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Sample records for active closed conformation

  1. Closed conformation of the active site loop of rabbit muscle triosephosphate isomerase in the absence of substrate: evidence of conformational heterogeneity.

    PubMed

    Aparicio, Ricardo; Ferreira, Sérgio T; Polikarpov, Igor

    2003-12-12

    The active site loop of triosephosphate isomerase (TIM) exhibits a hinged-lid motion, alternating between the two well defined "open" and "closed" conformations. Until now the closed conformation had only been observed in protein complexes with substrate analogues. Here, we present the first rabbit muscle apo TIM structure, refined to 1.5A resolution, in which the active site loop is either in the open or in the closed conformation in different subunits of the enzyme. In the closed conformation described here, the lid loop residues participate in stabilizing hydrogen bonds characteristic of holo TIM structures, whereas chemical interactions observed in the open loop conformation are similar to those found in the apo structures of TIM. In the closed conformation, a number of water molecules are observed at the projected ligand atom positions that are hydrogen bonded to the active site residues. Additives used during crystallization (DMSO and Tris molecules and magnesium atoms) were modeled in the electron density maps. However, no specific binding of these molecules is observed at, or close to, the active site and the lid loop. To further investigate this unusual closed conformation of the apo enzyme, two more rabbit muscle TIM structures, one in the same and another in a different crystal form, were determined. These structures present the open lid conformation only, indicating that the closed conformation cannot be explained by crystal contact effects. To rationalize why the active site loop is closed in the absence of ligand in one of the subunits, extensive comparison with previously solved TIM structures was carried out, supported by the bulk of available experimental information about enzyme kinetics and reaction mechanism of TIM. The observation of both open and closed lid conformations in TIM crystals might be related to a persistent conformational heterogeneity of this protein in solution.

  2. High resolution crystal structures of triosephosphate isomerase complexed with its suicide inhibitors: The conformational flexibility of the catalytic glutamate in its closed, liganded active site

    PubMed Central

    Venkatesan, Rajaram; Alahuhta, Markus; Pihko, Petri M; Wierenga, Rik K

    2011-01-01

    The key residue of the active site of triosephosphate isomerase (TIM) is the catalytic glutamate, which is proposed to be important (i) as a catalytic base, for initiating the reaction, as well as (ii) for the subsequent proton shuttling steps. The structural properties of this glutamate in the liganded complex have been investigated by studying the high resolution crystal structures of typanosomal TIM, complexed with three suicide inhibitors: (S)-glycidol phosphate ((S)-GOP, at 0.99 Å resolution), (R)-glycidol phosphate, ((R)-GOP, at 1.08 Å resolution), and bromohydroxyacetone phosphate (BHAP, at 1.97 Å resolution). The structures show that in the (S)-GOP active site this catalytic glutamate is in the well characterized, competent conformation. However, an unusual side chain conformation is observed in the (R)-GOP and BHAP complexes. In addition, Glu97, salt bridged to the catalytic lysine in the competent active site, adopts an unusual side chain conformation in these two latter complexes. The higher chemical reactivity of (S)-GOP compared with (R)-GOP, as known from solution studies, can be understood: the structures indicate that in the case of (S)-GOP, Glu167 can attack the terminal carbon of the epoxide in a stereoelectronically favored, nearly linear O–C–O arrangement, but this is not possible for the (R)-GOP isomer. These structures confirm the previously proposed conformational flexibility of the catalytic glutamate in its closed, liganded state. The importance of this conformational flexibility for the proton shuttling steps in the TIM catalytic cycle, which is apparently achieved by a sliding motion of the side chain carboxylate group above the enediolate plane, is also discussed. PMID:21633986

  3. Insights into open/closed conformations of the catalytically active human guanylate kinase as investigated by small-angle X-ray scattering.

    PubMed

    Jain, Rohit; Khan, Nazimuddin; Menzel, Andreas; Rajkovic, Ivan; Konrad, Manfred; Techert, Simone

    2016-01-01

    Bio-catalysis is the outcome of a subtle interplay between internal motions in enzymes and chemical kinetics. Small-angle X-ray scattering (SAXS) investigation of an enzyme's internal motions during catalysis offers an integral view of the protein's structural plasticity, dynamics, and function, which is useful for understanding allosteric effects and developing novel medicines. Guanylate kinase (GMPK) is an essential enzyme involved in the guanine nucleotide metabolism of unicellular and multicellular organisms. It is also required for the intracellular activation of numerous antiviral and anticancer purine nucleoside analog prodrugs. Catalytically active recombinant human GMPK (hGMPK) was purified for the first time and changes in the size and shape of open/closed hGMPK were tracked by SAXS. The binding of substrates (GMP + AMPPNP or Ap5G or GMP + ADP) resulted in the compaction of size and shape of hGMPK. The structural changes between open and completely closed hGMPK conformation were confirmed by observing differences in the hGMPK secondary structures with circular dichroism spectroscopy. PMID:26446352

  4. Leu85 in the beta1-beta2 linker of ASIC1 slows activation and decreases the apparent proton affinity by stabilizing a closed conformation.

    PubMed

    Li, Tianbo; Yang, Youshan; Canessa, Cecilia M

    2010-07-16

    Acid-sensing ion channels (ASICs) are proton-activated channels expressed in neurons of the central and peripheral nervous systems where they modulate neuronal activity in response to external increases in proton concentration. The size of ASIC1 currents evoked by a given local acidification is determined by the number of channels in the plasma membrane and by the apparent proton affinities for activation and steady-state desensitization of the channel. Thus, the magnitude of the pH drop and the value of the baseline pH both are functionally important. Recent characterization of ASIC1s from an increasing number of species has made evident that proton affinities of these channels vary across vertebrates. We found that in species with high baseline plasma pH, e.g. frog, shark, and fish, ASIC1 has high proton affinity compared with the mammalian channel. The beta1-beta2 linker in the extracellular domain, specifically by the substitution M85L, determines the interspecies differences in proton affinities and also the time course of ASIC1 macroscopic currents. The mechanism underlying these observations is a delay in channel opening after application of protons, most likely by stabilizing a closed conformation that decreases the apparent affinity to protons and also slows the rise and decay phases of the current. Together, the results suggest evolutionary adaptation of ASIC1 to match the value of the species-specific plasma pH. At the molecular level, adaptation is achieved by substitutions of nonionizable residues rather than by modification of the channel proton sensor. PMID:20479002

  5. Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases.

    PubMed

    Viviani, V R; Simões, A; Bevilaqua, V R; Gabriel, G V M; Arnoldi, F G C; Hirano, T

    2016-08-30

    Beetle luciferases elicit the emission of different bioluminescence colors from green to red. Whereas firefly luciferases emit yellow-green light and are pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures and in the presence of divalent heavy metals, click beetle and railroadworm luciferases emit a wider range of colors from green to red but are pH-independent. Despite many decades of study, the structural determinants and mechanisms of bioluminescence colors and pH sensitivity remain enigmatic. Here, through modeling studies, site-directed mutagenesis, and spectral and kinetic studies using recombinant luciferases from the three main families of bioluminescent beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm, and Pyrearinus termitilluminans click beetle), we investigated the role of E311 and R337 in bioluminescence color determination. All mutations of these residues in firefly luciferase produced red mutants, indicating that the preservation of opposite charges and the lengths of the side chains of E311 and R337 are essential for keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission. Kinetic studies indicate that residue R337 is important for binding luciferin and creating a positively charged environment around excited oxyluciferin phenolate. In Pyrearinus green-emitting luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes a blue-shift that is no longer affected by guanidine. These results provide compelling evidence that the presence of arginine at position 334 is essential for blue-shifting the emission spectra of most beetle luciferases. Therefore, residues E311 and R337 play both structural and catalytic roles in bioluminescence color determination, by stabilizing a closed hydrophobic conformation favorable for green light emission, and also

  6. Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases.

    PubMed

    Viviani, V R; Simões, A; Bevilaqua, V R; Gabriel, G V M; Arnoldi, F G C; Hirano, T

    2016-08-30

    Beetle luciferases elicit the emission of different bioluminescence colors from green to red. Whereas firefly luciferases emit yellow-green light and are pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures and in the presence of divalent heavy metals, click beetle and railroadworm luciferases emit a wider range of colors from green to red but are pH-independent. Despite many decades of study, the structural determinants and mechanisms of bioluminescence colors and pH sensitivity remain enigmatic. Here, through modeling studies, site-directed mutagenesis, and spectral and kinetic studies using recombinant luciferases from the three main families of bioluminescent beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm, and Pyrearinus termitilluminans click beetle), we investigated the role of E311 and R337 in bioluminescence color determination. All mutations of these residues in firefly luciferase produced red mutants, indicating that the preservation of opposite charges and the lengths of the side chains of E311 and R337 are essential for keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission. Kinetic studies indicate that residue R337 is important for binding luciferin and creating a positively charged environment around excited oxyluciferin phenolate. In Pyrearinus green-emitting luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes a blue-shift that is no longer affected by guanidine. These results provide compelling evidence that the presence of arginine at position 334 is essential for blue-shifting the emission spectra of most beetle luciferases. Therefore, residues E311 and R337 play both structural and catalytic roles in bioluminescence color determination, by stabilizing a closed hydrophobic conformation favorable for green light emission, and also

  7. Pilot Non-Conformance to Alerting System Commands During Closely Spaced Parallel Approaches

    NASA Technical Reports Server (NTRS)

    Pritchett, Amy R.; Hansman, R. John

    1997-01-01

    Pilot non-conformance to alerting system commands has been noted in general and to a TCAS-like collision avoidance system in a previous experiment. This paper details two experiments studying collision avoidance during closely-spaced parallel approaches in instrument meteorological conditions (IMC), and specifically examining possible causal factors of, and design solutions to, pilot non-conformance.

  8. Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization

    PubMed Central

    Lavoie, Hugo; Thevakumaran, Neroshan; Gavory, Gwenaëlle; Li, John; Padeganeh, Abbas; Guiral, Sébastien; Duchaine, Jean; Mao, Daniel Y. L.; Bouvier, Michel; Sicheri, Frank; Therrien, Marc

    2016-01-01

    RAF kinases play a prominent role in cancer. Their mode of activation is complex, but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and as a result undesirably stimulate RAS/ERK-mediated cell growth. The mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe BRET-based biosensors for the extended RAF family enabling the detection of RAF dimerization in living cells. Notably, we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization as well as for probing structural determinants of RAF dimerization in vivo. Our findings, which appear generalizable to other kinase families allosterically regulated by kinase domain dimerization, suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain. PMID:23685672

  9. A closed conformation of the Caenorhabditis elegans separase–securin complex

    PubMed Central

    Bachmann, Gudrun; Richards, Mark W.; Winter, Anja; Beuron, Fabienne; Morris, Edward

    2016-01-01

    The protease separase plays a key role in sister chromatid disjunction and centriole disengagement. To maintain genomic stability, separase activity is strictly regulated by binding of an inhibitory protein, securin. Despite its central role in cell division, the separase and securin complex is poorly understood at the structural level. This is partly owing to the difficulty of generating a sufficient quantity of homogeneous, stable protein. Here, we report the production of Caenorhabditis elegans separase–securin complex, and its characterization using biochemical methods and by negative staining electron microscopy. Single particle analysis generated a density map at a resolution of 21–24 Å that reveals a close, globular structure of complex connectivity harbouring two lobes. One lobe matches closely a homology model of the N-terminal HEAT repeat domain of separase, whereas the second lobe readily accommodates homology models of the separase C-terminal death and caspase-like domains. The globular structure of the C. elegans separase–securin complex contrasts with the more elongated structure previously described for the Homo sapiens complex, which could represent a different functional state of the complex, suggesting a mechanism for the regulation of separase activity through conformational change. PMID:27249343

  10. A closed conformation of the Caenorhabditis elegans separase-securin complex.

    PubMed

    Bachmann, Gudrun; Richards, Mark W; Winter, Anja; Beuron, Fabienne; Morris, Edward; Bayliss, Richard

    2016-04-01

    The protease separase plays a key role in sister chromatid disjunction and centriole disengagement. To maintain genomic stability, separase activity is strictly regulated by binding of an inhibitory protein, securin. Despite its central role in cell division, the separase and securin complex is poorly understood at the structural level. This is partly owing to the difficulty of generating a sufficient quantity of homogeneous, stable protein. Here, we report the production of Caenorhabditis elegans separase-securin complex, and its characterization using biochemical methods and by negative staining electron microscopy. Single particle analysis generated a density map at a resolution of 21-24 Å that reveals a close, globular structure of complex connectivity harbouring two lobes. One lobe matches closely a homology model of the N-terminal HEAT repeat domain of separase, whereas the second lobe readily accommodates homology models of the separase C-terminal death and caspase-like domains. The globular structure of the C. elegans separase-securin complex contrasts with the more elongated structure previously described for the Homo sapiens complex, which could represent a different functional state of the complex, suggesting a mechanism for the regulation of separase activity through conformational change.

  11. Pilot Non-Conformance to Alerting System Commands During Closely Spaced Parallel Approaches

    NASA Technical Reports Server (NTRS)

    Pritchett, Amy Ruth; Hansman, R. John; Corker, Kevin (Technical Monitor)

    1997-01-01

    Cockpit alerting systems monitor potentially hazardous situations, both inside and outside the aircraft. When a hazard is projected to occur, the alerting system displays alerts and/or command decisions to the pilot. However, pilots have been observed to not conform to alerting system commands by delaying their response or by not following the automatic commands exactly. This non-conformance to the automatic alerting system can reduce its benefit. Therefore, a need exists to understand the causes and effects of pilot non-conformance in order to develop automatic alerting systems whose commands the pilots are more likely to follow. These considerations were examined through flight simulator evaluations of the collision avoidance task during closely spaced parallel approaches. This task provided a useful case-study because the effects of non-conformance can be significant, given the time-critical nature of the task. A preliminary evaluation of alerting systems identified non-conformance in over 40% of the cases and a corresponding drop in collision avoidance performance. A follow-on experiment found subjects' alerting and maneuver selection criteria were consistent with different strategies than those used by automatic systems, indicating the pilot may potentially disagree with the alerting system if the pilot attempts to verify automatic alerts and commanded avoidance maneuvers. A final experiment found supporting automatic alerts with the explicit display of its underlying criteria resulted in more consistent subject reactions. In light of these experimental results, a general discussion of pilot non-conformance is provided. Contributing factors in pilot non-conformance include a lack of confidence in the automatic system and mismatches between the alerting system's commands and the pilots' own decisions based on the information available to them. The effects of non-conformance on system performance are discussed. Possible methods of reconciling mismatches are

  12. The Crystal Structures of the Open and Catalytically Competent Closed Conformation of Escherichia coli Glycogen Synthase

    SciTech Connect

    Sheng, Fang; Jia, Xiaofei; Yep, Alejandra; Preiss, Jack; Geiger, James H.

    2009-07-06

    Escherichia coli glycogen synthase (EcGS, EC 2.4.1.21) is a retaining glycosyltransferase (GT) that transfers glucose from adenosine diphosphate glucose to a glucan chain acceptor with retention of configuration at the anomeric carbon. EcGS belongs to the GT-B structural superfamily. Here we report several EcGS x-ray structures that together shed considerable light on the structure and function of these enzymes. The structure of the wild-type enzyme bound to ADP and glucose revealed a 15.2 degrees overall domain-domain closure and provided for the first time the structure of the catalytically active, closed conformation of a glycogen synthase. The main chain carbonyl group of His-161, Arg-300, and Lys-305 are suggested by the structure to act as critical catalytic residues in the transglycosylation. Glu-377, previously thought to be catalytic is found on the alpha-face of the glucose and plays an electrostatic role in the active site and as a glucose ring locator. This is also consistent with the structure of the EcGS(E377A)-ADP-HEPPSO complex where the glucose moiety is either absent or disordered in the active site

  13. Crystal structure of full-length KcsA in its closed conformation

    SciTech Connect

    Uysal, Serdar; Vásquez, Valeria; Tereshko, Valentina; Esaki, Kaori; Fellouse, Frederic A.; Sidhu, Sachdev S.; Koide, Shohei; Perozo, Eduardo; Kossiakoff, Anthony; UC; Genentech

    2009-05-21

    KcsA is a proton-activated, voltage-modulated K(+) channel that has served as the archetype pore domain in the Kv channel superfamily. Here, we have used synthetic antigen-binding fragments (Fabs) as crystallographic chaperones to determine the structure of full-length KcsA at 3.8 A, as well as that of its isolated C-terminal domain at 2.6 A. The structure of the full-length KcsA-Fab complex reveals a well-defined, 4-helix bundle that projects approximately 70 A toward the cytoplasm. This bundle promotes a approximately 15 degree bending in the inner bundle gate, tightening its diameter and shifting the narrowest point 2 turns of helix below. Functional analysis of the full-length KcsA-Fab complex suggests that the C-terminal bundle remains whole during gating. We suggest that this structure likely represents the physiologically relevant closed conformation of KcsA.

  14. Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex.

    PubMed

    Llácer, Jose L; Hussain, Tanweer; Marler, Laura; Aitken, Colin Echeverría; Thakur, Anil; Lorsch, Jon R; Hinnebusch, Alan G; Ramakrishnan, V

    2015-08-01

    Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5' end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition.

  15. Identification of mutations by RNA conformational polymorphism {open_quotes}bar code{close_quotes} analysis

    SciTech Connect

    Lenz, H.J.; Danenberg, K.D.; Schnieders, B. |

    1995-11-01

    DNA single-strand conformational polymorphism (SSCP) analysis is widely used for detection of point mutations in clinical specimens. Performing SSCP analysis with cRNA instead of DNA has been shown to improve mutation detection frequency. RNA can exist in numerous metastable conformations, which appear as patterns of bands on nondenaturing electrophoresis gels. Single base mutations can cause not only mobility shifts of major bands, but also loss of some conformations and appearance of new conformations. Unique RNA SSCP patterns associated with specific base sequences in many cases allow visual identification of point mutations. However, in some cases, the RNA SSCP pattern of a single base change in a sequence is not sufficiently different for a positive identification of the mutation. Improvement in the detection capability of RNA SSCP was obtained by adding 3{prime}-deoxy-nucleotides to the transcription reaction. The presence of chain-terminating nucleotides in the transcription reaction formed numerous new RNA fragments, thereby generating complex band patterns ({open_quotes}bar codes{close_quotes}) unique to each RNA sequence. This method was applied to analyzing p53 mutations in patients with colon cancer. 8 refs., 3 figs.

  16. Stabilizing a flexible interdomain hinge region harboring the SMB binding site drives uPAR into its closed conformation.

    PubMed

    Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai; Luo, Zhipu; Li, Rui; Gårdsvoll, Henrik; de Lorenzi, Valentina; Sidenius, Nicolai; Huang, Mingdong; Ploug, Michael

    2015-03-27

    The urokinase-type plasminogen activator receptor (uPAR) is a multidomain glycolipid-anchored membrane protein, which facilitates extracellular matrix remodeling by focalizing plasminogen activation to cell surfaces via its high-affinity interaction with uPA. The modular assembly of its three LU (Ly6/uPAR-like) domains is inherently flexible and binding of uPA drives uPAR into its closed conformation, which presents the higher-affinity state for vitronectin thus providing an allosteric regulatory mechanism. Using a new class of epitope-mapped anti-uPAR monoclonal antibodies (mAbs), we now demonstrate that the reciprocal stabilization is indeed also possible. By surface plasmon resonance studies, we show that these mAbs and vitronectin have overlapping binding sites on uPAR and that they share Arg91 as hotspot residue in their binding interfaces. The crystal structure solved for one of these uPAR·mAb complexes at 3.0Å clearly shows that this mAb preselects the closed uPAR conformation with an empty but correctly assembled large hydrophobic binding cavity for uPA. Accordingly, these mAbs inhibit the uPAR-dependent lamellipodia formation and migration on vitronectin-coated matrices irrespective of the conformational status of uPAR and its occupancy with uPA. This is the first study to the best of our knowledge, showing that the dynamic assembly of the three LU domains in uPARwt can be driven toward the closed form by an external ligand, which is not engaging the hydrophobic uPA binding cavity. As this binding interface is also exploited by the somatomedin B domain of vitronectin, therefore, this relationship should be taken into consideration when exploring uPAR-dependent cell adhesion and migration in vitronectin-rich environments. PMID:25659907

  17. Energetics of the Cleft Closing Transition and the Role of Electrostatic Interactions in Conformational Rearrangements of the Glutamate Receptor Ligand Binding Domain

    PubMed Central

    Mamonova, Tatyana; Yonkunas, Michael J.; Kurnikova, Maria G.

    2009-01-01

    The ionotropic glutamate receptors are localized in the pre- and postsynaptic membrane of neurons in the brain. Activation by the principal excitatory neurotransmitter glutamate allows the ligand binding domain to change conformation, communicating opening of the channel for ion conduction. The free energy of the GluR2 S1S2 ligand binding domain (S1S2) closure transition was computed using a combination of thermodynamic integration and umbrella sampling modeling methods. A path that involves lowering the charge on E705 was chosen to clarify the role of this binding site residue. A continuum electrostatic approach in S1S2 is used to show E705, located in the ligand binding cleft, stabilizes the closed conformation of S1S2. In the closed conformation, in the absence of a ligand, S1S2 is somewhat more closed than reported from X-ray structures. A semi-open conformation has been identified which is characterized by disruption of a single cross-cleft interaction and differs only slightly in energy from the fully closed S1S2. The fully open S1S2 conformation exhibits a wide energy well and shares structural similarity to the apo S1S2 crystal structure. Hybrid continuum electrostatics/MD calculations along the chosen closure transition pathway reveal solvation energies, as well as electrostatic interaction energies between two lobes of the protein increase the relative energetic difference between the open and the closed conformational states. By analyzing the role of several cross-cleft contacts as well as other binding site residues we demonstrate how S1S2 interactions facilitate formation of the closed conformation of the ligand binding domain. PMID:18823129

  18. The longin SNARE VAMP7/TI-VAMP adopts a closed conformation.

    PubMed

    Vivona, Sandro; Liu, Corey W; Strop, Pavel; Rossi, Valeria; Filippini, Francesco; Brunger, Axel T

    2010-06-01

    SNARE protein complexes are key mediators of exocytosis by juxtaposing opposing membranes, leading to membrane fusion. SNAREs generally consist of one or two core domains that can form a four-helix bundle with other SNARE core domains. Some SNAREs, such as syntaxin target-SNAREs and longin vesicular-SNAREs, have independent, folded N-terminal domains that can interact with their respective SNARE core domains and thereby affect the kinetics of SNARE complex formation. This autoinhibition mechanism is believed to regulate the role of the longin VAMP7/TI-VAMP in neuronal morphogenesis. Here we use nuclear magnetic resonance spectroscopy to study the longin-SNARE core domain interaction for VAMP7. Using complete backbone resonance assignments, chemical shift perturbations analysis, and hydrogen/deuterium exchange experiments, we conclusively show that VAMP7 adopts a preferentially closed conformation in solution. Taken together, the closed conformation of longins is conserved, in contrast to the syntaxin family of SNAREs for which mixtures of open and closed states have been observed. This may indicate different regulatory mechanisms for SNARE complexes containing syntaxins and longins, respectively.

