. Conclusions/Significance We have quantified essential aspects of the metabolic core functionality. The always active enzymatic reactions form a hub –with a high degree of effective connectivity- exhibiting a wide range of functional information values being able to act either as a source or as a sink of bio-molecular causal interactions. Likewise, we have found that the metabolic core is an essential part of an emergent functional structure characterized by catalytic modules and metabolic switches which allow critical transitions in enzymatic activity. Both, the metabolic core and the catalytic switches in which also intermittently-active enzymes are involved seem to be fundamental elements in the self-regulation of the Systemic Metabolic Structure. PMID:22125607

New biochemical insight of conserved water molecules at catalytic and structural Zn2+ ions in human matrix metalloproteinase-I: a study by MD-simulation.

PubMed

Chakrabarti, Bornali; Bairagya, Hridoy R; Mukhopadhyay, Bishnu P; Sekar, K

2017-02-01

Human matrix metalloproteinase (MMP)-1 or collagenase-1 plays a significant role in embryonic development, tissue remodeling, and is also involved in several diseases like arthritis, metastasis, etc. Molecular dynamics simulation studies on hMMP-1 X-ray structures (PDB Id. 1CGE, 1CGF, 1CGL, 1HFC, and 2TCL) suggest that the three conserved water molecules (W H/1 , W I , W S ) are coordinated with catalytic zinc (Zn C ), and one water molecule (W) is associated at structural zinc ion (Zn S ). Transition of the coordination geometry around Zn C from tetrahedral to octahedral and tetrahedral to trigonal bipyramidal at Zn S are also observed during the dynamics. Recognition of two zinc ions through water mediated bridges (Zn C - W H (W 1 )…W 2 ….H 183 - Zn S ) and stabilization of secondary coordination zone around the metal ions indicates the possibility of Zn C …Zn S coupled catalytic mechanism in hMMP-I. This study not only reveals a functionally important role of conserved water molecules in hMMP-I but also highlights the involvement of other non catalytic residues, such as S172 and D170 in the catalytic mechanism. The results obtained in this study could be relevant for importance of conserved water mediated recognition site of the sequence residue id. 202(RWTNNFREY)210, interaction of W(tryptophan)203 to zinc bound histidine, their influence on the water molecules that are involved in bridging between Zn C and Zn S , and structure-based design of specific hMMP inhibitors. Graphical abstract Water mediated recognition of structural and catalytic zinc ions of hMMP-1 structure (MD simulatated conformation).

On the dynamical nature of the active center in a single-site photocatalyst visualized by 4D ultrafast electron microscopy

PubMed Central

Yoo, Byung-Kuk; Su, Zixue; Thomas, John Meurig; Zewail, Ahmed H.

2016-01-01

Understanding the dynamical nature of the catalytic active site embedded in complex systems at the atomic level is critical to developing efficient photocatalytic materials. Here, we report, using 4D ultrafast electron microscopy, the spatiotemporal behaviors of titanium and oxygen in a titanosilicate catalytic material. The observed changes in Bragg diffraction intensity with time at the specific lattice planes, and with a tilted geometry, provide the relaxation pathway: the Ti4+=O2− double bond transformation to a Ti3+−O1− single bond via the individual atomic displacements of the titanium and the apical oxygen. The dilation of the double bond is up to 0.8 Å and occurs on the femtosecond time scale. These findings suggest the direct catalytic involvement of the Ti3+−O1− local structure, the significance of nonthermal processes at the reactive site, and the efficient photo-induced electron transfer that plays a pivotal role in many photocatalytic reactions. PMID:26729878

Three parameters optimizing closed-loop control in sequential segmental neuromuscular stimulation.

PubMed

Zonnevijlle, E D; Somia, N N; Perez Abadia, G; Stremel, R W; Maldonado, C J; Werker, P M; Kon, M; Barker, J H

1999-05-01

In conventional dynamic myoplasties, the force generation is poorly controlled. This causes unnecessary fatigue of the transposed/transplanted electrically stimulated muscles and causes damage to the involved tissues. We introduced sequential segmental neuromuscular stimulation (SSNS) to reduce muscle fatigue by allowing part of the muscle to rest periodically while the other parts work. Despite this improvement, we hypothesize that fatigue could be further reduced in some applications of dynamic myoplasty if the muscles were made to contract according to need. The first necessary step is to gain appropriate control over the contractile activity of the dynamic myoplasty. Therefore, closed-loop control was tested on a sequentially stimulated neosphincter to strive for the best possible control over the amount of generated pressure. A selection of parameters was validated for optimizing control. We concluded that the frequency of corrections, the threshold for corrections, and the transition time are meaningful parameters in the controlling algorithm of the closed-loop control in a sequentially stimulated myoplasty.

Understanding social forces involved in diabetes outcomes: a systems science approach to quality-of-life research.

PubMed

Lounsbury, David W; Hirsch, Gary B; Vega, Chawntel; Schwartz, Carolyn E

2014-04-01

The field of quality-of-life (QOL) research would benefit from learning about and integrating systems science approaches that model how social forces interact dynamically with health and affect the course of chronic illnesses. Our purpose is to describe the systems science mindset and to illustrate the utility of a system dynamics approach to promoting QOL research in chronic disease, using diabetes as an example. We build a series of causal loop diagrams incrementally, introducing new variables and their dynamic relationships at each stage. These causal loop diagrams demonstrate how a common set of relationships among these variables can generate different disease and QOL trajectories for people with diabetes and also lead to a consideration of non-clinical (psychosocial and behavioral) factors that can have implications for program design and policy formulation. The policy implications of the causal loop diagrams are discussed, and empirical next steps to validate the diagrams and quantify the relationships are described.

System identification of dynamic closed-loop control of total peripheral resistance by arterial and cardiopulmonary baroreceptors

NASA Technical Reports Server (NTRS)

Aljuri, A. N.; Bursac, N.; Marini, R.; Cohen, R. J.

2001-01-01

Prolonged exposure to microgravity in space flight missions (days) impairs the mechanisms responsible for defense of arterial blood pressure (ABP) and cardiac output (CO) against orthostatic stress in the post-flight period. The mechanisms responsible for the observed orthostatic intolerance are not yet completely understood. Additionally, effective counter measures to attenuate this pathophysiological response are not available. The aim of this study was to investigate the ability of our proposed system identification method to predict closed-loop dynamic changes in TPR induced by changes in mean arterial pressure (MAP) and right atrial pressure (RAP). For this purpose we designed and employed a novel experimental animal model for the examination of arterial and cardiopulmonary baroreceptors in the dynamic closed-loop control of total peripheral resistance (TPR), and applied system identification to the analysis of beat-to-beat fluctuations in the measured signals. Grant numbers: NAG5-4989. c 2001. Elsevier Science Ltd. All rights reserved.