  19. The Longin SNARE VAMP7/TI-VAMP Adopts a Closed Conformation*

    PubMed Central

    Vivona, Sandro; Liu, Corey W.; Strop, Pavel; Rossi, Valeria; Filippini, Francesco; Brunger, Axel T.

    2010-01-01

    SNARE protein complexes are key mediators of exocytosis by juxtaposing opposing membranes, leading to membrane fusion. SNAREs generally consist of one or two core domains that can form a four-helix bundle with other SNARE core domains. Some SNAREs, such as syntaxin target-SNAREs and longin vesicular-SNAREs, have independent, folded N-terminal domains that can interact with their respective SNARE core domains and thereby affect the kinetics of SNARE complex formation. This autoinhibition mechanism is believed to regulate the role of the longin VAMP7/TI-VAMP in neuronal morphogenesis. Here we use nuclear magnetic resonance spectroscopy to study the longin-SNARE core domain interaction for VAMP7. Using complete backbone resonance assignments, chemical shift perturbations analysis, and hydrogen/deuterium exchange experiments, we conclusively show that VAMP7 adopts a preferentially closed conformation in solution. Taken together, the closed conformation of longins is conserved, in contrast to the syntaxin family of SNAREs for which mixtures of open and closed states have been observed. This may indicate different regulatory mechanisms for SNARE complexes containing syntaxins and longins, respectively. PMID:20378544

  20. Modulation of a pre-existing conformational equilibrium tunes adenylate kinase activity.

    PubMed

    Ådén, Jörgen; Verma, Abhinav; Schug, Alexander; Wolf-Watz, Magnus

    2012-10-10

    Structural plasticity is often required for distinct microscopic steps during enzymatic reaction cycles. Adenylate kinase from Escherichia coli (AK(eco)) populates two major conformations in solution; the open (inactive) and closed (active) state, and the overall turnover rate is inversely proportional to the lifetime of the active conformation. Therefore, structural plasticity is intimately coupled to enzymatic turnover in AK(eco). Here, we probe the open to closed conformational equilibrium in the absence of bound substrate with NMR spectroscopy and molecular dynamics simulations. The conformational equilibrium in absence of substrate and, in turn, the turnover number can be modulated with mutational- and osmolyte-driven perturbations. Removal of one hydrogen bond between the ATP and AMP binding subdomains results in a population shift toward the open conformation and a resulting increase of k(cat). Addition of the osmolyte TMAO to AK(eco) results in population shift toward the closed conformation and a significant reduction of k(cat). The Michaelis constants (K(M)) scale with the change in k(cat), which follows from the influence of the population of the closed conformation for substrate binding affinity. Hence, k(cat) and K(M) are mutually dependent, and in the case of AK(eco), any perturbation that modulates k(cat) is mirrored with a proportional response in K(M). Thus, our results demonstrate that the equilibrium constant of a pre-existing conformational equilibrium directly affects enzymatic catalysis. From an evolutionary perspective, our findings suggest that, for AK(eco), there exists ample flexibility to obtain a specificity constant (k(cat)/K(M)) that commensurate with the exerted cellular selective pressure.

  1. Conformation-Activity Relationships of Polyketide Natural Products

    PubMed Central

    Larsen, Erik M.; Wilson, Matthew R.; Taylor, Richard E.

    2015-01-01

    Polyketides represent an important class of secondary metabolites that interact with biological targets connected to a variety of disease-associated pathways. Remarkably, nature’s assembly lines, polyketide synthases, manufacture these privileged structures through a combinatorial mixture of just a few structural units. This review highlights the role of these structural elements in shaping a polyketide’s conformational preferences, the use of computer-based molecular modeling and solution NMR studies in the identification of low-energy conformers, and the importance of conformational analogues in probing the bound conformation. In particular, this review covers several examples wherein conformational analysis complements classic structure-activity relationships in the design of biologically active natural product analogues. PMID:25974024

  2. Protein Conformational Landscapes and Catalysis. Influence of Active Site Conformations in the Reaction Catalyzed by L-Lactate Dehydrogenase

    PubMed Central

    Świderek, Katarzyna; Tuñón, Iñaki; Martí, Sergio; Moliner, Vicent

    2015-01-01

    In the last decade L-Lactate Dehydrogenase (LDH) has become an extremely useful marker in both clinical diagnosis and in monitoring the course of many human diseases. It has been assumed from the 80s that the full catalytic process of LDH starts with the binding of the cofactor and the substrate followed by the enclosure of the active site by a mobile loop of the protein before the reaction to take place. In this paper we show that the chemical step of the LDH catalyzed reaction can proceed within the open loop conformation, and the different reactivity of the different protein conformations would be in agreement with the broad range of rate constants measured in single molecule spectrometry studies. Starting from a recently solved X-ray diffraction structure that presented an open loop conformation in two of the four chains of the tetramer, QM/MM free energy surfaces have been obtained at different levels of theory. Depending on the level of theory used to describe the electronic structure, the free energy barrier for the transformation of pyruvate into lactate with the open conformation of the protein varies between 12.9 and 16.3 kcal/mol, after quantizing the vibrations and adding the contributions of recrossing and tunneling effects. These values are very close to the experimentally deduced one (14.2 kcal·mol−1) and ~2 kcal·mol−1 smaller than the ones obtained with the closed loop conformer. Calculation of primary KIEs and IR spectra in both protein conformations are also consistent with our hypothesis and in agreement with experimental data. Our calculations suggest that the closure of the active site is mainly required for the inverse process; the oxidation of lactate to pyruvate. According to this hypothesis H4 type LDH enzyme molecules, where it has been propose that lactate is transformed into pyruvate, should have a better ability to close the mobile loop than the M4 type LDH molecules. PMID:25705562

  3. Conformational heterogeneity in closed and open states of the KcsA potassium channel in lipid bicelles.

    PubMed

    Kim, Dorothy M; Dikiy, Igor; Upadhyay, Vikrant; Posson, David J; Eliezer, David; Nimigean, Crina M

    2016-08-01

    The process of ion channel gating-opening and closing-involves local and global structural changes in the channel in response to external stimuli. Conformational changes depend on the energetic landscape that underlies the transition between closed and open states, which plays a key role in ion channel gating. For the prokaryotic, pH-gated potassium channel KcsA, closed and open states have been extensively studied using structural and functional methods, but the dynamics within each of these functional states as well as the transition between them is not as well understood. In this study, we used solution nuclear magnetic resonance (NMR) spectroscopy to investigate the conformational transitions within specific functional states of KcsA. We incorporated KcsA channels into lipid bicelles and stabilized them into a closed state by using either phosphatidylcholine lipids, known to favor the closed channel, or mutations designed to trap the channel shut by disulfide cross-linking. A distinct state, consistent with an open channel, was uncovered by the addition of cardiolipin lipids. Using selective amino acid labeling at locations within the channel that are known to move during gating, we observed at least two different slowly interconverting conformational states for both closed and open channels. The pH dependence of these conformations and the predictable disruptions to this dependence observed in mutant channels with altered pH sensing highlight the importance of conformational heterogeneity for KcsA gating. PMID:27432996

  4. Cooperative binding of ATP and RNA induces a closed conformation in a DEAD box RNA helicase.

    PubMed

    Theissen, Bettina; Karow, Anne R; Köhler, Jürgen; Gubaev, Airat; Klostermeier, Dagmar

    2008-01-15

    RNA helicases couple the energy from ATP hydrolysis with structural changes of their RNA substrates. DEAD box helicases form the largest class of RNA helicases and share a helicase core comprising two RecA-like domains. An opening and closing of the interdomain cleft during RNA unwinding has been postulated but not shown experimentally. Single-molecule FRET experiments with the Bacillus subtilis DEAD box helicase YxiN carrying donor and acceptor fluorophores on different sides of the interdomain cleft reveal an open helicase conformation in the absence of nucleotides, or in the presence of ATP, or ADP, or RNA. In the presence of ADP and RNA, the open conformation is retained. By contrast, cooperative binding of ATP and RNA leads to a compact helicase structure, proving that the ATP- and ADP-bound states of RNA helicases display substantially different structures only when the RNA substrate is bound. These results establish a closure of the interdomain cleft in the helicase core at the beginning of the unwinding reaction, and suggest a conserved mechanism of energy conversion among DEAD box helicases across kingdoms.

  5. Outdoor Education: Opening and Closing Activities.

    ERIC Educational Resources Information Center

    Mingie, Walter, Comp.

    Opening activites (to create an atmosphere of cooperation and a desire to work, explore, and learn together) and closing activities (to summarize what has happened or been learned) for outdoor education programs are described. All activities are intended to incite enthusiasm to learn and make the learning activity a desired, joyful experience.…

  6. NMR-based conformational ensembles explain pH-gated opening and closing of OmpG channel.

    PubMed

    Zhuang, Tiandi; Chisholm, Christina; Chen, Min; Tamm, Lukas K

    2013-10-01

    The outer membrane protein G (OmpG) is a monomeric 33 kDa 14-stranded β-barrel membrane protein functioning as a nonspecific porin for the uptake of oligosaccharides in Escherichia coli. Two different crystal structures of OmpG obtained at different values of pH suggest a pH-gated pore opening mechanism. In these structures, extracellular loop 6 extends away from the barrel wall at neutral pH but is folded back into the pore lumen at low pH, blocking transport through the pore. Loop 6 was invisible in a previously published solution NMR structure of OmpG in n-dodecylphosphocholine micelles, presumably due to conformational exchange on an intermediate NMR time scale. Here we present an NMR paramagnetic relaxation enhancement (PRE)-based approach to visualize the conformational dynamics of loop 6 and to calculate conformational ensembles that explain the pH-gated opening and closing of the OmpG channel. The different loop conformers detected by the PRE ensemble calculations were validated by disulfide cross-linking of strategically engineered cysteines and electrophysiological single channel recordings. The results indicate a more dynamically regulated channel opening and closing than previously thought and reveal additional membrane-associated conformational ensembles at pH 6.3 and 7.0. We anticipate this approach to be generally applicable to detect and characterize functionally important conformational ensembles of membrane proteins.

  7. Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition

    PubMed Central

    Fang, Jian; Cheng, Jingdong; Wang, Jiaolong; Zhang, Qiao; Liu, Mengjie; Gong, Rui; Wang, Ping; Zhang, Xiaodan; Feng, Yangyang; Lan, Wenxian; Gong, Zhou; Tang, Chun; Wong, Jiemin; Yang, Huirong; Cao, Chunyang; Xu, Yanhui

    2016-01-01

    UHRF1 is an important epigenetic regulator for maintenance DNA methylation. UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD–PHD. The Spacer also facilitates UHRF1–DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA. When TTD–PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1. PMID:27045799

  8. Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition

    NASA Astrophysics Data System (ADS)

    Fang, Jian; Cheng, Jingdong; Wang, Jiaolong; Zhang, Qiao; Liu, Mengjie; Gong, Rui; Wang, Ping; Zhang, Xiaodan; Feng, Yangyang; Lan, Wenxian; Gong, Zhou; Tang, Chun; Wong, Jiemin; Yang, Huirong; Cao, Chunyang; Xu, Yanhui

    2016-04-01

    UHRF1 is an important epigenetic regulator for maintenance DNA methylation. UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD-PHD. The Spacer also facilitates UHRF1-DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA. When TTD-PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1.

  9. Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition.

    PubMed

    Fang, Jian; Cheng, Jingdong; Wang, Jiaolong; Zhang, Qiao; Liu, Mengjie; Gong, Rui; Wang, Ping; Zhang, Xiaodan; Feng, Yangyang; Lan, Wenxian; Gong, Zhou; Tang, Chun; Wong, Jiemin; Yang, Huirong; Cao, Chunyang; Xu, Yanhui

    2016-01-01

    UHRF1 is an important epigenetic regulator for maintenance DNA methylation. UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD-PHD. The Spacer also facilitates UHRF1-DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA. When TTD-PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1.

  10. Protons stabilize the closed conformation of gain-of-function mutants of the TRPV1 channel.

    PubMed

    Boukalova, Stepana; Teisinger, Jan; Vlachova, Viktorie

    2013-03-01

    The vanilloid transient receptor potential channel TRPV1 is a molecular integrator of noxious stimuli, including capsaicin, heat and protons. Despite clear similarities between the overall architecture of TRPV1 and voltage-dependent potassium (Kv) channels, the extent of conservation in the molecular logic for gating is unknown. In Kv channels, a small contact surface between S1 and the pore-helix is required for channel functioning. To explore the function of S1 in TRPV1, we used tryptophan-scanning mutagenesis and characterized the responses to capsaicin and protons. Wild-type-like currents were generated in 9 out of 17 mutants; three mutants (M445W, A452W, R455W) were non-functional. The conservative mutation R455K in the extracellular extent of S1 slowed down capsaicin-induced activation and prevented normal channel closure. This mutant was neither activated nor potentiated by protons, on the contrary, protons promoted a rapid deactivation of its currents. Similar phenotypes were found in two other gain-of-function mutants and also in the pore-helix mutant T633A, known to uncouple proton activation. We propose that the S1 domain contains a functionally important region that may be specifically involved in TRPV1 channel gating, and thus be important for the energetic coupling between S1-S4 sensor activation and gate opening. Analogous to Kv channels, the S1-pore interface might serve to stabilize conformations associated with TRPV1 channel gating.

  11. Pyruvate dehydrogenase kinase-4 structures reveal a metastable open conformation fostering robust core-free basal activity.

    PubMed

    Wynn, R Max; Kato, Masato; Chuang, Jacinta L; Tso, Shih-Chia; Li, Jun; Chuang, David T

    2008-09-12

    Human pyruvate dehydrogenase complex (PDC) is down-regulated by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. PDK4 is overexpressed in skeletal muscle in type 2 diabetes, resulting in impaired glucose utilization. Here we show that human PDK4 has robust core-free basal activity, which is considerably higher than activity levels of other PDK isoforms stimulated by the PDC core. PDK4 binds the L3 lipoyl domain, but its activity is not significantly stimulated by any individual lipoyl domains or the core of PDC. The 2.0-A crystal structures of the PDK4 dimer with bound ADP reveal an open conformation with a wider active-site cleft, compared with that in the closed conformation epitomized by the PDK2-ADP structure. The open conformation in PDK4 shows partially ordered C-terminal cross-tails, in which the conserved DW (Asp(394)-Trp(395)) motif from one subunit anchors to the N-terminal domain of the other subunit. The open conformation fosters a reduced binding affinity for ADP, facilitating the efficient removal of product inhibition by this nucleotide. Alteration or deletion of the DW-motif disrupts the C-terminal cross-tail anchor, resulting in the closed conformation and the nearly complete inactivation of PDK4. Fluorescence quenching and enzyme activity data suggest that compounds AZD7545 and dichloroacetate lock PDK4 in the open and the closed conformational states, respectively. We propose that PDK4 with bound ADP exists in equilibrium between the open and the closed conformations. The favored metastable open conformation is responsible for the robust basal activity of PDK4 in the absence of the PDC core. PMID:18658136

  12. Saturated Activity: Very Close, Detached Binary Stars

    NASA Astrophysics Data System (ADS)

    Rucinski, Slavek M.

    It is proposed to obtain EUVE spectra of 4 close, synchronized, late-type binary stars with orbital/rotational periods shorter than 1.2 day, to study stellar coronal activity at very high, saturated levels. Among stars of spectral types between late-F to mid-K, only components of very close binary systems (and very rare young stars) can have such short rotational periods. Together with the EGO-1 and EGO-2 results for DH Leo and TZ CrB obtained by others, the spectra will be utilized in a comprehensive discussion of the saturated stellar activity, in relation to and in contrast with, the previously obtained by us spectra of the single, rapidly-rotating young star, AB Dor (P=0.51 day, EGO-1) and of two contact binary systems, 44i Boo (P=0.27 day) and VW Cep (P=0.28 day, EGO-2).

  13. Structures of trehalose synthase from Deinococcus radiodurans reveal that a closed conformation is involved in catalysis of the intramolecular isomerization

    PubMed Central

    Wang, Yung-Lin; Chow, Sih-Yao; Lin, Yi-Ting; Hsieh, Yu-Chiao; Lee, Guan-Chiun; Liaw, Shwu-Huey

    2014-01-01

    Trehalose synthase catalyzes the simple conversion of the inexpensive maltose into trehalose with a side reaction of hydrolysis. Here, the crystal structures of the wild type and the N253A mutant of Deinococcus radiodurans trehalose synthase (DrTS) in complex with the inhibitor Tris are reported. DrTS consists of a catalytic (β/α)8 barrel, subdomain B, a C-terminal β domain and two TS-unique subdomains (S7 and S8). The C-terminal domain and S8 contribute the majority of the dimeric interface. DrTS shares high structural homology with sucrose hydrolase, amylosucrase and sucrose isomerase in complex with sucrose, in particular a virtually identical active-site architecture and a similar substrate-induced rotation of subdomain B. The inhibitor Tris was bound and mimics a sugar at the −1 subsite. A maltose was modelled into the active site, and subsequent mutational analysis suggested that Tyr213, Glu320 and Glu324 are essential within the +1 subsite for the TS activity. In addition, the interaction networks between subdomains B and S7 seal the active-site entrance. Disruption of such networks through the replacement of Arg148 and Asn253 with alanine resulted in a decrease in isomerase activity by 8–9-fold and an increased hydrolase activity by 1.5–1.8-fold. The N253A structure showed a small pore created for water entry. Therefore, our DrTS-Tris may represent a substrate-induced closed conformation that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis. PMID:25478833

  14. Structures of trehalose synthase from Deinococcus radiodurans reveal that a closed conformation is involved in catalysis of the intramolecular isomerization.

    PubMed

    Wang, Yung Lin; Chow, Sih Yao; Lin, Yi Ting; Hsieh, Yu Chiao; Lee, Guan Chiun; Liaw, Shwu Huey

    2014-12-01

    Trehalose synthase catalyzes the simple conversion of the inexpensive maltose into trehalose with a side reaction of hydrolysis. Here, the crystal structures of the wild type and the N253A mutant of Deinococcus radiodurans trehalose synthase (DrTS) in complex with the inhibitor Tris are reported. DrTS consists of a catalytic (β/α)8 barrel, subdomain B, a C-terminal β domain and two TS-unique subdomains (S7 and S8). The C-terminal domain and S8 contribute the majority of the dimeric interface. DrTS shares high structural homology with sucrose hydrolase, amylosucrase and sucrose isomerase in complex with sucrose, in particular a virtually identical active-site architecture and a similar substrate-induced rotation of subdomain B. The inhibitor Tris was bound and mimics a sugar at the -1 subsite. A maltose was modelled into the active site, and subsequent mutational analysis suggested that Tyr213, Glu320 and Glu324 are essential within the +1 subsite for the TS activity. In addition, the interaction networks between subdomains B and S7 seal the active-site entrance. Disruption of such networks through the replacement of Arg148 and Asn253 with alanine resulted in a decrease in isomerase activity by 8-9-fold and an increased hydrolase activity by 1.5-1.8-fold. The N253A structure showed a small pore created for water entry. Therefore, our DrTS-Tris may represent a substrate-induced closed conformation that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis.

  15. How Closely Related Are Conformations of Protein Ions Sampled by IM-MS to Native Solution Structures?

    PubMed

    Chen, Shu-Hua; Russell, David H

    2015-09-01

    Here, we critically evaluate the effects of changes in the ion internal energy (E(int)) on ion-neutral collision cross sections (CCS) of ions of two structurally diverse proteins, specifically the [M + 6H](6+) ion of ubiquitin (ubq(6+)), the [M + 5H](5+) ion of the intrinsically disordered protein (IDP) apo-metallothionein-2A (MT), and its partially- and fully-metalated isoform, the [CdiMT](5+) ion. The ion-neutral CCS for ions formed by "native-state" ESI show a strong dependence on E(int). Collisional activation is used to increase E(int) prior to the ions entering and within the traveling wave (TW) ion mobility analyzer. Comparisons of experimental CCSs with those generated by molecular dynamics (MD) simulation for solution-phase ions and solvent-free ions as a function of temperature provide new insights about conformational preferences and retention of solution conformations. The E(int)-dependent CCSs, which reveal increased conformational diversity of the ion population, are discussed in terms of folding/unfolding of solvent-free ions. For example, ubiquitin ions that have low internal energies retain native-like conformations, whereas ions that are heated by collisional activation possess higher internal energies and yield a broader range of CCS owing to increased conformational diversity due to losses of secondary and tertiary structures. In contrast, the CCS profile for the IDP apoMT is consistent with kinetic trapping of an ion population composed of a wide range of conformers, and as the E(int) is increased, these structurally labile conformers unfold to an elongated conformation.

  16. How Closely Related Are Conformations of Protein Ions Sampled by IM-MS to Native Solution Structures?

    PubMed

    Chen, Shu-Hua; Russell, David H

    2015-09-01

    Here, we critically evaluate the effects of changes in the ion internal energy (E(int)) on ion-neutral collision cross sections (CCS) of ions of two structurally diverse proteins, specifically the [M + 6H](6+) ion of ubiquitin (ubq(6+)), the [M + 5H](5+) ion of the intrinsically disordered protein (IDP) apo-metallothionein-2A (MT), and its partially- and fully-metalated isoform, the [CdiMT](5+) ion. The ion-neutral CCS for ions formed by "native-state" ESI show a strong dependence on E(int). Collisional activation is used to increase E(int) prior to the ions entering and within the traveling wave (TW) ion mobility analyzer. Comparisons of experimental CCSs with those generated by molecular dynamics (MD) simulation for solution-phase ions and solvent-free ions as a function of temperature provide new insights about conformational preferences and retention of solution conformations. The E(int)-dependent CCSs, which reveal increased conformational diversity of the ion population, are discussed in terms of folding/unfolding of solvent-free ions. For example, ubiquitin ions that have low internal energies retain native-like conformations, whereas ions that are heated by collisional activation possess higher internal energies and yield a broader range of CCS owing to increased conformational diversity due to losses of secondary and tertiary structures. In contrast, the CCS profile for the IDP apoMT is consistent with kinetic trapping of an ion population composed of a wide range of conformers, and as the E(int) is increased, these structurally labile conformers unfold to an elongated conformation. PMID:26115967

  17. Conformational and Thermodynamic Landscape of GPCR Activation from Theory and Computation.

    PubMed

    Dong, Sijia S; Goddard, William A; Abrol, Ravinder

    2016-06-21

    We present a hybrid computational methodology to predict multiple energetically accessible conformations for G protein-coupled receptors (GPCRs) that might play a role in binding to ligands and different signaling partners. To our knowledge, this method, termed ActiveGEnSeMBLE, enables the first quantitative energy profile for GPCR activation that is consistent with the qualitative profile deduced from experiments. ActiveGEnSeMBLE starts with a systematic coarse grid sampling of helix tilts/rotations (∼13 trillion transmembrane-domain conformations) and selects the conformational landscape based on energy. This profile identifies multiple potential active-state energy wells, with the TM3-TM6 intracellular distance as an approximate activation coordinate. These energy wells are then sampled locally using a finer grid to find locally minimized conformation in each energy well. We validate this strategy using the inactive and active experimental structures of β2 adrenergic receptor (hβ2AR) and M2 muscarinic acetylcholine receptor. Structures of membrane-embedded hβ2AR along its activation coordinate are subjected to molecular-dynamics simulations for relaxation and interaction energy analysis to generate a quantitative energy landscape for hβ2AR activation. This landscape reveals several metastable states along this coordinate, indicating that for hβ2AR, the agonist alone is not enough to stabilize the active state and that the G protein is necessary, consistent with experimental observations. The method's application to somatostatin receptor SSTR5 (no experimental structure available) shows that to predict an active conformation it is better to start from an inactive structure template based on a close homolog than to start from an active template based on a distant homolog. The energy landscape for hSSTR5 activation is consistent with hβ2AR in the role of the G protein. These results demonstrate the utility of the ActiveGEnSeMBLE method for predicting

  18. Conformational and Thermodynamic Landscape of GPCR Activation from Theory and Computation.

    PubMed

    Dong, Sijia S; Goddard, William A; Abrol, Ravinder

    2016-06-21

    We present a hybrid computational methodology to predict multiple energetically accessible conformations for G protein-coupled receptors (GPCRs) that might play a role in binding to ligands and different signaling partners. To our knowledge, this method, termed ActiveGEnSeMBLE, enables the first quantitative energy profile for GPCR activation that is consistent with the qualitative profile deduced from experiments. ActiveGEnSeMBLE starts with a systematic coarse grid sampling of helix tilts/rotations (∼13 trillion transmembrane-domain conformations) and selects the conformational landscape based on energy. This profile identifies multiple potential active-state energy wells, with the TM3-TM6 intracellular distance as an approximate activation coordinate. These energy wells are then sampled locally using a finer grid to find locally minimized conformation in each energy well. We validate this strategy using the inactive and active experimental structures of β2 adrenergic receptor (hβ2AR) and M2 muscarinic acetylcholine receptor. Structures of membrane-embedded hβ2AR along its activation coordinate are subjected to molecular-dynamics simulations for relaxation and interaction energy analysis to generate a quantitative energy landscape for hβ2AR activation. This landscape reveals several metastable states along this coordinate, indicating that for hβ2AR, the agonist alone is not enough to stabilize the active state and that the G protein is necessary, consistent with experimental observations. The method's application to somatostatin receptor SSTR5 (no experimental structure available) shows that to predict an active conformation it is better to start from an inactive structure template based on a close homolog than to start from an active template based on a distant homolog. The energy landscape for hSSTR5 activation is consistent with hβ2AR in the role of the G protein. These results demonstrate the utility of the ActiveGEnSeMBLE method for predicting

  19. Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site.

    PubMed

    Gajadeera, Chathurada S; Zhang, Xinyi; Wei, Yinan; Tsodikov, Oleg V

    2015-02-01

    Cytoplasmic inorganic pyrophosphatase (PPiase) is an enzyme essential for survival of organisms, from bacteria to human. PPiases are divided into two structurally distinct families: family I PPiases are Mg(2+)-dependent and present in most archaea, eukaryotes and prokaryotes, whereas the relatively less understood family II PPiases are Mn(2+)-dependent and present only in some archaea, bacteria and primitive eukaryotes. Staphylococcus aureus (SA), a dangerous pathogen and a frequent cause of hospital infections, contains a family II PPiase (PpaC), which is an attractive potential target for development of novel antibacterial agents. We determined a crystal structure of SA PpaC in complex with catalytic Mn(2+) at 2.1Å resolution. The active site contains two catalytic Mn(2+) binding sites, each half-occupied, reconciling the previously observed 1:1 Mn(2+):enzyme stoichiometry with the presence of two divalent metal ion sites in the apo-enzyme. Unexpectedly, despite the absence of the substrate or products in the active site, the two domains of SA PpaC form a closed active site, a conformation observed in structures of other family II PPiases only in complex with substrate or product mimics. A region spanning residues 295-298, which contains a conserved substrate binding RKK motif, is flipped out of the active site, an unprecedented conformation for a PPiase. Because the mutant of Arg295 to an alanine is devoid of activity, this loop likely undergoes an induced-fit conformational change upon substrate binding and product dissociation. This closed conformation of SA PPiase may serve as an attractive target for rational design of inhibitors of this enzyme. PMID:25576794

  20. Conformation analysis of a surface loop that controls active site access in the GH11 xylanase A from Bacillus subtilis.

    PubMed

    Vieira, Davi Serradella; Ward, Richard John

    2012-04-01

    Xylanases (EC 3.2.1.8 endo-1,4-glycosyl hydrolase) catalyze the hydrolysis of xylan, an abundant hemicellulose of plant cell walls. Access to the catalytic site of GH11 xylanases is regulated by movement of a short β-hairpin, the so-called thumb region, which can adopt open or closed conformations. A crystallographic study has shown that the D11F/R122D mutant of the GH11 xylanase A from Bacillus subtilis (BsXA) displays a stable "open" conformation, and here we report a molecular dynamics simulation study comparing this mutant with the native enzyme over a range of temperatures. The mutant open conformation was stable at 300 and 328 K, however it showed a transition to the closed state at 338 K. Analysis of dihedral angles identified thumb region residues Y113 and T123 as key hinge points which determine the open-closed transition at 338 K. Although the D11F/R122D mutations result in a reduction in local inter-intramolecular hydrogen bonding, the global energies of the open and closed conformations in the native enzyme are equivalent, suggesting that the two conformations are equally accessible. These results indicate that the thumb region shows a broader degree of energetically permissible conformations which regulate the access to the active site region. The R122D mutation contributes to the stability of the open conformation, but is not essential for thumb dynamics, i.e., the wild type enzyme can also adapt to the open conformation.

  1. Quantitative structure-activity relationships for polychlorinated hydroxybiphenyl estrogen receptor binding affinity: An assessment of conformer flexibility

    SciTech Connect

    Bradbury, S.P.; Ankley, G.T.; Mekenyan, O.G.

    1996-11-01

    A diverse group of xenobiotics has a high binding affinity to the estrogen receptor (ER), suggesting that it can accommodate large variability in ligand structure. Relationships between xenobiotic surface, binding affinity, and estrogenic response have been suggested to be dependent on the conformational structures of the ligands. To explore the influence of conformational flexibility on ER binding affinity, a quantitative structure-activity relationship (QSAR) study was undertaken with estradiol, diethylstilbestrol, and a set of polychlorinated hydroxybiphenyls (PCHBs) of environmental concern. Although the low-energy minima of the PCHB congeners suggested that interconversions among conformers were likely, the electronic parameters associated with the conformer geometries for a specific PCHB congener could vary significantly. The results of the QSAR analysis suggested that among the PCHBs studied, the most polarizable conformers (lower absolute volume polarizability values) were most closely associated with ER binding affinity. Across the set of polarizable conformers, which did not include the low-energy gas-phase conformers, the electron donating properties of the hydroxy moiety and the aromatic component of the estradiol A ring analogue in the PCHBs were found to be correlated with higher ER binding affinity.

  2. Open and closed conformations of two SpoIIAA-like proteins (YP_749275.1 and YP_001095227.1) provide insights into membrane association and ligand binding.

    PubMed

    Kumar, Abhinav; Lomize, Andrei; Jin, Kevin K; Carlton, Dennis; Miller, Mitchell D; Jaroszewski, Lukasz; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Chiu, Hsiu Ju; Clayton, Thomas; Das, Debanu; Deller, Marc C; Duan, Lian; Feuerhelm, Julie; Grant, Joanna C; Grzechnik, Anna; Han, Gye Won; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Krishna, S Sri; Marciano, David; McMullan, Daniel; Morse, Andrew T; Nigoghossian, Edward; Okach, Linda; Reyes, Ron; Rife, Christopher L; Sefcovic, Natasha; Tien, Henry J; Trame, Christine B; van den Bedem, Henry; Weekes, Dana; Xu, Qingping; Hodgson, Keith O; Wooley, John; Elsliger, Marc André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

    2010-10-01

    The crystal structures of the proteins encoded by the YP_749275.1 and YP_001095227.1 genes from Shewanella frigidimarina and S. loihica, respectively, have been determined at 1.8 and 2.25 Å resolution, respectively. These proteins are members of a novel family of bacterial proteins that adopt the α/β SpoIIAA-like fold found in STAS and CRAL-TRIO domains. Despite sharing 54% sequence identity, these two proteins adopt distinct conformations arising from different dispositions of their α2 and α3 helices. In the `open' conformation (YP_001095227.1), these helices are 15 Å apart, leading to the creation of a deep nonpolar cavity. In the `closed' structure (YP_749275.1), the helices partially unfold and rearrange, occluding the cavity and decreasing the solvent-exposed hydrophobic surface. These two complementary structures are reminiscent of the conformational switch in CRAL-TRIO carriers of hydrophobic compounds. It is suggested that both proteins may associate with the lipid bilayer in their `open' monomeric state by inserting their amphiphilic helices, α2 and α3, into the lipid bilayer. These bacterial proteins may function as carriers of nonpolar substances or as interfacially activated enzymes.

  3. Cyclic AMP Analog Blocks Kinase Activation by Stabilizing Inactive Conformation: Conformational Selection Highlights a New Concept in Allosteric Inhibitor Design*

    PubMed Central

    Badireddy, Suguna; Yunfeng, Gao; Ritchie, Mark; Akamine, Pearl; Wu, Jian; Kim, Choel W.; Taylor, Susan S.; Qingsong, Lin; Swaminathan, Kunchithapadam; Anand, Ganesh S.

    2011-01-01

    The regulatory (R) subunit of protein kinase A serves to modulate the activity of protein kinase A in a cAMP-dependent manner and exists in two distinct and structurally dissimilar, end point cAMP-bound “B” and C-subunit-bound “H”-conformations. Here we report mechanistic details of cAMP action as yet unknown through a unique approach combining x-ray crystallography with structural proteomics approaches, amide hydrogen/deuterium exchange and ion mobility mass spectrometry, applied to the study of a stereospecific cAMP phosphorothioate analog and antagonist((Rp)-cAMPS). X-ray crystallography shows cAMP-bound R-subunit in the B form but surprisingly the antagonist Rp-cAMPS-bound R-subunit crystallized in the H conformation, which was previously assumed to be induced only by C-subunit-binding. Apo R-subunit crystallized in the B form as well but amide exchange mass spectrometry showed large differences between apo, agonist and antagonist-bound states of the R-subunit. Further ion mobility reveals the apo R-subunit as an ensemble of multiple conformations with collisional cross-sectional areas spanning both the agonist and antagonist-bound states. Thus contrary to earlier studies that explained the basis for cAMP action through “induced fit” alone, we report evidence for conformational selection, where the ligand-free apo form of the R-subunit exists as an ensemble of both B and H conformations. Although cAMP preferentially binds the B conformation, Rp-cAMPS interestingly binds the H conformation. This reveals the unique importance of the equatorial oxygen of the cyclic phosphate in mediating conformational transitions from H to B forms highlighting a novel approach for rational structure-based drug design. Ideal inhibitors such as Rp-cAMPS are those that preferentially “select” inactive conformations of target proteins by satisfying all “binding” constraints alone without inducing conformational changes necessary for activation. PMID:21081668

  4. Protein Conformational Gating of Enzymatic Activity in Xanthine Oxidoreductase

    SciTech Connect

    Ishikita, Hiroshi; Eger, Bryan T.; Okamoto, Ken; Nishino, Takeshi; Pai, Emil F.

    2012-05-24

    In mammals, xanthine oxidoreductase can exist as xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. In spite of identical protein primary structures, the redox potential difference between XDH and XO for the flavin semiquinone/hydroquinone pair (E{sub sq/hq}) is {approx}170 mV, a striking difference. The former greatly prefers NAD{sup +} as ultimate substrate for ET from the iron-sulfur cluster FeS-II via flavin while the latter only accepts dioxygen. In XDH (without NAD{sup +}), however, the redox potential of the electron donor FeS-II is 180 mV higher than that for the acceptor flavin, yielding an energetically uphill ET. On the basis of new 1.65, 2.3, 1.9, and 2.2 {angstrom} resolution crystal structures for XDH, XO, the NAD{sup +}- and NADH-complexed XDH, E{sub sq/hq} were calculated to better understand how the enzyme activates an ET from FeS-II to flavin. The majority of the E{sub sq/hq} difference between XDH and XO originates from a conformational change in the loop at positions 423-433 near the flavin binding site, causing the differences in stability of the semiquinone state. There was no large conformational change observed in response to NAD{sup +} binding at XDH. Instead, the positive charge of the NAD{sup +} ring, deprotonation of Asp429, and capping of the bulk surface of the flavin by the NAD{sup +} molecule all contribute to altering E{sub sq/hq} upon NAD{sup +} binding to XDH.

  5. Conformational intermediates and fusion activity of influenza virus hemagglutinin.

    PubMed

    Korte, T; Ludwig, K; Booy, F P; Blumenthal, R; Herrmann, A

    1999-06-01

    Three strains of influenza virus (H1, H2, and H3) exhibited similar characteristics in the ability of their hemagglutinin (HA) to induce membrane fusion, but the HAs differed in their susceptibility to inactivation. The extent of inactivation depended on the pH of preincubation and was lowest for A/Japan (H2 subtype), in agreement with previous studies (A. Puri, F. Booy, R. W. Doms, J. M. White, and R. Blumenthal, J. Virol. 64:3824-3832, 1990). While significant inactivation of X31 (H3 subtype) was observed at 37 degrees C at pH values corresponding to the maximum of fusion (about pH 5.0), no inactivation was seen at preincubation pH values 0.2 to 0.4 pH units higher. Surprisingly, low-pH preincubation under those conditions enhanced the fusion rates and extents of A/Japan as well as those of X31. For A/PR 8/34 (H1 subtype), neither a shift of the pH (to >5.0) nor a decrease of the temperature to 20 degrees C was sufficient to prevent inactivation. We provide evidence that the activated HA is a conformational intermediate distinct from the native structure and from the final structure associated with the conformational change of HA, which is implicated by the high-resolution structure of the soluble trimeric fragment TBHA2 (P. A. Bullough, F. M. Hughson, J. J. Skehel, and D. C. Wiley, Nature 371:37-43, 1994). PMID:10233915

  6. Structural Analysis of an Open Active Site Conformation of Nonheme Iron Halogenase CytC3

    SciTech Connect

    Wong, Cintyu; Galonic Fujimori, Danica; Walsh, Christopher T.; Drennan, Catherine L.

    2009-04-28

    CytC3, a member of the recently discovered class of nonheme Fe(II) and {alpha}-ketoglutarate ({alpha}KG)-dependent halogenases, catalyzes the double chlorination of l-2-aminobutyric acid (Aba) to produce a known Streptomyces antibiotic, {gamma},{gamma}-dichloroaminobutyrate. Unlike the majority of the Fe(II)-{alpha}KG-dependent enzymes that catalyze hydroxylation reactions, halogenases catalyze a transfer of halides. To examine the important enzymatic features that discriminate between chlorination and hydroxylation, the crystal structures of CytC3 both with and without {alpha}KG/Fe(II) have been solved to 2.2 {angstrom} resolution. These structures capture CytC3 in an open active site conformation, in which no chloride is bound to iron. Comparison of the open conformation of CytC3 with the closed conformation of another nonheme iron halogenase, SyrB2, suggests two important criteria for creating an enzyme-bound FeCl catalyst: (1) the presence of a hydrogen-bonding network between the chloride and surrounding residues, and (2) the presence of a hydrophobic pocket in which the chloride resides.

  7. 12 CFR 225.129 - Activities closely related to banking.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 3 2011-01-01 2011-01-01 false Activities closely related to banking. 225.129 Section 225.129 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Holding Companies Interpretations § 225.129 Activities closely related to banking. Courier activities....

  8. 12 CFR 225.129 - Activities closely related to banking.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Activities closely related to banking. 225.129 Section 225.129 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Holding Companies Interpretations § 225.129 Activities closely related to banking. Courier activities....

  9. 12 CFR 225.129 - Activities closely related to banking.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 12 Banks and Banking 3 2012-01-01 2012-01-01 false Activities closely related to banking. 225.129 Section 225.129 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Holding Companies Interpretations § 225.129 Activities closely related to banking. Courier activities....

  10. Structural Insights into NEDD8 Activation of Cullin-RING Ligases: Conformational Control of Conjugation

    SciTech Connect

    Duda,D.; Borg, L.; Scott, D.; Hunt, H.; Hammel, M.; Schulman, B.

    2008-01-01

    Cullin-RING ligases (CRLs) comprise the largest ubiquitin E3 subclass, in which a central cullin subunit links a substrate-binding adaptor with an E2-binding RING. Covalent attachment of the ubiquitin-like protein NEDD8 to a conserved C-terminal domain (ctd) lysine stimulates CRL ubiquitination activity and prevents binding of the inhibitor CAND1. Here we report striking conformational rearrangements in the crystal structure of NEDD8{approx}Cul5ctd-Rbx1 and SAXS analysis of NEDD8{approx}Cul1ctd-Rbx1 relative to their unmodified counterparts. In NEDD8ylated CRL structures, the cullin WHB and Rbx1 RING subdomains are dramatically reoriented, eliminating a CAND1-binding site and imparting multiple potential catalytic geometries to an associated E2. Biochemical analyses indicate that the structural malleability is important for both CRL NEDD8ylation and subsequent ubiquitination activities. Thus, our results point to a conformational control of CRL activity, with ligation of NEDD8 shifting equilibria to disfavor inactive CAND1-bound closed architectures, and favor dynamic, open forms that promote polyubiquitination.

  11. FXR agonist activity of conformationally constrained analogs of GW 4064

    SciTech Connect

    Akwabi-Ameyaw, Adwoa; Bass, Jonathan Y.; Caldwell, Richard D.; Caravella, Justin A.; Chen, Lihong; Creech, Katrina L.; Deaton, David N.; Madauss, Kevin P.; Marr, Harry B.; McFadyen, Robert B.; Miller, Aaron B.; Navas, III, Frank; Parks, Derek J.; Spearing, Paul K.; Todd, Dan; Williams, Shawn P.; Wisely, G. Bruce

    2010-09-27

    Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Replacement of the metabolically labile stilbene with either benzothiophene or naphthalene rings led to the identification of potent full agonists 2a and 2g.

  12. FXR agonist activity of conformationally constrained analogs of GW 4064.

    PubMed

    Akwabi-Ameyaw, Adwoa; Bass, Jonathan Y; Caldwell, Richard D; Caravella, Justin A; Chen, Lihong; Creech, Katrina L; Deaton, David N; Madauss, Kevin P; Marr, Harry B; McFadyen, Robert B; Miller, Aaron B; Navas, Frank; Parks, Derek J; Spearing, Paul K; Todd, Dan; Williams, Shawn P; Bruce Wisely, G

    2009-08-15

    Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Replacement of the metabolically labile stilbene with either benzothiophene or naphthalene rings led to the identification of potent full agonists 2a and 2g.

  13. Crystal structure of plasminogen activator inhibitor-1 in an active conformation with normal thermodynamic stability.

    PubMed

    Jensen, Jan K; Thompson, Lawrence C; Bucci, Joel C; Nissen, Poul; Gettins, Peter G W; Peterson, Cynthia B; Andreasen, Peter A; Morth, J Preben

    2011-08-26

    The serpin plasminogen activator inhibitor-1 (PAI-1) is a crucial regulator in fibrinolysis and tissue remodeling. PAI-1 has been associated with several pathological conditions and is a validated prognostic marker in human cancers. However, structural information about the native inhibitory form of PAI-1 has been elusive because of its inherent conformational instability and rapid conversion to a latent, inactive structure. Here we report the crystal structure of PAI-1 W175F at 2.3 Å resolution as the first model of the metastable native molecule. Structural comparison with a quadruple mutant (14-1B) previously used as representative of the active state uncovered key differences. The most striking differences occur near the region that houses three of the four mutations in the 14-1B PAI-1 structure. Prominent changes are localized within a loop connecting β-strand 3A with the F helix, in which a previously observed 3(10)-helix is absent in the new structure. Notably these structural changes are found near the binding site for the cofactor vitronectin. Because vitronectin is the only known physiological regulator of PAI-1 that slows down the latency conversion, the structure of this region is important. Furthermore, the previously identified chloride-binding site close to the F-helix is absent from the present structure and likely to be artifactual, because of its dependence on the 14-1B mutations. Instead we found a different chlorine-binding site that is likely to be present in wild type PAI-1 and that more satisfactorily accounts for the chlorine stabilizing effect on PAI-1.

  14. Crystal Structure of Plasminogen Activator Inhibitor-1 in an Active Conformation with Normal Thermodynamic Stability*

    PubMed Central

    Jensen, Jan K.; Thompson, Lawrence C.; Bucci, Joel C.; Nissen, Poul; Gettins, Peter G. W.; Peterson, Cynthia B.; Andreasen, Peter A.; Morth, J. Preben

    2011-01-01

    The serpin plasminogen activator inhibitor-1 (PAI-1) is a crucial regulator in fibrinolysis and tissue remodeling. PAI-1 has been associated with several pathological conditions and is a validated prognostic marker in human cancers. However, structural information about the native inhibitory form of PAI-1 has been elusive because of its inherent conformational instability and rapid conversion to a latent, inactive structure. Here we report the crystal structure of PAI-1 W175F at 2.3 Å resolution as the first model of the metastable native molecule. Structural comparison with a quadruple mutant (14-1B) previously used as representative of the active state uncovered key differences. The most striking differences occur near the region that houses three of the four mutations in the 14-1B PAI-1 structure. Prominent changes are localized within a loop connecting β-strand 3A with the F helix, in which a previously observed 310-helix is absent in the new structure. Notably these structural changes are found near the binding site for the cofactor vitronectin. Because vitronectin is the only known physiological regulator of PAI-1 that slows down the latency conversion, the structure of this region is important. Furthermore, the previously identified chloride-binding site close to the F-helix is absent from the present structure and likely to be artifactual, because of its dependence on the 14-1B mutations. Instead we found a different chlorine-binding site that is likely to be present in wild type PAI-1 and that more satisfactorily accounts for the chlorine stabilizing effect on PAI-1. PMID:21697084

  15. 12 CFR 225.123 - Activities closely related to banking.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 3 2011-01-01 2011-01-01 false Activities closely related to banking. 225.123 Section 225.123 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Holding Companies Interpretations § 225.123 Activities closely related to banking. (a) Effective June...

  16. 12 CFR 225.123 - Activities closely related to banking.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Activities closely related to banking. 225.123 Section 225.123 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Holding Companies Interpretations § 225.123 Activities closely related to banking. (a) Effective June...

  17. 12 CFR 225.131 - Activities closely related to banking.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 3 2011-01-01 2011-01-01 false Activities closely related to banking. 225.131 Section 225.131 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Holding Companies Interpretations § 225.131 Activities closely related to banking. (a) Bank...

  18. 12 CFR 225.131 - Activities closely related to banking.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Activities closely related to banking. 225.131 Section 225.131 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Holding Companies Interpretations § 225.131 Activities closely related to banking. (a) Bank...

  19. 12 CFR 225.131 - Activities closely related to banking.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 12 Banks and Banking 3 2014-01-01 2014-01-01 false Activities closely related to banking. 225.131 Section 225.131 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Financial Holding Companies Interpretations § 225.131 Activities closely related to banking. (a)...

  20. 12 CFR 225.123 - Activities closely related to banking.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 12 Banks and Banking 3 2013-01-01 2013-01-01 false Activities closely related to banking. 225.123 Section 225.123 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Financial Holding Companies Interpretations § 225.123 Activities closely related to banking. (a)...

  1. 12 CFR 225.123 - Activities closely related to banking.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 12 Banks and Banking 3 2012-01-01 2012-01-01 false Activities closely related to banking. 225.123 Section 225.123 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Holding Companies Interpretations § 225.123 Activities closely related to banking. (a) Effective June...

  2. 12 CFR 225.131 - Activities closely related to banking.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 12 Banks and Banking 3 2013-01-01 2013-01-01 false Activities closely related to banking. 225.131 Section 225.131 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Financial Holding Companies Interpretations § 225.131 Activities closely related to banking. (a)...

  3. 12 CFR 225.129 - Activities closely related to banking.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 12 Banks and Banking 3 2014-01-01 2014-01-01 false Activities closely related to banking. 225.129 Section 225.129 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Financial Holding Companies Interpretations § 225.129 Activities closely related to banking....

  4. 12 CFR 225.131 - Activities closely related to banking.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 12 Banks and Banking 3 2012-01-01 2012-01-01 false Activities closely related to banking. 225.131 Section 225.131 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Holding Companies Interpretations § 225.131 Activities closely related to banking. (a) Bank...

  5. 12 CFR 225.123 - Activities closely related to banking.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 12 Banks and Banking 3 2014-01-01 2014-01-01 false Activities closely related to banking. 225.123 Section 225.123 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Financial Holding Companies Interpretations § 225.123 Activities closely related to banking. (a)...

  6. 12 CFR 225.129 - Activities closely related to banking.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 12 Banks and Banking 3 2013-01-01 2013-01-01 false Activities closely related to banking. 225.129 Section 225.129 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL... Financial Holding Companies Interpretations § 225.129 Activities closely related to banking....

  7. Ligand Docking to Intermediate and Close-To-Bound Conformers Generated by an Elastic Network Model Based Algorithm for Highly Flexible Proteins.

    PubMed

    Kurkcuoglu, Zeynep; Doruker, Pemra

    2016-01-01

    Incorporating receptor flexibility in small ligand-protein docking still poses a challenge for proteins undergoing large conformational changes. In the absence of bound structures, sampling conformers that are accessible by apo state may facilitate docking and drug design studies. For this aim, we developed an unbiased conformational search algorithm, by integrating global modes from elastic network model, clustering and energy minimization with implicit solvation. Our dataset consists of five diverse proteins with apo to complex RMSDs 4.7-15 Å. Applying this iterative algorithm on apo structures, conformers close to the bound-state (RMSD 1.4-3.8 Å), as well as the intermediate states were generated. Dockings to a sequence of conformers consisting of a closed structure and its "parents" up to the apo were performed to compare binding poses on different states of the receptor. For two periplasmic binding proteins and biotin carboxylase that exhibit hinge-type closure of two dynamics domains, the best pose was obtained for the conformer closest to the bound structure (ligand RMSDs 1.5-2 Å). In contrast, the best pose for adenylate kinase corresponded to an intermediate state with partially closed LID domain and open NMP domain, in line with recent studies (ligand RMSD 2.9 Å). The docking of a helical peptide to calmodulin was the most challenging case due to the complexity of its 15 Å transition, for which a two-stage procedure was necessary. The technique was first applied on the extended calmodulin to generate intermediate conformers; then peptide docking and a second generation stage on the complex were performed, which in turn yielded a final peptide RMSD of 2.9 Å. Our algorithm is effective in producing conformational states based on the apo state. This study underlines the importance of such intermediate states for ligand docking to proteins undergoing large transitions. PMID:27348230

  8. Ligand Docking to Intermediate and Close-To-Bound Conformers Generated by an Elastic Network Model Based Algorithm for Highly Flexible Proteins

    PubMed Central

    Kurkcuoglu, Zeynep; Doruker, Pemra

    2016-01-01

    Incorporating receptor flexibility in small ligand-protein docking still poses a challenge for proteins undergoing large conformational changes. In the absence of bound structures, sampling conformers that are accessible by apo state may facilitate docking and drug design studies. For this aim, we developed an unbiased conformational search algorithm, by integrating global modes from elastic network model, clustering and energy minimization with implicit solvation. Our dataset consists of five diverse proteins with apo to complex RMSDs 4.7–15 Å. Applying this iterative algorithm on apo structures, conformers close to the bound-state (RMSD 1.4–3.8 Å), as well as the intermediate states were generated. Dockings to a sequence of conformers consisting of a closed structure and its “parents” up to the apo were performed to compare binding poses on different states of the receptor. For two periplasmic binding proteins and biotin carboxylase that exhibit hinge-type closure of two dynamics domains, the best pose was obtained for the conformer closest to the bound structure (ligand RMSDs 1.5–2 Å). In contrast, the best pose for adenylate kinase corresponded to an intermediate state with partially closed LID domain and open NMP domain, in line with recent studies (ligand RMSD 2.9 Å). The docking of a helical peptide to calmodulin was the most challenging case due to the complexity of its 15 Å transition, for which a two-stage procedure was necessary. The technique was first applied on the extended calmodulin to generate intermediate conformers; then peptide docking and a second generation stage on the complex were performed, which in turn yielded a final peptide RMSD of 2.9 Å. Our algorithm is effective in producing conformational states based on the apo state. This study underlines the importance of such intermediate states for ligand docking to proteins undergoing large transitions. PMID:27348230

  9. Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin*

    PubMed Central

    Richard, Benjamin; Swanson, Richard; Schedin-Weiss, Sophia; Ramirez, Ben; Izaguirre, Gonzalo; Gettins, Peter G. W.; Olson, Steven T.

    2008-01-01

    A conformationally altered prelatent form of antithrombin that possesses both anticoagulant and antiangiogenic activities is produced during the conversion of native to latent antithrombin (Larsson, H., Akerud, P., Nordling, K., Raub-Segall, E., Claesson-Welsh, L., and Björk, I. (2001) J. Biol. Chem. 276, 11996–12002). Here, we show that the previously characterized prelatent antithrombin is a mixture of native antithrombin and a modified, true prelatent antithrombin that are resolvable by heparin-agarose chromatography. Kinetic analyses revealed that prelatent antithrombin is an intermediate in the conversion of native to latent antithrombin whose formation is favored by stabilizing anions of the Hofmeister series. Purified prelatent antithrombin had reduced anticoagulant function compared with native antithrombin, due to a reduced heparin affinity and consequent impaired ability of heparin to either bridge prelatent antithrombin and coagulation proteases in a ternary complex or to induce full conformational activation of the serpin. Significantly, prelatent antithrombin possessed an antiangiogenic activity more potent than that of latent antithrombin, based on the relative abilities of the two forms to inhibit endothelial cell growth. The prelatent form was conformationally altered from native antithrombin as judged from an attenuation of tryptophan fluorescence changes following heparin activation and a reduced thermal stability. The alterations are consistent with the limited structural changes involving strand 1C observed in a prelatent form of plasminogen activator inhibitor-1 (Dupont, D. M., Blouse, G. E., Hansen, M., Mathiasen, L., Kjelgaard, S., Jensen, J. K., Christensen, A., Gils, A., Declerck, P. J., Andreasen, P. A., and Wind, T. (2006) J. Biol. Chem. 281, 36071–36081), since the 1H NMR spectrum, electrophoretic mobility, and proteolytic susceptibility of prelatent antithrombin most resemble those of native rather than those of latent antithrombin

  10. Conformation of the C1 phorbol-ester-binding domain participates in the activating conformational change of protein kinase C.

    PubMed Central

    Ho, C; Slater, S J; Stagliano, B A; Stubbs, C D

    1999-01-01

    The fluorescent phorbol ester 12-N-methylanthraniloylphorbol 13-acetate [sapintoxin D (SAPD)] was used as both the activator and the probe for the activating conformational change of the C1 domain of recombinant protein kinase C (PKC)alpha. Fluorescence emission spectra and steady-state anisotropy measurements of SAPD in fully active membrane-associated PKC show that there is a relatively hydrophobic environment and restricted motional freedom characterizing the phorbol-ester-binding site. SAPD also interacts with the membrane lipids so that it was necessary to resort to time-resolved anisotropy measurements to resolve the signals corresponding to PKC-bound SAPD from that associated with buffer and lipid. In the presence of membrane lipids (unilamellar vesicles of phosphatidylcholine and phosphatidylserine, 4:1 molar ratio) and Ca(2+), at a concentration sufficient to activate the enzyme fully, a long correlation time characteristic of highly restricted motion was observed for PKC-associated SAPD. The fraction of SAPD molecules displaying this restricted motion, in comparison with the total SAPD including that in lipids and in buffer, increased with increasing concentrations of Ca(2+) and paralleled the appearance of enzyme activity, whereas the rotational correlation time remained constant. This could be rationalized as an increase in the number of active PKC conformers in the total population of PKC molecules. It therefore seems that there is a distinct conformation of the C1 activator-binding domain associated with the active form of PKC. The addition of SAPD and dioleoyl-sn-glycerol together produced an activity higher than that achievable by either activator alone both at concentrations that alone induced maximal activity for the respective activator; this higher activity was associated with a further restriction in SAPD motion. Increasing the cholesterol concentration, the phosphatidylethanolamine concentration, the sn-2 unsaturation in phosphatidylcholine

  11. Metal-Dependent Conformational Activation Explains Highly Promutagenic Replication across O6-Methylguanine by Human DNA Polymerase β

    PubMed Central

    2015-01-01

    Human DNA polymerase β (polβ) inserts, albeit slowly, T opposite the carcinogenic lesion O6-methylguanine (O6MeG) ∼30-fold more frequently than C. To gain insight into this promutagenic process, we solved four ternary structures of polβ with an incoming dCTP or dTTP analogue base-paired with O6MeG in the presence of active-site Mg2+ or Mn2+. The Mg2+-bound structures show that both the O6MeG·dCTP/dTTP–Mg2+ complexes adopt an open protein conformation, staggered base pair, and one active-site metal ion. The Mn2+-bound structures reveal that, whereas the O6Me·dCTP–Mn2+ complex assumes the similar altered conformation, the O6MeG·dTTP–Mn2+ complex adopts a catalytically competent state with a closed protein conformation and pseudo-Watson–Crick base pair. On the basis of these observations, we conclude that polβ slows nucleotide incorporation opposite O6MeG by inducing an altered conformation suboptimal for catalysis and promotes mutagenic replication by allowing Watson–Crick-mode for O6MeG·T but not for O6MeG·C in the enzyme active site. The O6MeG·dTTP–Mn2+ ternary structure, which represents the first structure of mismatched polβ ternary complex with a closed protein conformation and coplanar base pair, the first structure of pseudo-Watson–Crick O6MeG·T formed in the active site of a DNA polymerase, and a rare, if not the first, example of metal-dependent conformational activation of a DNA polymerase, indicate that catalytic metal-ion coordination is utilized as a kinetic checkpoint by polβ and is crucial for the conformational activation of polβ. Overall, our structural studies not only explain the promutagenic polβ catalysis across O6MeG but also provide new insights into the replication fidelity of polβ. PMID:24694247

  12. Monopolar Spindle 1 (MPS1) Kinase Promotes Production of Closed MAD2 (C-MAD2) Conformer and Assembly of the Mitotic Checkpoint Complex*

    PubMed Central

    Tipton, Aaron R.; Ji, Wenbin; Sturt-Gillespie, Brianne; Bekier, Michael E.; Wang, Kexi; Taylor, William R.; Liu, Song-Tao

    2013-01-01

    MPS1 kinase is an essential component of the spindle assembly checkpoint (SAC), but its functioning mechanisms are not fully understood. We have shown recently that direct interaction between BUBR1 and MAD2 is critical for assembly and function of the human mitotic checkpoint complex (MCC), the SAC effector. Here we report that inhibition of MPS1 kinase activity by reversine disrupts BUBR1-MAD2 as well as CDC20-MAD2 interactions, causing premature activation of the anaphase-promoting complex/cyclosome. The effect of MPS1 inhibition is likely due to reduction of closed MAD2 (C-MAD2), as expressing a MAD2 mutant (MAD2L13A) that is locked in the C conformation rescued the checkpoint defects. In the presence of reversine, exogenous C-MAD2 does not localize to unattached kinetochores but is still incorporated into the MCC. Contrary to a previous report, we found that sustained MPS1 activity is required for maintaining both the MAD1·C-MAD2 complex and open MAD2 (O-MAD2) at unattached kinetochores to facilitate C-MAD2 production. Additionally, mitotic phosphorylation of BUBR1 is also affected by MPS1 inhibition but seems dispensable for MCC assembly. Our results support the notion that MPS1 kinase promotes C-MAD2 production and subsequent MCC assembly to activate the SAC. PMID:24151075

  13. Conformation and activity of lipase B from Candida antarctica in bicontinuous microemulsions.

    PubMed

    Subinya, Mireia; Steudle, Anne K; Jurkowski, Tomasz P; Stubenrauch, Cosima

    2015-07-01

    The paper at hand deals with the influence of the pH-value on the conformation and activity of the lipase B from Candida antarctica (CalB) which is incorporated in a bicontinuous microemulsion. The microemulsion used for this purpose consists of water/NaCl, n-octane, and the non-ionic surfactant penthaethylene glycol monodecylether (C10E5). The conformational study clearly shows (1) that CalB molecules are partitioned between the interfacial monolayer and the water domains and (2) that the pH-value of the microemulsion's water domains strongly influences the conformation of CalB at the interfacial monolayer. From these observations we conclude that there is a continuous exchange between the CalB molecules, which are located at the interfacial monolayer and those which are located in the water domains of the microemulsion. This exchange strongly influences the CalB conformation in both regions. In addition to the conformation, we also studied the catalytic activity of CalB. The catalytic measurements revealed a bell-shaped dependence between the CalB activity and the pH-value. The maximum catalytic activity of CalB in bicontinuous microemulsions was observed at pH=5.5. At this pH we observed the highest amount of α-helix conformation of the CalB molecules that are located at the interfacial monolayer, which, in turn, allows connecting the activity with the conformation.

  14. The Redox State Regulates the Conformation of Rv2466c to Activate the Antitubercular Prodrug TP053.

    PubMed

    Albesa-Jové, David; Comino, Natalia; Tersa, Montse; Mohorko, Elisabeth; Urresti, Saioa; Dainese, Elisa; Chiarelli, Laurent R; Pasca, Maria Rosalia; Manganelli, Riccardo; Makarov, Vadim; Riccardi, Giovanna; Svergun, Dmitri I; Glockshuber, Rudi; Guerin, Marcelo E

    2015-12-25

    Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin-fold with a Cys(19)-Pro(20)-Trp(21)-Cys(22) motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle x-ray scattering showed that the fully reduced form of Rv2466c adopts a "closed" compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an "open" conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical, and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.

  15. The Redox State Regulates the Conformation of Rv2466c to Activate the Antitubercular Prodrug TP053.

    PubMed

    Albesa-Jové, David; Comino, Natalia; Tersa, Montse; Mohorko, Elisabeth; Urresti, Saioa; Dainese, Elisa; Chiarelli, Laurent R; Pasca, Maria Rosalia; Manganelli, Riccardo; Makarov, Vadim; Riccardi, Giovanna; Svergun, Dmitri I; Glockshuber, Rudi; Guerin, Marcelo E

    2015-12-25

    Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin-fold with a Cys(19)-Pro(20)-Trp(21)-Cys(22) motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle x-ray scattering showed that the fully reduced form of Rv2466c adopts a "closed" compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an "open" conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical, and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily. PMID:26546681

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

    SciTech Connect

    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 that 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.

  17. Structure of a mitochondrial cytochrome c conformer competent for peroxidase activity

    PubMed Central

    McClelland, Levi J.; Mou, Tung-Chung; Jeakins-Cooley, Margaret E.; Sprang, Stephen R.; Bowler, Bruce E.

    2014-01-01

    At the onset of apoptosis, the peroxidation of cardiolipin at the inner mitochondrial membrane by cytochrome c requires an open coordination site on the heme. We report a 1.45-Å resolution structure of yeast iso-1-cytochrome c with the Met80 heme ligand swung out of the heme crevice and replaced by a water molecule. This conformational change requires modest adjustments to the main chain of the heme crevice loop and is facilitated by a trimethyllysine 72-to-alanine mutation. This mutation also enhances the peroxidase activity of iso-1-cytochrome c. The structure shows a buried water channel capable of facilitating peroxide access to the active site and of moving protons produced during peroxidase activity to the protein surface. Alternate positions of the side chain of Arg38 appear to mediate opening and closing of the buried water channel. In addition, two buried water molecules can adopt alternate positions that change the network of hydrogen bonds in the buried water channel. Taken together, these observations suggest that low and high proton conductivity states may mediate peroxidase function. Comparison of yeast and mammalian cytochrome c sequences, in the context of the steric factors that permit opening of the heme crevice, suggests that higher organisms have evolved to inhibit peroxidase activity, providing a more stringent barrier to the onset of apoptosis. PMID:24760830

  18. An allolactose trapped at the lacZ β-galactosidase active site with its galactosyl moiety in a (4)H3 conformation provides insights into the formation, conformation, and stabilization of the transition state.

    PubMed

    Wheatley, Robert W; Huber, Reuben E

    2015-12-01

    When lactose was incubated with G794A-β-galactosidase (a variant with a "closed" active site loop that binds transition state analogs well) an allolactose was trapped with its Gal moiety in a (4)H3 conformation, similar to the oxocarbenium ion-like conformation expected of the transition state. The numerous interactions formed between the (4)H3 structure and β-galactosidase indicate that this structure is representative of the transition state. This conformation is also very similar to that of d-galactono-1,5-lactone, a good transition state analog. Evidence indicates that substrates take up the (4)H3 conformation during migration from the shallow to the deep mode. Steric forces utilizing His418 and other residues are important for positioning the O1 leaving group into a quasi-axial position. An electrostatic interaction between the O5 of the distorted Gal and Tyr503 as well as C-H-π bonds with Trp568 are also significant. Computational studies of the energy of sugar ring distortion show that the β-galactosidase reaction itinerary is driven by energetic considerations in utilization of a (4)H3 transition state with a novel (4)C1-(4)H3-(4)C1 conformation itinerary. To our knowledge, this is the first X-ray crystallographic structural demonstration that the transition state of a natural substrate of a glycosidase has a (4)H3 conformation.

  19. An allolactose trapped at the lacZ β-galactosidase active site with its galactosyl moiety in a (4)H3 conformation provides insights into the formation, conformation, and stabilization of the transition state.

    PubMed

    Wheatley, Robert W; Huber, Reuben E

    2015-12-01

    When lactose was incubated with G794A-β-galactosidase (a variant with a "closed" active site loop that binds transition state analogs well) an allolactose was trapped with its Gal moiety in a (4)H3 conformation, similar to the oxocarbenium ion-like conformation expected of the transition state. The numerous interactions formed between the (4)H3 structure and β-galactosidase indicate that this structure is representative of the transition state. This conformation is also very similar to that of d-galactono-1,5-lactone, a good transition state analog. Evidence indicates that substrates take up the (4)H3 conformation during migration from the shallow to the deep mode. Steric forces utilizing His418 and other residues are important for positioning the O1 leaving group into a quasi-axial position. An electrostatic interaction between the O5 of the distorted Gal and Tyr503 as well as C-H-π bonds with Trp568 are also significant. Computational studies of the energy of sugar ring distortion show that the β-galactosidase reaction itinerary is driven by energetic considerations in utilization of a (4)H3 transition state with a novel (4)C1-(4)H3-(4)C1 conformation itinerary. To our knowledge, this is the first X-ray crystallographic structural demonstration that the transition state of a natural substrate of a glycosidase has a (4)H3 conformation. PMID:26291713

  20. Active site conformational changes of prostasin provide a new mechanism of protease regulation by divalent cations

    SciTech Connect

    Spraggon, Glen; Hornsby, Michael; Shipway, Aaron; Tully, David C.; Bursulaya, Badry; Danahay, Henry; Harris, Jennifer L.; Lesley, Scott A.

    2010-01-12

    Prostasin or human channel-activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d-FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca{sup +2}. Comparison of the structures with each other and with other members of the trypsin-like serine protease family reveals unique structural features of prostasin and a large degree of conformational variation within specificity determining loops. Of particular interest is the S1 subsite loop which opens and closes in response to basic residues or divalent ions, directly binding Ca{sup +2} cations. This induced fit active site provides a new possible mode of regulation of trypsin-like proteases adapted in particular to extracellular regions with variable ionic concentrations such as the outer membrane layer of the epithelial cell.

  1. Mapping the conformational transition in Src activation by cumulating the information from multiple molecular dynamics trajectories

    PubMed Central

    Yang, Sichun; Banavali, Nilesh K.; Roux, Benoît

    2009-01-01

    The Src-family kinases are allosteric enzymes that play a key role in the regulation of cell growth and proliferation. In response to cellular signals, they undergo large conformational changes to switch between distinct inactive and active states. A computational strategy for characterizing the conformational transition pathway is presented to bridge the inactive and active states of the catalytic domain of Hck. The information from a large number (78) of independent all-atom molecular dynamics trajectories with explicit solvent is combined together to assemble a connectivity map of the conformational transition. Two intermediate states along the activation pathways are identified, and their structural features are characterized. A coarse free-energy landscape is built in terms of the collective motions corresponding to the opening of the activation loop (A-loop) and the rotation of the αC helix. This landscape shows that the protein can adopt a multitude of conformations in which the A-loop is partially open, while the αC helix remains in the orientation characteristic of the inactive conformation. The complete transition leading to the active conformation requires a concerted movement involving further opening of the A-loop, the relative alignment of N-lobe and C-lobe, and the rotation of the αC helix needed to recruit the residues necessary for catalysis in the active site. The analysis leads to a dynamic view of the full-length kinase activation, whereby transitions of the catalytic domain to intermediate configurations with a partially open A-loop are permitted, even while the SH2-SH3 clamp remains fully engaged. These transitions would render Y416 available for the transphosphorylation event that ultimately locks down the active state. The results provide a broad framework for picturing the conformational transitions leading to kinase activation. PMID:19225111

  2. Antigen conformation determines processing requirements for T-cell activation.

    PubMed Central

    Streicher, H Z; Berkower, I J; Busch, M; Gurd, F R; Berzofsky, J A

    1984-01-01

    We studied the difference in requirements for processing and presentation to a single T-cell clone of four different forms of the same epitope of sperm whale myoglobin--namely, on the native protein, on two conformationally altered forms of the protein, or as a 22-residue antigenic peptide fragment. The T-cell clone was I-Ed-restricted and specific for an epitope on the CNBr fragment 132-153 involving Lys-140. As inhibitors of macrophage processing of antigen, we used several agents that inhibit lysosomal function: the weak bases chloroquine and NH4Cl, the cationic ionophore monensin, and the competitive protease inhibitor leupeptin. When these agents were used to inhibit processing of antigen by presenting cells and then washed out before T cells were added to culture, they inhibited the presentation of native antigen but not of fragment 132-153. To our surprise, the intact but denatured form, S-methylmyoglobin, behaved like the fragment not like the native protein. Apomyoglobin was intermediate in susceptibility to inhibition. Thus, native myoglobin requires a processing step that appears to involve lysosomal proteolysis, which is not required by fragment 132-153 or the denatured unfolded forms. For an antigen the size of myoglobin (Mr 17,800), it appears that unfolding of the native conformation, rather than further reduction in size, is the critical parameter determining the need for processing. Since a major difference between native myoglobin and the other forms is the greater accessibility in the latter of sites, such as hydrophobic residues, buried in the native protein, we propose that processing may be necessary to expose these sites, perhaps for interaction with the cell membrane or the Ia of the antigen-presenting cell. PMID:6333686

  3. Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation

    DOE PAGES

    Colbert, Karen N.; Hattendorf, Douglas A.; Weiss, Thomas M.; Burkhardt, Pawel; Fasshauer, Dirk; Weis, William I.

    2013-07-15

    In neurons, soluble N-ethylmaleimide–sensitive factor attachment receptor (SNARE) proteins drive the fusion of synaptic vesicles to the plasma membrane through the formation of a four-helix SNARE complex. Members of the Sec1/Munc18 protein family regulate membrane fusion through interactions with the syntaxin family of SNARE proteins. The neuronal protein Munc18a interacts with a closed conformation of the SNARE protein syntaxin1a (Syx1a) and with an assembled SNARE complex containing Syx1a in an open conformation. The N-peptide of Syx1a (amino acids 1–24) has been implicated in the transition of Munc18a-bound Syx1a to Munc18a-bound SNARE complex, but the underlying mechanism is not understood. Inmore » addition, we report the X-ray crystal structures of Munc18a bound to Syx1a with and without its native N-peptide (Syx1aΔN), along with small-angle X-ray scattering (SAXS) data for Munc18a bound to Syx1a, Syx1aΔN, and Syx1a L165A/E166A (LE), a mutation thought to render Syx1a in a constitutively open conformation. We show that all three complexes adopt the same global structure, in which Munc18a binds a closed conformation of Syx1a. We also identify a possible structural connection between the Syx1a N-peptide and SNARE domain that might be important for the transition of closed-to-open Syx1a in SNARE complex assembly. Although the role of the N-peptide in Munc18a-mediated SNARE complex assembly remains unclear, our results demonstrate that the N-peptide and LE mutation have no effect on the global conformation of the Munc18a–Syx1a complex.« less

  4. Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation

    SciTech Connect

    Colbert, Karen N.; Hattendorf, Douglas A.; Weiss, Thomas M.; Burkhardt, Pawel; Fasshauer, Dirk; Weis, William I.

    2013-07-15

    In neurons, soluble N-ethylmaleimide–sensitive factor attachment receptor (SNARE) proteins drive the fusion of synaptic vesicles to the plasma membrane through the formation of a four-helix SNARE complex. Members of the Sec1/Munc18 protein family regulate membrane fusion through interactions with the syntaxin family of SNARE proteins. The neuronal protein Munc18a interacts with a closed conformation of the SNARE protein syntaxin1a (Syx1a) and with an assembled SNARE complex containing Syx1a in an open conformation. The N-peptide of Syx1a (amino acids 1–24) has been implicated in the transition of Munc18a-bound Syx1a to Munc18a-bound SNARE complex, but the underlying mechanism is not understood. In addition, we report the X-ray crystal structures of Munc18a bound to Syx1a with and without its native N-peptide (Syx1aΔN), along with small-angle X-ray scattering (SAXS) data for Munc18a bound to Syx1a, Syx1aΔN, and Syx1a L165A/E166A (LE), a mutation thought to render Syx1a in a constitutively open conformation. We show that all three complexes adopt the same global structure, in which Munc18a binds a closed conformation of Syx1a. We also identify a possible structural connection between the Syx1a N-peptide and SNARE domain that might be important for the transition of closed-to-open Syx1a in SNARE complex assembly. Although the role of the N-peptide in Munc18a-mediated SNARE complex assembly remains unclear, our results demonstrate that the N-peptide and LE mutation have no effect on the global conformation of the Munc18a–Syx1a complex.

  5. Activation loop 3 and the 170 loop interact in the active conformation of coagulation factor VIIa.

    PubMed

    Persson, Egon; Olsen, Ole H

    2009-06-01

    The initiation of blood coagulation involves tissue factor (TF)-induced allosteric activation of factor VIIa (FVIIa), which circulates in a zymogen-like state. In addition, the (most) active conformation of FVIIa presumably relies on a number of intramolecular interactions. We have characterized the role of Gly372(223) in FVIIa, which is the sole residue in activation loop 3 that is capable of forming backbone hydrogen bonds with the unusually long 170 loop and with activation loop 2, by studying the effects of replacement with Ala [G372(223)A]. G372A-FVIIa, both in the free and TF-bound form, exhibited reduced cleavage of factor X (FX) and of peptidyl substrates, and had increased K(m) values compared with wild-type FVIIa. Inhibition of G372A-FVIIa.sTF by p-aminobenzamidine was characterized by a seven-fold higher K(i) than obtained with FVIIa.sTF. Crystallographic and modelling data suggest that the most active conformation of FVIIa depends on the backbone hydrogen bond between Gly372(223) and Arg315(170C) in the 170 loop. Despite the reduced activity and inhibitor susceptibility, native and active site-inhibited G372A-FVIIa bound sTF with the same affinity as the corresponding forms of FVIIa, and burial of the N-terminus of the protease domain increased similarly upon sTF binding to G372A-FVIIa and FVIIa. Thus Gly372(223) in FVIIa appears to play a critical role in maturation of the S1 pocket and adjacent subsites, but does not appear to be of importance for TF binding and the ensuing allostery. PMID:19490111

  6. Asymmetry of the active site loop conformation between subunits of glutamate-1-semialdehyde aminomutase in solution.

    PubMed

    Campanini, Barbara; Bettati, Stefano; di Salvo, Martino Luigi; Mozzarelli, Andrea; Contestabile, Roberto

    2013-01-01

    Glutamate-1-semialdehyde aminomutase (GSAM) is a dimeric, pyridoxal 5'-phosphate (PLP)- dependent enzyme catalysing in plants and some bacteria the isomerization of L-glutamate-1-semialdehyde to 5-aminolevulinate, a common precursor of chlorophyll, haem, coenzyme B12, and other tetrapyrrolic compounds. During the catalytic cycle, the coenzyme undergoes conversion from pyridoxamine 5'-phosphate (PMP) to PLP. The entrance of the catalytic site is protected by a loop that is believed to switch from an open to a closed conformation during catalysis. Crystallographic studies indicated that the structure of the mobile loop is related to the form of the cofactor bound to the active site, allowing for asymmetry within the dimer. Since no information on structural and functional asymmetry of the enzyme in solution is available in the literature, we investigated the active site accessibility by determining the cofactor fluorescence quenching of PMP- and PLP-GSAM forms. PLP-GSAM is partially quenched by potassium iodide, suggesting that at least one catalytic site is accessible to the anionic quencher and therefore confirming the asymmetry observed in the crystal structure. Iodide induces release of the cofactor from PMP-GSAM, apparently from only one catalytic site, therefore suggesting an asymmetry also in this form of the enzyme in solution, in contrast with the crystallographic data.

  7. Active conformation control of unfolded proteins by hyperthermal collision with a metal surface.

    PubMed

    Rinke, Gordon; Rauschenbach, Stephan; Harnau, Ludger; Albarghash, Alyazan; Pauly, Matthias; Kern, Klaus

    2014-10-01

    The physical and chemical properties of macromolecules like proteins are strongly dependent on their conformation. The degrees of freedom of their chemical bonds generate a huge conformational space, of which, however, only a small fraction is accessible in thermal equilibrium. Here we show that soft-landing electrospray ion beam deposition (ES-IBD) of unfolded proteins allows to control their conformation. The dynamics and result of the deposition process can be actively steered by selecting the molecular ion beam's charge state or tuning the incident energy. Using these parameters, protein conformations ranging from fully extended to completely compact can be prepared selectively on a surface, as evidenced on the subnanometer/amino acid resolution level by scanning tunneling microscopy (STM). Supported by molecular dynamics (MD) simulations, our results demonstrate that the final conformation on the surface is reached through a mechanical deformation during the hyperthermal ion surface collision. Our experimental results independently confirm the findings of ion mobility spectrometry (IMS) studies of protein gas phase conformations. Moreover, we establish a new route for the processing of macromolecular materials, with the potential to reach conformations that would be inaccessible otherwise. PMID:25198655

  8. The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation.

    PubMed Central

    Bassi, G S; Murchie, A I; Lilley, D M

    1996-01-01

    The hammerhead ribozyme undergoes an ion-dependent folding process into the active conformation. We find that the folding can be blocked at specific stages by changes of sequence or functionality within the core. In the the absence of added metal ions, the global structure of the hammerhead is extended, with a large angle subtended between stems I and II. No core sequence changes appear to alter this geometry, consistent with an unstructured core under these conditions. Upon addition of low concentrations of magnesium ions, the hammerhead folds by an association of stems II and III, to include a large angle between them. This stage is inhibited or altered by mutations within the oligopurine sequence lying between stems II and III, and folding is completely prevented by an A14G mutation. Further increase in magnesium ion concentration brings about a second stage of folding in the natural sequence hammerhead, involving a reorientation of stem I, which rotates around into the same direction of stem II. Because this transition occurs over the same range of magnesium ion concentration over which the hammerhead ribozyme becomes active, it is likely that the final conformation is most closely related to the active form of the structure. Magnesium ion-dependent folding into this conformation is prevented by changes at G5, notably removal of the 2'-hydroxyl group and replacement of the base by cytidine. The ability to dissect the folding process by means of sequence changes suggests that two separate ion-dependent stages are involved in the folding of the hammerhead ribozyme into the active conformation. PMID:8752086

  9. 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

  10. Insights into mechanism of glucokinase activation: observation of multiple distinct protein conformations.

    PubMed

    Liu, Shenping; Ammirati, Mark J; Song, Xi; Knafels, John D; Zhang, Jeff; Greasley, Samantha E; Pfefferkorn, Jeffrey A; Qiu, Xiayang

    2012-04-20

    Human glucokinase (GK) is a principal regulating sensor of plasma glucose levels. Mutations that inactivate GK are linked to diabetes, and mutations that activate it are associated with hypoglycemia. Unique kinetic properties equip GK for its regulatory role: although it has weak basal affinity for glucose, positive cooperativity in its binding of glucose causes a rapid increase in catalytic activity when plasma glucose concentrations rise above euglycemic levels. In clinical trials, small molecule GK activators (GKAs) have been efficacious in lowering plasma glucose and enhancing glucose-stimulated insulin secretion, but they carry a risk of overly activating GK and causing hypoglycemia. The theoretical models proposed to date attribute the positive cooperativity of GK to the existence of distinct protein conformations that interconvert slowly and exhibit different affinities for glucose. Here we report the respective crystal structures of the catalytic complex of GK and of a GK-glucose complex in a wide open conformation. To assess conformations of GK in solution, we also carried out small angle x-ray scattering experiments. The results showed that glucose dose-dependently converts GK from an apo conformation to an active open conformation. Compared with wild type GK, activating mutants required notably lower concentrations of glucose to be converted to the active open conformation. GKAs decreased the level of glucose required for GK activation, and different compounds demonstrated distinct activation profiles. These results lead us to propose a modified mnemonic model to explain cooperativity in GK. Our findings may offer new approaches for designing GKAs with reduced hypoglycemic risk.

  11. Autophosphorylation Activity of a Soluble Hexameric Histidine Kinase Correlates with the Shift in Protein Conformational Equilibrium

    PubMed Central

    Wojnowska, Marta; Yan, Jun; Sivalingam, Ganesh N.; Cryar, Adam; Gor, Jayesh; Thalassinos, Konstantinos; Djordjevic, Snezana

    2013-01-01

    Summary In a commonly accepted model, in response to stimuli, bacterial histidine kinases undergo a conformational transition between an active and inactive form. Structural information on histidine kinases is limited. By using ion mobility-mass spectrometry (IM-MS), we demonstrate an exchange between two conformational populations of histidine kinase ExsG that are linked to different levels of kinase activity. ExsG is an atypical signaling protein that incorporates an uncommon histidine kinase catalytic core at the C terminus preceded by an N-terminal “receiver domain” that is normally associated with the response regulator proteins in two-component signal transduction systems. IM-MS analysis and enzymatic assays indicate that phosphorylation of the ExsG receiver domain stabilizes the “compact” form of the protein and inhibits kinase core activity; in contrast, nucleotide binding required for kinase activity is associated with the more open conformation of ExsG. PMID:24210218

  12. Summary of Closed Circuit Television Activities in Medical Education.

    ERIC Educational Resources Information Center

    London Univ. (England). Inst. of Education.

    This 1967 summary of closed circuit television (CCTV) activities in medical education presents descriptive information on 35 different medical institutions in Great Britain. Specific data on CCTV are offered by institution, equipment, and uses under each medical field: anatomy, anaesthetics, geriatrics, medicine, obstretrics and gynaecology,…

  13. Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

    PubMed

    Zhang, Yu; An, Jiao; Yang, Guang-Yu; Bai, Aixi; Zheng, Baisong; Lou, Zhiyong; Wu, Geng; Ye, Wei; Chen, Hai-Feng; Feng, Yan; Manco, Giuseppe

    2015-01-01

    Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a "hot spot" in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

  14. Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

    PubMed Central

    Zhang, Yu; An, Jiao; Yang, Guang-Yu; Bai, Aixi; Zheng, Baisong; Lou, Zhiyong; Wu, Geng; Ye, Wei; Chen, Hai-Feng; Feng, Yan; Manco, Giuseppe

    2015-01-01

    Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity. PMID:25706379

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

    PubMed

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

    2013-12-20

    Over the past 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, such as 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.

  16. Activity and Conformation of Yeast Alcohol Dehydrogenase (YADH) Entrapped in Reverse Micelles.

    PubMed

    Das; Mozumdar; Maitra

    2000-10-15

    Yeast alcohol dehydrogenase (YADH) solubilized in reverse micelles of aerosol OT (i.e., AOT or sodium bis (2-ethyl hexyl) sulfosuccinate) in isooctane has been shown to be catalytically more active than that in aqueous buffer under optimum conditions of pH, temperature, and water content in reverse micelles. Studies of the secondary structure conformational changes of the enzyme in reverse micelles have been made from circular dichroism spectroscopy. It has been seen that the conformation of YADH in reverse micelles is extremely sensitive to pH, temperature, and water content. A comparison has been made between the catalytic activity of the enzyme and the alpha-helix content in the conformation and it has been observed that the enzyme is most active at the maximum alpha-helix content. While the beta-sheet content in the conformation of the entrapped enzyme was found to be dependent on the enzyme-micelle interface interaction, the alpha-helix and random coil conformations are governed by the degree of entrapment and the extent of rigidity provided by the micelle core to the enzyme structure. Copyright 2000 Academic Press.

  17. Conformational change path between closed and open forms of C2 domain of coagulation factor V on a two-dimensional free-energy surface

    NASA Astrophysics Data System (ADS)

    Wu, Sangwook; Lee, Chang Jun; Pedersen, Lee G.

    2009-04-01

    We test a hypothesis that the closed form of the C2 domain of coagulation factor V is more stable than the open form in an aqueous environment using a two-dimensional free-energy calculation with a simple dielectric solvent model. Our result shows that while the free-energy difference between two forms is small, favoring the closed form, a two-dimensional free-energy surface (FES) reveals that a transition state (1.53 kcal/mol) exists between the two conformations. By mapping the one-dimensional order parameter ΔQ onto the two-dimensional FES, we search the conformational change path with the highest Boltzmann weighting factor between the closed and open form of the factor V C2 domain. The predicted transition path from the closed to open form is not that of simple side chain movements, but instead concerted movements of several loops. We also present a one-dimensional free-energy profile using a collective order parameter, which in a coarse manner locates the energy barriers found on the two-dimensional FES.

  18. Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation

    PubMed Central

    Lee, Tai-Sung; Giambaşu, George M.; Sosa, Carlos P.; Martick, Monika; Scott, William G.; York, Darrin M.

    2009-01-01

    The relationship between formation of active in-line attack conformations and monovalent (Na+) and divalent (Mg2+) metal ion binding in the hammerhead ribozyme has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence or absence of a Mg2+ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the hammerhead ribozyme folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of hammerhead ribozyme catalysis and support the supposition that Mg2+, in addition to stabilizing active conformations, plays a specific chemical role in catalysis. PMID:19265710

  19. Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction.

    PubMed

    Zeymer, Cathleen; Werbeck, Nicolas D; Zimmermann, Sabine; Reinstein, Jochen; Hansen, D Flemming

    2016-09-12

    States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side-chains were quantified by NMR spin-relaxation methods. In addition to apo and ligand-bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side-chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions. PMID:27534930

  20. Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction

    PubMed Central

    Zeymer, Cathleen; Werbeck, Nicolas D.; Zimmermann, Sabine

    2016-01-01

    Abstract States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side‐chains were quantified by NMR spin‐relaxation methods. In addition to apo and ligand‐bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side‐chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions. PMID:27534930

  1. (19)F NMR studies of the leucine-isoleucine-valine binding protein: evidence that a closed conformation exists in solution.

    PubMed

    Salopek-Sondi, Branka; Vaughan, Mark D; Skeels, Matthew C; Honek, John F; Luck, Linda A

    2003-10-01

    The leucine-isoleucine-valine binding protein (LIV) found in the periplasmic space of E. coli has been used as a structural model for a number of neuronal receptors. This "venus fly trap" type protein has been characterized by crystallography in only the open form. Herein we have labeled LIV with 5-fluorotryptophan (5F-Trp) and difluoromethionine (DFM) in order to explore the structural dynamics of this protein and the application of DFM as a potential (19)F NMR structural probe for this family of proteins. Based on mass spectrometric analysis of the protein overproduced in the presence of DFM, approximately 30% of the five LIV methionine residues were randomly substituted with the fluorinated analog. Urea denaturation experiments imply a slight decrease in protein stability when DFM is incorporated into LIV. However, the fluorinated methionine did not alter leucine-binding activity upon its incorporation into the protein. Binding of L-leucine stabilizes both the unlabeled and DFM-labeled LIV, and induces the protein to adopt a three-state unfolding model in place of the two-state process observed for the free protein. The (19)F NMR spectrum of DFM-labeled LIV gave distinct resonances for the five Met residues found in LIV. 5F-Trp labeled LIV gave a well resolved spectrum for the three Trp residues. Trp to Phe mutants defined the resonances in the spectrum. The distinct narrowing in line width of the resonances when ligand was added identified the closed form of the protein.

  2. The effect of citric acid on the activity, thermodynamics and conformation of mushroom polyphenoloxidase.

    PubMed

    Liu, Wei; Zou, Li-qiang; Liu, Jun-ping; Zhang, Zhao-qin; Liu, Cheng-mei; Liang, Rui-hong

    2013-09-01

    Few reports have focused on the effect of citric acid on thermodynamics and conformation of polyphenoloxidase (PPO). In this study, variations on activity, thermodynamics and conformation of mushroom PPO induced by citric acid (1-60mM) and relationships among these were investigated. It showed that with the increasing concentration of citric acid, the activity of PPO decreased gradually to an inactivity condition; inactivation rate constant (k) of PPO increased and the activation energy (Ea) as well as thermodynamic parameters (ΔG, ΔH, ΔS) decreased, which indicated that the thermosensitivity, stability and number of non-covalent bonds of PPO decreased. The conformation was gradually unfolded, which was reflected in the decrease of α-helix contents, increase of β-sheet and exposure of aromatic amino acid residuals. Moreover, two linear relationships of relative activities, enthalpies (ΔH) against α-helix contents were obtained. It indicated that changes of activity and thermodynamics might correlate to the unfolding of conformation.

  3. The uncoupled ATPase activity of the ABC transporter BtuC2D2 leads to a hysteretic conformational change, conformational memory, and improved activity

    PubMed Central

    Livnat-Levanon, Nurit; I. Gilson, Amy; Ben-Tal, Nir; Lewinson, Oded

    2016-01-01

    ABC transporters comprise a large and ubiquitous family of proteins. From bacteria to man they translocate solutes at the expense of ATP hydrolysis. Unlike other enzymes that use ATP as an energy source, ABC transporters are notorious for having high levels of basal ATPase activity: they hydrolyze ATP also in the absence of their substrate. It is unknown what are the effects of such prolonged and constant activity on the stability and function of ABC transporters or any other enzyme. Here we report that prolonged ATP hydrolysis is beneficial to the ABC transporter BtuC2D2. Using ATPase assays, surface plasmon resonance interaction experiments, and transport assays we observe that the constantly active transporter remains stable and functional for much longer than the idle one. Remarkably, during extended activity the transporter undergoes a slow conformational change (hysteresis) and gradually attains a hyperactive state in which it is more active than it was to begin with. This phenomenon is different from stabilization of enzymes by ligand binding: the hyperactive state is only reached through ATP hydrolysis, and not ATP binding. BtuC2D2 displays a strong conformational memory for this excited state, and takes hours to return to its basal state after catalysis terminates. PMID:26905293

  4. Exploring the Role of Conformational Heterogeneity in cis-Autoproteolytic Activation of ThnT

    PubMed Central

    2015-01-01

    In the past decade, there have been major achievements in understanding the relationship between enzyme catalysis and protein structural plasticity. In autoprocessing systems, however, there is a sparsity of direct evidence of the role of conformational dynamics, which are complicated by their intrinsic chemical reactivity. ThnT is an autoproteolytically activated enzyme involved in the biosynthesis of the β-lactam antibiotic thienamycin. Conservative mutation of ThnT results in multiple conformational states that can be observed via X-ray crystallography, establishing ThnT as a representative and revealing system for studing how conformational dynamics control autoactivation at a molecular level. Removal of the nucleophile by mutation to Ala disrupts the population of a reactive state and causes widespread structural changes from a conformation that promotes autoproteolysis to one associated with substrate catalysis. Finer probing of the active site polysterism was achieved by EtHg derivatization of the nucleophile, which indicates the active site and a neighboring loop have coupled dynamics. Disruption of these interactions by mutagenesis precludes the ability to observe a reactive state through X-ray crystallography, and application of this insight to other autoproteolytically activated enzymes offers an explanation for the widespread crystallization of inactive states. We suggest that the N → O(S) acyl shift in cis-autoproteolysis might occur through a si-face attack, thereby unifying the fundamental chemistry of these enzymes through a common mechanism. PMID:24933323

  5. (19)F NMR studies of the leucine-isoleucine-valine binding protein: evidence that a closed conformation exists in solution.

    PubMed

    Salopek-Sondi, Branka; Vaughan, Mark D; Skeels, Matthew C; Honek, John F; Luck, Linda A

    2003-10-01

    The leucine-isoleucine-valine binding protein (LIV) found in the periplasmic space of E. coli has been used as a structural model for a number of neuronal receptors. This "venus fly trap" type protein has been characterized by crystallography in only the open form. Herein we have labeled LIV with 5-fluorotryptophan (5F-Trp) and difluoromethionine (DFM) in order to explore the structural dynamics of this protein and the application of DFM as a potential (19)F NMR structural probe for this family of proteins. Based on mass spectrometric analysis of the protein overproduced in the presence of DFM, approximately 30% of the five LIV methionine residues were randomly substituted with the fluorinated analog. Urea denaturation experiments imply a slight decrease in protein stability when DFM is incorporated into LIV. However, the fluorinated methionine did not alter leucine-binding activity upon its incorporation into the protein. Binding of L-leucine stabilizes both the unlabeled and DFM-labeled LIV, and induces the protein to adopt a three-state unfolding model in place of the two-state process observed for the free protein. The (19)F NMR spectrum of DFM-labeled LIV gave distinct resonances for the five Met residues found in LIV. 5F-Trp labeled LIV gave a well resolved spectrum for the three Trp residues. Trp to Phe mutants defined the resonances in the spectrum. The distinct narrowing in line width of the resonances when ligand was added identified the closed form of the protein. PMID:12956607

  6. Activation Barrier-Limited Folding and Conformational Sampling of a Dynamic Protein Domain.

    PubMed

    Dogan, Jakob; Toto, Angelo; Andersson, Eva; Gianni, Stefano; Jemth, Per

    2016-09-20

    Folding reaction mechanisms of globular protein domains have been extensively studied by both experiment and simulation and found to be highly concerted chemical reactions in which numerous noncovalent bonds form in an apparent two-state fashion. However, less is known regarding intrinsically disordered proteins because their folding can usually be studied only in conjunction with binding to a ligand. We have investigated by kinetics the folding mechanism of such a disordered protein domain, the nuclear coactivator-binding domain (NCBD) from CREB-binding protein. While a previous computational study suggested that NCBD folds without an activation free energy barrier, our experimental data demonstrate that NCBD, despite its highly dynamic structure, displays relatively slow folding (∼10 ms at 277 K) consistent with a barrier-limited process. Furthermore, the folding kinetics corroborate previous nuclear magnetic resonance data showing that NCBD exists in two folded conformations and one more denatured conformation at equilibrium and, thus, that the folding mechanism is a three-state mechanism. The refolding kinetics is limited by unfolding of the less populated folded conformation, suggesting that the major route for interconversion between the two folded states is via the denatured state. Because the two folded conformations have been suggested to bind distinct ligands, our results have mechanistic implications for conformational sampling in protein-protein interactions. PMID:27542287

  7. Passive and active EO sensing close to the sea surface

    NASA Astrophysics Data System (ADS)

    Steinvall, Ove; Persson, Rolf; Berglund, Folke; Öhgren, Johan; Gustafsson, Frank

    2014-10-01

    The present paper investigates the use of an eye-safe laser rangefinder at 1.5 μm and TV/IR imaging to obtain information on atmospheric properties at various paths close to the sea surface. On one day active/passive imaging NIR and SWIR systems were also used. The paper will describe the experimental equipment and the results from measurements of atmospheric backscatter as well as TV and IR images of test targets along a 1.8 km path close to the Baltic Sea. The site also contained a weather station and a scintillometer for logging weather and turbulence parameters. Results correlating the lidar attenuation with the imaging performance will be given and compared with models.

  8. Insights into the conformation of aminofluorene-deoxyguanine adduct in a DNA polymerase active site.

    PubMed

    Vaidyanathan, Vaidyanathan G; Liang, Fengting; Beard, William A; Shock, David D; Wilson, Samuel H; Cho, Bongsup P

    2013-08-01

    The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo(-)) and DNA polymerase β (pol β) using (19)F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol β. The addition of a non-hydrolysable 2'-deoxycytosine-5'-[(α,β)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo(-) complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol β, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with (19)F NMR data. Surface plasmon resonance binding kinetics revealed that pol β binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential.

  9. Sugar binding effects on the enzymatic reaction and conformation near the active site of pokeweed antiviral protein revealed by fluorescence spectroscopy.

    PubMed

    Nakashima, Hiromichi; Fukunaga, Yukihiro; Ueno, Ryosuke; Nishimoto, Etsuko

    2014-05-01

    In various trials for elucidating the physiological function of pokeweed antiviral protein (PAP), studies on the interaction with sugar are essential. The fluorescence titration curves showed that PAP retained the strong affinity against N-acetylglucosamine (NAG) and two sites in one PAP molecule co-operatively participated in the binding. In the complex of PAP with NAG, Trp208 located at the entrance lid site of substrate came closer to Tyr72 about 0.3 Å. Furthermore, the fluorescence anisotropy decay measurement demonstrated that the segmental rotation of Trp208 was enlarged by the binding of PAP with NAG. Such conformational changes around the active site closely correlate with the enzymatic activity of PAP. The N-glycosidase activity of PAP was enhanced more than two times in the presence of NAG. The obtained results consistently suggested the enzymatic activity of PAP would be regulated through the conformation change near the active site induced by the binding with NAG.

  10. NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site

    PubMed Central

    Hoffmann, Bernd; Mitchell, G. Thomas; Gendron, Patrick; Major, François; Andersen, Angela A.; Collins, Richard A.; Legault, Pascale

    2003-01-01

    Substrate cleavage by the Neurospora Varkud satellite (VS) ribozyme involves a structural change in the stem-loop I substrate from an inactive to an active conformation. We have determined the NMR solution structure of a mutant stem-loop I that mimics the active conformation of the cleavage site internal loop. This structure shares many similarities, but also significant differences, with the previously determined structures of the inactive internal loop. The active internal loop displays different base-pairing interactions and forms a novel RNA fold composed exclusively of sheared G-A base pairs. From chemical-shift mapping we identified two Mg2+ binding sites in the active internal loop. One of the Mg2+ binding sites forms in the active but not the inactive conformation of the internal loop and is likely important for catalysis. Using the structure comparison program mc-search, we identified the active internal loop fold in other RNA structures. In Thermus thermophilus 16S rRNA, this RNA fold is directly involved in a long-range tertiary interaction. An analogous tertiary interaction may form between the active internal loop of the substrate and the catalytic domain of the VS ribozyme. The combination of NMR and bioinformatic approaches presented here has identified a novel RNA fold and provides insights into the structural basis of catalytic function in the Neurospora VS ribozyme. PMID:12782785

  11. Conformational Stability and Catalytic Activity of PTEN Variants Linked to Cancers and Autism Spectrum Disorders

    PubMed Central

    Johnston, Sean B.; Raines, Ronald T.

    2015-01-01

    Phosphoinositides are membrane components that play critical regulatory roles in mammalian cells. The enzyme PTEN, which catalyzes the dephosphorylation of the phosphoinositide PIP3, is damaged in most sporadic tumors. Mutations in the PTEN gene have also been linked to autism spectrum disorders and other forms of delayed development. Here, human PTEN is shown to be on the cusp of unfolding under physiological conditions. Variants of human PTEN linked to somatic cancers and disorders on the autism spectrum are shown to be impaired in their conformational stability, catalytic activity, or both. Those variants linked only to autism have higher activity than those linked to cancers. PTEN-L, which is a secreted trans-active isoform, has greater conformational stability than does the wild-type enzyme. These data indicate that PTEN is a fragile enzyme cast in a crucial role in cellular metabolism, and suggest that PTEN-L is a repository for a critical catalytic activity. PMID:25647146

  12. Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

    PubMed Central

    Yanamala, Naveena; Tirupula, Kalyan C; Klein-Seetharaman, Judith

    2008-01-01

    Metabotropic glutamate receptors (mGluRs) are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects – enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%), the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%), the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86%) and 8/9 (89%) for ArgusLab and 10/14 (71%) and 7/9 (78%) for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by photochemical isomerization

  13. Conformational variability of the glycine receptor M2 domain in response to activation by different agonists.

    PubMed

    Pless, Stephan A; Dibas, Mohammed I; Lester, Henry A; Lynch, Joseph W

    2007-12-01

    Models describing the structural changes mediating Cys loop receptor activation generally give little attention to the possibility that different agonists may promote activation via distinct M2 pore-lining domain structural rearrangements. We investigated this question by comparing the effects of different ligands on the conformation of the external portion of the homomeric alpha1 glycine receptor M2 domain. Conformational flexibility was assessed by tethering a rhodamine fluorophore to cysteines introduced at the 19' or 22' positions and monitoring fluorescence and current changes during channel activation. During glycine activation, fluorescence of the label attached to R19'C increased by approximately 20%, and the emission peak shifted to lower wavelengths, consistent with a more hydrophobic fluorophore environment. In contrast, ivermectin activated the receptors without producing a fluorescence change. Although taurine and beta-alanine were weak partial agonists at the alpha1R19'C glycine receptor, they induced large fluorescence changes. Propofol, which drastically enhanced these currents, did not induce a glycine-like blue shift in the spectral emission peak. The inhibitors strychnine and picrotoxin elicited fluorescence and current changes as expected for a competitive antagonist and an open channel blocker, respectively. Glycine and taurine (or beta-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to the nearby L22'C residue. Thus, results from two separate labeled residues support the conclusion that the glycine receptor M2 domain responds with distinct conformational changes to activation by different agonists. PMID:17911099

  14. Non-enzymatic Glycation of Almond Cystatin Leads to Conformational Changes and Altered Activity.

    PubMed

    Siddiqui, Azad A; Sohail, Aamir; Bhat, Sheraz A; Rehman, Md T; Bano, Bilqees

    2015-01-01

    The non-enzymatic reaction between proteins and reducing sugars, known as glycation, leads to the formation of inter and intramolecular cross-links of proteins. Stable end products called as advanced Maillard products or advanced glycation end products (AGEs) have received tremendous attention since last decades. It was suggested that the formation of AGEs not only modify the conformation of proteins but also induces altered biological activity. In this study, cystatin purified from almond was incubated with three different sugars namely D-ribose, fructose and lactose to monitor the glycation process. Structural changes induced in cystatin on glycation were studied using UV-visible spectroscopy, fluorescence spectroscopy, CD and FTIR techniques. Glycated cystatin was found to migrate slower on electrophoresis as compared to control cystatin. Biological activity data of glycated cystatin showed that D-ribose was most effective in inducing conformational changes with maximum altered activity.

  15. Small-angle neutron and X-ray scattering reveal conformational changes in rhodopsin activation

    NASA Astrophysics Data System (ADS)

    Shrestha, Utsab R.; Bhowmik, Debsindhu; Perera, Suchitrhanga M. C. D.; Chawla, Udeep; Struts, Andrey V.; Graziono, Vito; Pingali, Sai Venkatesh; Heller, William T.; Qian, Shuo; Brown, Michael F.; Chu, Xiang-Qiang

    2015-03-01

    Understanding G-protein-coupled receptor (GPCR) activation plays a crucial role in the development of novel improved molecular drugs. During photo-activation, the retinal chromophore of the visual GPCR rhodopsin isomerizes from 11-cis to all-trans conformation, yielding an equilibrium between inactive Meta-I and active Meta-II states. The principal goals of this work are to address whether the activation of rhodopsin leads to a single state or a conformational ensemble, and how protein organizational structure changes with detergent environment in solution. We use both small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) techniques to answer the above questions. For the first time we observe the change in protein conformational ensemble upon photo-activation by SANS with contrast variation, which enables the separate study of the protein structure within the detergent assembly. In addition, SAXS study of protein structure within detergent assembly suggests that the detergent molecules form a belt of monolayer (micelle) around protein with different geometrical shapes to keep the protein in folded state.

  16. Branched conformational properties of macromolecules in close relation to chemical synthesis. II. Influence of excluded volume interactions.

    PubMed

    Burchard, Walther; Schweins, Ralf; Werner, Marcel

    2015-09-21

    The description of perturbed particle conformations needs as a prerequisite the algorithm of unperturbed chains which is outlined in Paper I [J. Chem. Phys. 143, 114906 (2015)]. The mean square segment length ⟨r(2)(n)⟩=b(2)n(2ν) with ν = 0.588 for linear chains in a good solvent is used as an approximation also for branched samples. The mean square radius of gyration is easily derived, but for the hydrodynamic, the segment distribution by Domb et al. [Proc. Phys. Soc., London 85, 624 (1965)] is required. Both radii can analytically be expressed by Gamma functions. For the angular dependence of scattered light, the Fourier transform of the Domb distribution for self-avoiding random walk is needed, which cannot be obtained as an analytical function and was derived by numerical integration. The summation over all segment length in the particle was performed with an analytic fit-curve for the Fourier transform and was carried out numerically. Results were derived (i) for uniform and polydisperse linear chains, (ii) or f-functional randomly branched polymers and their monodisperse fractions, (iii) for random A3B2 co-polymers, and (iv) for AB2 hyper-branched samples. The deviation of the Gaussian approximation with the variance of ⟨r(2)(n)⟩=b(2)n(2ν) slightly overestimates the excluded volume interaction but still remains a fairly good approximation for region of qR(g) < 10.

  17. New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined H/D Exchange Approach and Molecular Dynamics Simulations

    NASA Astrophysics Data System (ADS)

    Kazazić, Saša; Bertoša, Branimir; Luić, Marija; Mikleušević, Goran; Tarnowski, Krzysztof; Dadlez, Michal; Narczyk, Marta; Bzowska, Agnieszka

    2016-01-01

    The biologically active form of purine nucleoside phosphorylase (PNP) from Escherichia coli (EC 2.4.2.1) is a homohexamer unit, assembled as a trimer of dimers. Upon binding of phosphate, neighboring monomers adopt different active site conformations, described as open and closed. To get insight into the functions of the two distinctive active site conformations, virtually inactive Arg24Ala mutant is complexed with phosphate; all active sites are found to be in the open conformation. To understand how the sites of neighboring monomers communicate with each other, we have combined H/D exchange (H/DX) experiments with molecular dynamics (MD) simulations. Both methods point to the mobility of the enzyme, associated with a few flexible regions situated at the surface and within the dimer interface. Although H/DX provides an average extent of deuterium uptake for all six hexamer active sites, it was able to indicate the dynamic mechanism of cross-talk between monomers, allostery. Using this technique, it was found that phosphate binding to the wild type (WT) causes arrest of the molecular motion in backbone fragments that are flexible in a ligand-free state. This was not the case for the Arg24Ala mutant. Upon nucleoside substrate/inhibitor binding, some release of the phosphate-induced arrest is observed for the WT, whereas the opposite effects occur for the Arg24Ala mutant. MD simulations confirmed that phosphate is bound tightly in the closed active sites of the WT; conversely, in the open conformation of the active site of the WT phosphate is bound loosely moving towards the exit of the active site. In Arg24Ala mutant binary complex Pi is bound loosely, too.

  18. A conformational switch in the active site of BT_2972, a methyltransferase from an antibiotic resistant pathogen B. thetaiotaomicron.

    PubMed

    Kumar, Veerendra; Sivaraman, J

    2011-01-01

    Methylation is one of the most common biochemical reactions involved in cellular and metabolic functions and is catalysed by the action of methyltransferases. Bacteroides thetaiotaomicron is an antibiotic-resistant bacterium that confers resistance through methylation, and as yet, there is no report on the structure of methyltransferases from this bacterium. Here, we report the crystal structure of an AdoMet-dependent methyltransferase, BT_2972 and its complex with AdoMet and AdoHcy for B. thetaiotaomicron VPI-5482 strain along with isothermal titration calorimetric assessment of the binding affinities. Comparison of the apo and complexed BT_2972 structures reveals a significant conformational change between open and closed forms of the active site that presumably regulates the association with cofactors and may aid interaction with substrate. Together, our analysis suggests that BT_2972 is a small molecule methyltransferase and might catalyze two O-methylation reaction steps involved in the ubiquinone biosynthesis pathway. PMID:22140448

  19. A neglected modulator of insulin-degrading enzyme activity and conformation: The pH.

    PubMed

    Grasso, Giuseppe; Satriano, Cristina; Milardi, Danilo

    2015-01-01

    Insulin-degrading enzyme (IDE), a ubiquitously expressed zinc metalloprotease, has multiple activities in addition to insulin degradation and its malfunction is believed to connect type 2 diabetes with Alzheimer's disease. IDE has been found in many different cellular compartments, where it may experience significant physio-pathological pH variations. However, the exact role of pH variations on the interplay between enzyme conformations, stability, oligomerization state and catalysis is not understood. Here, we use ESI mass spectrometry, atomic force microscopy, surface plasmon resonance and circular dichroism to investigate the structure-activity relationship of IDE at different pH values. We show that acidic pH affects the ability of the enzyme to bind the substrate and decrease the stability of the protein by inducing an α-helical bundle conformation with a concomitant dissociation of multi-subunit IDE assemblies into monomeric units and loss of activity. These effects suggest a major role played by electrostatic forces in regulating multi-subunit enzyme assembly and function. Our results clearly indicate a pH dependent coupling among enzyme conformation, assembly and stability and suggest that cellular acidosis can have a large effect on IDE oligomerization state, inducing an enzyme inactivation and an altered insulin degradation that could have an impact on insulin signaling.

  20. Role of the C-terminus in the activity, conformation, and stability of interleukin-6.

    PubMed Central

    Ward, L. D.; Hammacher, A.; Zhang, J. G.; Weinstock, J.; Yasukawa, K.; Morton, C. J.; Norton, R. S.; Simpson, R. J.

    1993-01-01

    Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was < 0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule. PMID:8401231

  1. Conformation-activity relationship of sweet molecules. Comparison of aspartame and naphthimidazolesulfonic acids.

    PubMed

    Castiglione-Morelli, M A; Lelj, F; Naider, F; Tallon, M; Tancredi, T; Temussi, P A

    1990-02-01

    The shape of the active site of the receptor for sweet molecules was previously defined on the basis of a combination of both rigid (saccharins) and flexible (aspartame) molds. In this paper, the sweetness receptor is refined with use of the shapes of 3-anilino-2-styryl-3H-naphtho[1,2-d]imidazolesulfonate (sweet) and of 3-anilino-2-phenyl-3H-naphtho[1,2-d]imidazolesulfonate (tasteless), two large and almost completely rigid tastants. The minimum-energy conformations of the flexible portions of these tastants have been determined by using a detailed conformational analysis based on ab initio calculations. The refined receptor site is still consistent with all previously examined sweet molecules. In order to unequivocally assign the prochiral beta-CH2 protons of the Phe moiety of aspartame, (2S,3S)-[2H]-alpha-L-Asp-L-PheOMe was synthesized and examined by 500-MHz 1H NMR spectroscopy. The results indicate that the minimum-energy conformation for aspartame in water, DMSO-d6, and CDCl3 (as a crown ether complex) is different from that originally proposed (FIIDII instead of FIDII, according to a notation referred to the side chains). Although this conformation is not directly consistent with the shape of the sweet receptor, the interconversion of FIIDII to FIDII was found to require only 1 kcal/mol. Furthermore, a 120-ps molecular dynamics simulation in vacuo confirms the high flexibility of aspartame and the accessibility of the FIDII conformer whose topology is fully consistent with our model. PMID:2299622

  2. Conformation-activity relationship of sweet molecules. Comparison of aspartame and naphthimidazolesulfonic acids.

    PubMed

    Castiglione-Morelli, M A; Lelj, F; Naider, F; Tallon, M; Tancredi, T; Temussi, P A

    1990-02-01

    The shape of the active site of the receptor for sweet molecules was previously defined on the basis of a combination of both rigid (saccharins) and flexible (aspartame) molds. In this paper, the sweetness receptor is refined with use of the shapes of 3-anilino-2-styryl-3H-naphtho[1,2-d]imidazolesulfonate (sweet) and of 3-anilino-2-phenyl-3H-naphtho[1,2-d]imidazolesulfonate (tasteless), two large and almost completely rigid tastants. The minimum-energy conformations of the flexible portions of these tastants have been determined by using a detailed conformational analysis based on ab initio calculations. The refined receptor site is still consistent with all previously examined sweet molecules. In order to unequivocally assign the prochiral beta-CH2 protons of the Phe moiety of aspartame, (2S,3S)-[2H]-alpha-L-Asp-L-PheOMe was synthesized and examined by 500-MHz 1H NMR spectroscopy. The results indicate that the minimum-energy conformation for aspartame in water, DMSO-d6, and CDCl3 (as a crown ether complex) is different from that originally proposed (FIIDII instead of FIDII, according to a notation referred to the side chains). Although this conformation is not directly consistent with the shape of the sweet receptor, the interconversion of FIIDII to FIDII was found to require only 1 kcal/mol. Furthermore, a 120-ps molecular dynamics simulation in vacuo confirms the high flexibility of aspartame and the accessibility of the FIDII conformer whose topology is fully consistent with our model.

  3. Modulation of plasminogen activator inhibitor 1 by Triton X-100--identification of two consecutive conformational transitions.

    PubMed

    Gils, A; Declerck, P J

    1998-08-01

    Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily because of its conformational and functional flexibility. In the present study, we have evaluated the influence of the non-ionic detergent Triton X-100 (TX-100) on the functional stability and conformational transitions of PAI-1. At 37 degrees C, TX-100 induced a concentration-dependent decrease of the functional half-life of PAI-1 resulting in half-lives of 177 +/- 54 min (mean +/- SD, n = 3), 19 +/- 2 min, 1.7 +/- 0.3 min and 0.53 +/- 0.03 min in the presence of 0.005, 0.010, 0.020 and 0.2% TX-100, respectively, compared to a half-life of 270 +/- 146 min in the absence of TX-100. Conformational analysis at various time points and at different temperatures (0 degrees C, 25 degrees C, 37 degrees C) revealed that this inactivation proceeds through the formation of a substrate-like intermediate followed by the formation of the latent form. Kinetic evaluation demonstrated that this conversion fits to two consecutive first-order transitions, i.e. active k1--> substrate k2--> latent. The k1 value was strongly dependent on the concentration of TX-100 (e.g. 0.002 +/- 0.0006 s(-1) and 0.029 +/- 0.003 s(-1) for 0.01% and 0.2% TX-100 at 37 degrees C) whereas the conversion of substrate to latent (k2) was virtually independent of the TX-100 concentration (i.e. 0.012 +/- 0.002 s(-1) and 0.011 +/- 0.001 s(-1) for 0.01 and 0.2% TX-100 at 37 degrees C). Experiments with a variety of other non-ionic amphiphilic compounds revealed that the amphiphilic character of the compound is, at least in part, responsible for the observed effects and strongly indicate that the currently reported mechanism of inactivation is of general importance for the conformational transitions in PAI-1. In conclusion, TX- 100 changes the initial conformation of PAI-1 resulting in altered functional properties. This observation allows us to develop a new model for the mechanism involved in the conformational flexibility of

  4. Conformational Transitions in Human AP Endonuclease 1 and Its Active Site Mutant during Abasic Site Repair†

    PubMed Central

    Kanazhevskaya, Lyubov Yu.; Koval, Vladimir V.; Zharkov, Dmitry O.; Strauss, Phyllis R.; Fedorova, Olga S.

    2010-01-01

    AP endonuclease 1 (APE 1) is a crucial enzyme of the base excision repair pathway (BER) in human cells. APE1 recognizes apurinic/apyrimidinic (AP) sites and makes a nick in the phosphodiester backbone 5′ to them. The conformational dynamics and presteady-state kinetics of wild-type APE1 and its active site mutant, Y171F-P173L-N174K, have been studied. To observe conformational transitions occurring in the APE1 molecule during the catalytic cycle, we detected intrinsic tryptophan fluorescence of the enzyme under single turnover conditions. DNA duplexes containing a natural AP site, its tetrahydrofuran analogue, or a 2′-deoxyguanosine residue in the same position were used as specific substrates or ligands. The stopped-flow experiments have revealed high flexibility of the APE1 molecule and the complexity of the catalytic process. The fluorescent traces indicate that wild-type APE1 undergoes at least four conformational transitions during the processing of abasic sites in DNA. In contrast, nonspecific interactions of APE1 with undamaged DNA can be described by a two-step kinetic scheme. Rate and equilibrium constants were extracted from the stopped-flow and fluorescence titration data for all substrates, ligands, and products. A replacement of three residues at the enzymatic active site including the replacement of tyrosine 171 with phenylalanine in the enzyme active site resulted in a 2 × 104-fold decrease in the reaction rate and reduced binding affinity. Our data indicate the important role of conformational changes in APE1 for substrate recognition and catalysis. PMID:20575528

  5. Structure of a Specialized Acyl Carrier Protein Essential for Lipid A Biosynthesis with Very Long-chain Fatty Acids in Open and Closed Conformations

    SciTech Connect

    Ramelot, Theresa A.; Rossi, Paolo M.; Forouhar, Farhad; Lee, Hsiau-Wei; Yang, Yunhuang; Ni, Shuisong; Unser, Sarah; Lew, Scott; Seetharaman, Jayaraman; Xiao, Rong; Acton, Thomas; Everett, John K.; Prestegard, James H.; Hunt, John F.; Montelione, Gaetano; Kennedy, Michael A.

    2012-09-18

    The solution nuclear magnetic resonance (NMR) structures and backbone (15)N dynamics of the specialized acyl carrier protein (ACP), RpAcpXL, from Rhodopseudomonas palustris, in both the apo form and holo form modified by covalent attachment of 4'-phosphopantetheine at S37, are virtually identical, monomeric, and correspond to the closed conformation. The structures have an extra α-helix compared to the archetypical ACP from Escherichia coli, which has four helices, resulting in a larger opening to the hydrophobic cavity. Chemical shift differences between apo- and holo-RpAcpXL indicated some differences in the hinge region between α2 and α3 and in the hydrophobic cavity environment, but corresponding changes in nuclear Overhauser effect cross-peak patterns were not detected. In contrast to the NMR structures, apo-RpAcpXL was observed in an open conformation in crystals that diffracted to 2.0 Å resolution, which resulted from movement of α3. On the basis of the crystal structure, the predicted biological assembly is a homodimer. Although the possible biological significance of dimerization is unknown, there is potential that the resulting large shared hydrophobic cavity could accommodate the very long-chain fatty acid (28-30 carbons) that this specialized ACP is known to synthesize and transfer to lipid A. These structures are the first representatives of the AcpXL family and the first to indicate that dimerization may be important for the function of these specialized ACPs.

  6. Calcium Activation of the Ca-ATPase Enhances Conformational Heterogeneity Between Nucleotide Binding and Phosphorylation Domains

    SciTech Connect

    Chen, Baowei; Squier, Thomas C.; Bigelow, Diana J.

    2004-04-13

    High-resolution crystal structures obtained in two conformations of the Ca-ATPase suggest that a large-scale rigid-body domain reorientation of approximately 50 involving the nucleotide-binding (N) domain is required to permit the transfer of the -phosphoryl group of ATP to Asp351 in the phosphorylation (P) domain during coupled calcium transport. However, variability observed in the orientation of the N-domain relative to the P-domain in both different crystal structures of the Ca-ATPase following calcium activation, and structures of other P-type ATPases, suggests the presence of conformational heterogeneity in solution which may be modulated by contact interactions within the crystal. Therefore, to address the extent of conformational heterogeneity between these domains in solution, we have used fluorescence resonance energy transfer (FRET) to measure the spatial separation and conformational heterogeneity between donor (i.e., 5-[[2-[(iodoacetyl)amino]ethyl]amino] naphthalene-1-sulfonic acid) and acceptor (i.e., fluorescein 5-isothiocyanate) chromophores covalently bound to the P- and N-domains, respectively, within the Ca-ATPase stabilized in different enzymatic states associated with the transport cycle. In comparison to the unliganded enzyme, the spatial separation and conformational heterogeneity between these domains is unaffected by enzyme phosphorylation. However, calcium-activation results in a 3.4 increase in the average spatial separation, which increases from 29.4 to 32.8 , in good agreement with the high-resolution structures where these sites are respectively separated by 31.6 (1 IWO.pdb) and 35.9 (1EUL.pdb). Thus, the crystal structures accurately reflect the average solution structures of the Ca-ATPase. However, there is substantial conformational heterogeneity for all enzyme states measured, indicating that formation of catalytically important transition states involves a subpopulation of enzyme intermediates. These results suggest that the

  7. Synthesis and biological activity of conformationally restricted gypsy moth pheromone mimics.

    PubMed

    Chen, Hao; Gong, Yongmei; Gries, Regine M; Plettner, Erika

    2010-04-15

    The design and synthesis of a series of conformationally constrained mimics of gypsy moth sex pheromone, (+)-disparlure (7R,8S)-2-methyl-7,8-epoxyoctadecane, are described. The core structure of the mimics is derived from 5-(2'-hydroxyethyl)cyclopent-2-en-1-ol. Substituent optimization of the analogs was accomplished through the synthesis of mini-libraries and pure individual compounds, followed by electrophysiological experiments with male gypsy moth antennae. The electroantennogram results show that the analogs elicited weak to no antennal responses themselves. There was a clear structure-activity pattern for odorant activity, with ethyl substituents being best. Further, when puffed simultaneously with the pheromone, some of the compounds gave a significant enhancement of the antennal depolarization, indicating an additive or synergistic effect. A pure pheromone stimulus following a mixed compound/pheromone stimulus was generally not affected, with two exceptions: one compound enhanced and another inhibited a subsequent stimulus. The compounds also prolonged the stimulation of the antenna, which manifested itself in widened electroantennogram peaks. We tested the hypothesis that this prolonged stimulation may be due to the stabilization of a particular conformer of the pheromone-binding protein (PBP). Compounds that caused PBP2 to adopt a similar conformation than in the presence of pheromone also caused peak widening. This was not the case with PBP1.

  8. Salivary histatin 5: dependence of sequence, chain length, and helical conformation for candidacidal activity.

    PubMed

    Raj, P A; Edgerton, M; Levine, M J

    1990-03-01

    Histatin 5 (Asp1-Ser-His-Ala4-Lys-Arg-His-His8-Gly-Tyr-Lys-Arg12-Lys-Ph e-His-Glu16-Lys-His - His-Ser20-His-Arg-Gly-Tyr24), one of the basic histidine-rich peptides present in human parotid saliva and several of its fragments, 1-16 (N16), 9-24 (C16), 11-24 (C14), 13-24 (C12), 15-24 (C10), and 7-16 (M10), were synthesized by solid-phase procedures. Native histatin 5 from human parotid saliva was also purified. Their antifungal activities on two strains of Candida albicans have been studied and their conformational preferences both in aqueous and non-aqueous solutions examined by circular dichroism. The synthetic histatin 5, C16, and C14 peptides were highly active and inhibited the growth of C. albicans. The candidacidal activity data of synthetic histatin 5 were comparable to the values of the native histatin 5 isolated from parotid saliva and those reported previously, although the assay system used and the strains examined were different. The C16 fragment was as active as the whole peptide itself, whereas the N16 fragment was far less active than C14, suggesting that the sequence at the C-terminal is important for its fungicidal activity. An increase in the chain length of the C-terminal sequence from 12 to 16 residues increased the candidacidal activity, thereby indicating that a peptide chain length of at least 12 residues is necessary to elicit optimum biological activity. The CD spectra of these linear peptides showed that they are structurally more flexible, and they adopt different conformations depending on the solvent environment. CD studies provided evidence that histatin 5 and the longer fragments, C16, N16, and C14 preferred alpha-helical conformations in non-aqueous solvents such as trifluoroethanol and methanol, while in water and pH 7.4 phosphate buffers, they favored random coil structures. The shorter sequences seemed to adopt either turn structures or unordered structures both in aqueous and non-aqueous solutions. It appears that the sequence at

  9. Metals affect the structure and activity of human plasminogen activator inhibitor-1. II. Binding affinity and conformational changes

    PubMed Central

    Thompson, Lawrence C; Goswami, Sumit; Peterson, Cynthia B

    2011-01-01

    Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. The lifespan of the active form of PAI-1 is modulated via interaction with the plasma protein, vitronectin, and various metal ions. These metal ions fall into two categories: Type I metals, including calcium, magnesium, and manganese, stabilize PAI-1 in the absence of vitronectin, whereas Type II metals, including cobalt, copper, and nickel, destabilize PAI-1 in the absence of vitronectin, but stabilize PAI-1 in its presence. To provide a mechanistic basis for understanding the unusual modulation of PAI-1 structure and activity, the binding characteristics and conformational effects of these two types of metals were further evaluated. Steady-state binding measurements using surface plasmon resonance indicated that both active and latent PAI-1 exhibit a dissociation constant in the low micromolar range for binding to immobilized nickel. Stopped-flow measurements of approach-to-equilibrium changes in intrinsic protein fluorescence indicated that the Type I and Type II metals bind in different modes that induce distinct conformational effects on PAI-1. Changes in the observed rate constants with varying concentrations of metal allowed accurate determination of binding affinities for cobalt, nickel, and copper, yielding dissociation constants of ∼40, 30, and 0.09 μM, respectively. Competition experiments that tested effects on PAI-1 stability were consistent with these measurements of affinity and indicate that copper binds tightly to PAI-1. PMID:21280128

  10. Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects.

    PubMed

    Yang, Gloria; Hong, Nansook; Baier, Florian; Jackson, Colin J; Tokuriki, Nobuhiko

    2016-08-16

    How remote mutations can lead to changes in enzyme function at a molecular level is a central question in evolutionary biochemistry and biophysics. Here, we combine laboratory evolution with biochemical, structural, genetic, and computational analysis to dissect the molecular basis for the functional optimization of phosphotriesterase activity in a bacterial lactonase (AiiA) from the metallo-β-lactamase (MBL) superfamily. We show that a 1000-fold increase in phosphotriesterase activity is caused by a more favorable catalytic binding position of the paraoxon substrate in the evolved enzyme that resulted from conformational tinkering of the active site through peripheral mutations. A nonmutated active site residue, Phe68, was displaced by ∼3 Å through the indirect effects of two second-shell trajectory mutations, allowing molecular interactions between the residue and paraoxon. Comparative mutational scanning, i.e., examining the effects of alanine mutagenesis on different genetic backgrounds, revealed significant changes in the functional roles of Phe68 and other nonmutated active site residues caused by the indirect effects of trajectory mutations. Our work provides a quantitative measurement of the impact of second-shell mutations on the catalytic contributions of nonmutated residues and unveils the underlying intramolecular network of strong epistatic mutational relationships between active site residues and more remote residues. Defining these long-range conformational and functional epistatic relationships has allowed us to better understand the subtle, but cumulatively significant, role of second- and third-shell mutations in evolution.

  11. Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects.

    PubMed

    Yang, Gloria; Hong, Nansook; Baier, Florian; Jackson, Colin J; Tokuriki, Nobuhiko

    2016-08-16

    How remote mutations can lead to changes in enzyme function at a molecular level is a central question in evolutionary biochemistry and biophysics. Here, we combine laboratory evolution with biochemical, structural, genetic, and computational analysis to dissect the molecular basis for the functional optimization of phosphotriesterase activity in a bacterial lactonase (AiiA) from the metallo-β-lactamase (MBL) superfamily. We show that a 1000-fold increase in phosphotriesterase activity is caused by a more favorable catalytic binding position of the paraoxon substrate in the evolved enzyme that resulted from conformational tinkering of the active site through peripheral mutations. A nonmutated active site residue, Phe68, was displaced by ∼3 Å through the indirect effects of two second-shell trajectory mutations, allowing molecular interactions between the residue and paraoxon. Comparative mutational scanning, i.e., examining the effects of alanine mutagenesis on different genetic backgrounds, revealed significant changes in the functional roles of Phe68 and other nonmutated active site residues caused by the indirect effects of trajectory mutations. Our work provides a quantitative measurement of the impact of second-shell mutations on the catalytic contributions of nonmutated residues and unveils the underlying intramolecular network of strong epistatic mutational relationships between active site residues and more remote residues. Defining these long-range conformational and functional epistatic relationships has allowed us to better understand the subtle, but cumulatively significant, role of second- and third-shell mutations in evolution. PMID:27444875

  12. Conformal optical elements for correcting wavefront distortions in YAG : Nd{sup 3+} active elements

    SciTech Connect

    Korolkov, V P; Nasyrov, R K; Poleshchuk, A G; Arapov, Yu D; Ivanov, A F

    2013-02-28

    Correction of the wavefront is studied for the light beam passing wide-aperture YAG : Nd3+ single-crystal rods, which are used as active elements in high-power solid-state lasers. A nonideal character of the crystal structure is responsible for the deformation of the wavefront of passing radiation. By using the halftone technology we have developed conformal aberration correctors capable of compensating rod nonuniformities and reducing the laser radiation divergence by an order of magnitude. The results obtained make it possible to employ optically nonuniform active elements in laser constructions. (laser optics 2012)

  13. Magnesium impacts myosin V motor activity by altering key conformational changes in the mechanochemical cycle.

    PubMed

    Trivedi, Darshan V; Muretta, Joseph M; Swenson, Anja M; Thomas, David D; Yengo, Christopher M

    2013-07-01

    We investigated how magnesium (Mg) impacts key conformational changes during the ADP binding/release steps in myosin V and how these alterations impact the actomyosin mechanochemical cycle. The conformation of the nucleotide binding pocket was examined with our established FRET system in which myosin V labeled with FlAsH in the upper 50 kDa domain participates in energy transfer with mant labeled nucleotides. We examined the maximum actin-activated ATPase activity of MV FlAsH at a range of free Mg concentrations (0.1-9 mM) and found that the highest activity occurs at low Mg (0.1-0.3 mM), while there is a 50-60% reduction in activity at high Mg (3-9 mM). The motor activity examined with the in vitro motility assay followed a similar Mg-dependence, and the trend was similar with dimeric myosin V. Transient kinetic FRET studies of mantdADP binding/release from actomyosin V FlAsH demonstrate that the transition between the weak and strong actomyosin.ADP states is coupled to movement of the upper 50 kDa domain and is dependent on Mg with the strong state stabilized by Mg. We find that the kinetics of the upper 50 kDa conformational change monitored by FRET correlates well with the ATPase and motility results over a wide range of Mg concentrations. Our results suggest the conformation of the upper 50 kDa domain is highly dynamic in the Mg free actomyosin.ADP state, which is in agreement with ADP binding being entropy driven in the absence of Mg. Overall, our results demonstrate that Mg is a key factor in coupling the nucleotide- and actin-binding regions. In addition, Mg concentrations in the physiological range can alter the structural transition that limits ADP dissociation from actomyosin V, which explains the impact of Mg on actin-activated ATPase activity and in vitro motility.

  14. Synthetic peptide models for the redox-active disulfide loop of glutaredoxin. Conformational studies

    SciTech Connect

    Kishore, R.; Raghothama, S.; Balaram, P.

    1988-04-05

    Two cyclic peptide disulfides have been synthesized as models for the 14-membered redox-active disulfide loop glutaredoxin. /sup 1/H NMR studies at 270 MHz in chloroform solutions establish a type I ..beta..-turn conformation for the Pro-X segment in both peptides, stabilized by a 4 ..-->.. 1 hydrogen bond between the Cys(1) CO and Cys(4) NH groups. Nuclear Overhauser effects establish that the aromatic ring in the X = Phe peptide is oriented over the central peptide unit. In dimethyl sulfoxide solutions two conformational species are observed in slow exchange on the NMR time scale, for both peptides. These are assigned to type I and type II ..beta..-turn structures with -Pro-Tyr(Phe)-as the corner resides. The structural assignments are based on correlation of NMR parameters with model 14-membered cyclic cystine peptides with Pro-X spacers. Circular dichroism studies based on the -S-S-n-sigma* transition suggest a structural change in the disulfide bridge with changing solvent polarity, establishing conformational coupling between the peptide backbone and the disulfide linkage in these systems.

  15. Search for Active-State Conformation of Drug Target GPCR Using Real-Coded Genetic Algorithm

    NASA Astrophysics Data System (ADS)

    Ishino, Yoko; Harada, Takanori; Aida, Misako

    G-Protein coupled receptors (GPCRs) comprise a large superfamily of proteins and are a target for nearly 50% of drugs in clinical use today. GPCRs have a unique structural motif, seven transmembrane helices, and it is known that agonists and antagonists dock with a GPCR in its ``active'' and ``inactive'' condition, respectively. Knowing conformations of both states is eagerly anticipated for elucidation of drug action mechanism. Since GPCRs are difficult to crystallize, the 3D structures of these receptors have not yet been determined by X-ray crystallography, except the inactive-state conformation of two proteins. The conformation of them enabled the inactive form of other GPCRs to be modeled by computer-aided homology modeling. However, to date, the active form of GPCRs has not been solved. This paper describes a novel method to predict the 3D structure of an active-state GPCR aiming at molecular docking-based virtual screening using real-coded genetic algorithm (real-coded GA), receptor-ligand docking simulations, and molecular dynamics (MD) simulations. The basic idea of the method is that the MD is first used to calculate an average 3D coordinates of all atoms of a GPCR protein against heat fluctuation on the pico- or nano- second time scale, and then real-coded GA involving receptor-ligand docking simulations functions to determine the rotation angle of each helix as a movement on wider time scale. The method was validated using human leukotriene B4 receptor BLT1 as a sample GPCR. Our study demonstrated that the established evolutionary search for the active state of the leukotriene receptor provided the appropriate 3D structure of the receptor to dock with its agonists.

  16. A Two-Metal-Ion-Mediated Conformational Switching Pathway for HDV Ribozyme Activation

    PubMed Central

    Lee, Tai-Sung; Radak, Brian K.; Harris, Michael E.; York, Darrin M.

    2016-01-01

    RNA enzymes serve as a potentially powerful platform from which to design catalysts and engineer new biotechnology. A fundamental understanding of these systems provides insight to guide design. The hepatitis delta virus ribozyme (HDVr) is a small, self-cleaving RNA motif widely distributed in nature, that has served as a paradigm for understanding basic principles of RNA catalysis. Nevertheless, questions remain regarding the precise roles of divalent metal ions and key nucleotides in catalysis. In an effort to establish a reaction mechanism model consistent with available experimental data, we utilize molecular dynamics simulations to explore different conformations and metal ion binding modes along the HDVr reaction path. Building upon recent crystallographic data, our results provide a dynamic model of the HDVr reaction mechanism involving a conformational switch between multiple non-canonical G25:U20 base pair conformations in the active site. These local nucleobase dynamics play an important role in catalysis by modulating the metal binding environments of two Mg2+ ions that support catalysis at different steps of the reaction pathway. The first ion plays a structural role by inducing a base pair flip necessary to obtain the catalytic fold in which C75 moves towards to the scissile phosphate in the active site. Ejection of this ion then permits a second ion to bind elsewhere in the active site and facilitate nucleophile activation. The simulations collectively describe a mechanistic scenario that is consistent with currently available experimental data from crystallography, phosphorothioate substitutions, and chemical probing studies. Avenues for further experimental verification are suggested. PMID:27774349

  17. Distinct Roles of the Active-site Mg2+ Ligands, Asp882 and Asp705, of DNA Polymerase I (Klenow Fragment) during the Prechemistry Conformational Transitions*

    PubMed Central

    Bermek, Oya; Grindley, Nigel D. F.; Joyce, Catherine M.

    2011-01-01

    DNA polymerases catalyze the incorporation of deoxynucleoside triphosphates into a growing DNA chain using a pair of Mg2+ ions, coordinated at the active site by two invariant aspartates, whose removal by mutation typically reduces the polymerase activity to barely detectable levels. Using two stopped-flow fluorescence assays that we developed previously, we have investigated the role of the carboxylate ligands, Asp705 and Asp882, of DNA polymerase I (Klenow fragment) in the early prechemistry steps that prepare the active site for catalysis. We find that neither carboxylate is required for an early conformational transition, reported by a 2-aminopurine probe, that takes place in the open ternary complex after binding of the complementary dNTP. However, the subsequent fingers-closing step requires Asp882; this step converts the open ternary complex into the closed conformation, creating the active-site geometry required for catalysis. Crystal structures indicate that the Asp882 position changes very little during fingers-closing; this side chain may therefore serve as an anchor point to receive the dNTP-associated metal ion as the nucleotide is delivered into the active site. The Asp705 carboxylate is not required until after the fingers-closing step, and we suggest that its role is to facilitate the entry of the second Mg2+ into the active site. The two early prechemistry steps that we have studied take place normally at very low Mg2+ concentrations, although higher concentrations are needed for covalent nucleotide addition, consistent with the second metal ion entering the ternary complex after fingers-closing. PMID:21084297

  18. A Summary of Closed Brayton Cycle Development Activities at NASA

    NASA Technical Reports Server (NTRS)

    Mason, Lee S.

    2009-01-01

    NASA has been involved in the development of Closed Brayton Cycle (CBC) power conversion technology since the 1960's. CBC systems can be coupled to reactor, isotope, or solar heat sources and offer the potential for high efficiency, long life, and scalability to high power. In the 1960's and 1970's, NASA and industry developed the 10 kW Brayton Rotating Unit (BRU) and the 2 kW mini-BRU demonstrating technical feasibility and performance, In the 1980's, a 25 kW CBC Solar Dynamic (SD) power system option was developed for Space Station Freedom and the technology was demonstrated in the 1990's as part of the 2 kW SO Ground Test Demonstration (GTD). Since the early 2000's, NASA has been pursuing CBC technology for space reactor applications. Before it was cancelled, the Jupiter Icy Moons Orbiter (HMO) mission was considering a 100 kWclass CBC system coupled to a gas-cooled fission reactor. Currently, CBC technology is being explored for Fission Surface Power (FSP) systems to provide base power on the moon and Mars. These recent activities have resulted in several CBC-related technology development projects including a 50 kW Alternator Test Unit, a 20 kW Dual Brayton Test Loop, a 2 kW Direct Drive Gas Brayton Test Loop, and a 12 kW FSP Power Conversion Unit design.

  19. Antigenicity, catalytic activity and conformation of Agaricus bisporus tyrosinase: interaction of conformation-directed antibodies with the native and irradiated enzyme.

    PubMed

    Khan, I A; Ali, R

    1986-02-01

    The antiferromagnetically spin-coupled Cu2+ pair present in the active center of tyrosinase was found to be indispensable for its catalytic function. However, the metal ion did not contribute to the conformational integrity or antigenicity of the enzyme molecule. Irradiation of tyrosinase with 254 nm light resulted in dose dependent, essentially irreversible losses of its catalytic and antigenic functions. The apparent first order rate constants for the two processes were 17.6 X 10(-2) min-1 and 28.1 X 10(-2) min-1, respectively. The approximately 1.6-fold difference between the two rate constants suggests that the sites of antigenic determinants in tyrosinase are distinguishable from the enzymic active site by their higher photosensitivity. Kinetic analysis of the data as to photoinactivation, and the UV induced losses of antigenicity and structural integrity revealed that UV radiation disrupts the short-range noncovalent interactions occurring within the enzyme molecule. The disruption of the noncovalent interactions results in partial unfolding of the tyrosinase structure which in turn leads to the progressive loss of its catalytic activity and antigenicity. The anti-tyrosinase antibodies raised in rabbits were found to be directed against the native conformation of the enzyme. It is speculated that these antibodies might be useful in exploring the tyrosinase conformation and in studying the effects of various factors on the enzyme surface and molecular structure. PMID:3084463

  20. Novel analogs of alloferon: Synthesis, conformational studies, pro-apoptotic and antiviral activity.

    PubMed

    Kuczer, Mariola; Czarniewska, Elżbieta; Majewska, Anna; Różanowska, Maria; Rosiński, Grzegorz; Lisowski, Marek

    2016-06-01

    In this study, we report the structure-activity relationships of novel derivatives of the insect peptide alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH). The peptide structure was modified by exchanging His at position 9 or 12 for natural or non-natural amino acids. Biological properties of these peptides were determined in antiviral in vitro test against Human Herpes Virus 1 McIntrie strain (HHV-1MC) using a Vero cell line. The peptides were also evaluated for the pro-apoptotic action in vivo on hemocytes of the Tenebrio molitor beetle. Additionally, the structural properties of alloferon analogs were examined by the circular dichroism in water and methanol. It was found that most of the evaluated peptides can reduce the HHV-1 titer in Vero cells. [Ala(9)]-alloferon exhibits the strongest antiviral activity among the analyzed compounds. However, no cytotoxic activity against Vero cell line was observed for all the studied peptides. In vivo assays with hemocytes of T. molitor showed that [Lys(9)]-, [Phg(9)]-, [Lys(12)]-, and [Phe(12)]-alloferon exhibit a twofold increase in caspases activity in comparison with the native peptide. The CD conformational studies indicate that the investigated peptides seem to prefer the unordered conformation. PMID:26986636

  1. eIF4B, eIF4G and RNA regulate eIF4A activity in translation initiation by modulating the eIF4A conformational cycle.

    PubMed

    Harms, Ulf; Andreou, Alexandra Zoi; Gubaev, Airat; Klostermeier, Dagmar

    2014-07-01

    Eukaryotic translation initiation factor eIF4A is a DEAD-box helicase that resolves secondary structure elements in the 5'-UTR of mRNAs during ribosome scanning. Its RNA-stimulated ATPase and ATP-dependent helicase activities are enhanced by other translation initiation factors, but the underlying mechanisms are unclear. DEAD-box proteins alternate between open and closed conformations during RNA unwinding. The transition to the closed conformation is linked to duplex destabilization. eIF4A is a special DEAD-box protein that can adopt three different conformations, an open state in the absence of ligands, a half-open state stabilized by the translation initiation factor eIF4G and a closed state in the presence of eIF4G and eIF4B. We show here that eIF4A alone does not measurably sample the closed conformation. The translation initiation factors eIF4B and eIF4G accelerate the eIF4A conformational cycle. eIF4G increases the rate of closing more than the opening rate, and eIF4B selectively increases the closing rate. Strikingly, the rate constants and the effect of eIF4B are different for different RNAs, and are related to the presence of single-stranded regions. Modulating the kinetics of the eIF4A conformational cycle is thus central for the multi-layered regulation of its activity, and for its role as a regulatory hub in translation initiation. PMID:24848014

  2. Mutation in the SH1 helix reduces the activation energy of the ATP-induced conformational transition of myosin.

    PubMed

    Iwai, Sosuke; Chaen, Shigeru

    2007-05-25

    The SH1 helix is a joint that links the converter subdomain to the rest of the myosin motor domain. Recently, we showed that a mutation within the SH1 helix in Dictyostelium myosin II (R689H) reduced the elasticity and thermal stability of the protein. To reveal the involvement of the SH1 helix in ATP-dependent conformational changes of the motor domain, we have investigated the effects of the R689H mutation on the conformational changes of the converter, using a GFP-based fluorescence resonance energy transfer method. Although the mutation does not seem to strongly affect conformations, we found that it significantly reduced the activation energy required for the ATP-induced conformational transition corresponding to the recovery stroke. Given the effects of the mutation on the mechanical properties of myosin, we propose that the SH1 helix plays an important role in the mechanochemical energy conversion underlying the conformational change of the myosin motor domain.

  3. Mechanical Activation of a Multimeric Adhesive Protein Through Domain Conformational Change

    NASA Astrophysics Data System (ADS)

    Wijeratne, Sithara S.; Botello, Eric; Yeh, Hui-Chun; Zhou, Zhou; Bergeron, Angela L.; Frey, Eric W.; Patel, Jay M.; Nolasco, Leticia; Turner, Nancy A.; Moake, Joel L.; Dong, Jing-fei; Kiang, Ching-Hwa

    2013-03-01

    The mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers.

  4. Force Activation of a Multimeric Adhesive Protein through Domain Conformational Change

    NASA Astrophysics Data System (ADS)

    Wijeratne Sithara S

    The force-induced activation of adhesive proteins such as von Willebrand factor (VWF), which experience high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force induced functional change is manifested in the multimeric VWF's crucial role in blood coagulation, when high fluid shear stress activates pVWF multimers to bind platelets. Here we showed that a pathological level of high shear flow exposure of pVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of multimeric VWF. We found that shear-activated pVWF multimers (spVWF) are more resistant to mechanical unfolding than non-sheared pVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of pVWF multimers.

  5. Retinal conformation governs pKa of protonated Schiff base in rhodopsin activation.

    PubMed

    Zhu, Shengshuang; Brown, Michael F; Feller, Scott E

    2013-06-26

    We have explored the relationship between conformational energetics and the protonation state of the Schiff base in retinal, the covalently bound ligand responsible for activating the G protein-coupled receptor rhodopsin, using quantum chemical calculations. Guided by experimental structural determinations and large-scale molecular simulations on this system, we examined rotation about each bond in the retinal polyene chain, for both the protonated and deprotonated states that represent the dark and photoactivated states, respectively. Particular attention was paid to the torsional degrees of freedom that determine the shape of the molecule, and hence its interactions with the protein binding pocket. While most torsional degrees of freedom in retinal are characterized by large energetic barriers that minimize structural fluctuations under physiological temperatures, the C6-C7 dihedral defining the relative orientation of the β-ionone ring to the polyene chain has both modest barrier heights and a torsional energy surface that changes dramatically with protonation of the Schiff base. This surprising coupling between conformational degrees of freedom and protonation state is further quantified by calculations of the pKa as a function of the C6-C7 dihedral angle. Notably, pKa shifts of greater than two units arise from torsional fluctuations observed in molecular dynamics simulations of the full ligand-protein-membrane system. It follows that fluctuations in the protonation state of the Schiff base occur prior to forming the activated MII state. These new results shed light on important mechanistic aspects of retinal conformational changes that are involved in the activation of rhodopsin in the visual process.

  6. Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling.

    PubMed

    Taylor, John C; Markham, George D

    2003-07-15

    S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, methionine adenosyltransferase, a.k.a. MAT) is one of numerous enzymes that have a flexible polypeptide loop that moves to gate access to the active site in a motion that is closely coupled to catalysis. Crystallographic studies of this tetrameric enzyme have shown that the loop is closed in the absence of bound substrates. However, the loop must open to allow substrate binding and a variety of data indicate that the loop is closed during the catalytic steps. Previous kinetic studies indicate that during turnover loop motion occurs on a time scale of 10(-2)s, ca. 10-fold faster than chemical transformations and turnover. Site-directed spin labeling has been used to introduce nitroxide groups at two positions in the loop to illuminate how the motion of the loop is affected by substrate binding. The two loop mutants constructed, G105C and D107C, retain wild type levels of MAT activity; attachment of a methanethiosulfonate spin label to convert the cysteine to the "R1" residue reduced the k(cat) only for the labeled D107R1 form (7-fold). The K(m) value for methionine increased 2- to 4-fold for the cysteine mutants and 2- to 7-fold for the labeled proteins, whereas the K(m) for ATP was changed by at most 2-fold. EPR spectra for both labeled proteins are nearly identical and show the presence of two major spin label environments with rotational diffusion rates differing by approximately 10-fold; the slower rate is ca. 4-fold faster than the estimated protein rotational rate. The spectra are not altered by addition of substrates or products. At both positions the less mobile conformation constitutes ca. 65% of the total species, indicating an equilibrium that only slightly favors one form, that in which the label is more immobilized. The equilibrium constant that relates the two forms is comparable to the equilibrium constant of 1.5 for a conformational change that was previously deduced from the

  7. [Synthesis, conformation, and spectroscopy of nucleoside analogues concerning their antiviral activity].

    PubMed

    Kuśmierek, Jarosław T; Stolarski, Ryszard

    2015-01-01

    Chemically modified analogues of nucleosides and nucleotides, have been thoroughly investigated since the discovery of DNA double helix by Watson and Crick in 1953 (Nature 171: 737). Chemical structures, first of all tautomerism, of the nucleic acid bases, as well as the conformations of the nucleic acids constituents, determine the secondary and tertiary structures of DNA and RNA polymers. Similarly, structural and dynamic parameters of nucleoside derivatives determine their biological activity in mutagenesis, neoplastic transformation, as well as antiviral or anticancer properties. In this review, a multidisciplinary approach of Prof. David Shugar's group is presented in the studies on nucleosides and nucleotides. It consists in chemical syntheses of suitable analogues, measurements of physicochemical and spectral parameters, conformational analysis by means of nuclear magnetic resonance (NMR) and X-ray diffraction, as well as characteristics of the nucleoside analogues as inhibitors of some selected, target enzymes, crucial in respect to antiviral activity of the analogues. These long-lasting studies follows upon the line of the main paradigm of molecular biophysics, i. e. structure-activity relationship. PMID:26677575

  8. Escherichia coli purine repressor: key residues for the allosteric transition between active and inactive conformations and for interdomain signaling.

    PubMed

    Lu, F; Brennan, R G; Zalkin, H

    1998-11-10

    The Escherichia coli purine repressor, PurR, exists in an equilibrium between open and closed conformations. Binding of a corepressor, hypoxanthine or guanine, shifts the allosteric equilibrium in favor of the closed conformation and increases the operator DNA binding affinity by 40-fold compared to aporepressor. Glu70 and Trp147 PurR mutations were isolated which perturb the allosteric equilibrium. Three lines of evidence indicate that the allosteric equilibrium of E70A and W147A aporepressors was shifted toward the closed conformation. First, compared to wild-type PurR, these mutant repressors had a 10-30-fold higher corepressor binding affinity. Second, the mutant aporepressors bound to operator DNA with an affinity that is characteristic of the wild-type PurR holorepressor. Third, binding of guanine to wild-type PurR resulted in a near-UV circular dichroism spectral change at 297-305 nm that is attributed to the closed conformation. The circular dichroism spectrum of the E70A aporepressor at 297-305 nm was that expected for the closed conformation, and it was not appreciably altered by corepressor binding. Mutational analysis was used to identify an Arg115-Ser46' interdomain intersubunit hydrogen bond that is necessary for transmitting the allosteric transition in the corepressor binding domain to the DNA binding domain. R115A and S46G PurR mutants were defective in DNA binding in vitro and repressor function in vivo although corepressor binding was identical to the wild type. These results establish that the hydrogen bond between the side chain NH2 of Arg115 and the main chain CO of Ser46' plays a critical role in interdomain signaling.

  9. tBid Undergoes Multiple Conformational Changes at the Membrane Required for Bax Activation*

    PubMed Central

    Shamas-Din, Aisha; Bindner, Scott; Zhu, Weijia; Zaltsman, Yehudit; Campbell, Clinton; Gross, Atan; Leber, Brian; Andrews, David W.; Fradin, Cécile

    2013-01-01

    Bid is a Bcl-2 family protein that promotes apoptosis by activating Bax and eliciting mitochondrial outer membrane permeabilization (MOMP). Full-length Bid is cleaved in response to apoptotic stimuli into two fragments, p7 and tBid (p15), that are held together by strong hydrophobic interactions until the complex binds to membranes. The detailed mechanism(s) of fragment separation including tBid binding to membranes and release of the p7 fragment to the cytoplasm remain unclear. Using liposomes or isolated mitochondria with fluorescently labeled proteins at physiological concentrations as in vitro models, we report that the two components of the complex quickly separate upon interaction with a membrane. Once tBid binds to the membrane, it undergoes slow structural rearrangements that result in an equilibrium between two major tBid conformations on the membrane. The conformational change of tBid is a prerequisite for interaction with Bax and is, therefore, a novel step that can be modulated to promote or inhibit MOMP. Using automated high-throughput image analysis in cells, we show that down-regulation of Mtch2 causes a significant delay between tBid and Bax relocalization in cells. We propose that by promoting insertion of tBid via a conformational change at the mitochondrial outer membrane, Mtch2 accelerates tBid-mediated Bax activation and MOMP. Thus the interaction of Mtch2 and tBid is a potential target for therapeutic control of Bid initiated cell death. PMID:23744079

  10. The VPS-20 Subunit of the Endosomal Sorting Complex ESCRT-III Exhibits an Open Conformation in the Absence of Upstream Activation

    PubMed Central

    Schuh, Amber L.; Hanna, Michael; Quinney, Kyle; Wang, Lei; Sarkeshik, Ali; Yates, John R.; Audhya, Anjon

    2015-01-01

    Members of the endosomal sorting complex required for transport (ESCRT) machinery function in membrane remodeling processes during multivesicular endosome biogenesis, cytokinesis, retroviral budding, and plasma membrane repair. During lumenal vesicle formation at endosomes, the ESCRT-II complex and the ESCRT-III subunit VPS-20 play a specific role in regulating assembly of ESCRT-III filaments, which promote vesicle scission. Previous work suggests that Vps20 isoforms, like other ESCRT-III subunits, exhibits an autoinhibited, closed conformation in solution, and its activation depends on an association with ESCRT-II specifically at membranes. However, we show here that C. elegans ESCRT-II and VPS-20 interact directly in solution, both in cytosolic cell extracts and using recombinant proteins in vitro. Moreover, we demonstrate that purified VPS-20 exhibits an open, extended conformation, irrespective of ESCRT-II binding, in contrast with the closed, autoinhibited architecture of another ESCRT-III subunit, VPS-24. Our data argue that individual ESCRT-III subunits adopt distinct conformations, which are tailored for their specific functions during ESCRT-mediated membrane reorganization events. PMID:25588614

  11. Regioselective sulfation of Artemisia sphaerocephala polysaccharide: Solution conformation and antioxidant activities in vitro.

    PubMed

    Wang, Junlong; Yang, Wen; Tang, YinYing; Xu, Qing; Huang, Shengli; Yao, Jian; Zhang, Ji; Lei, Ziqiang

    2016-01-20

    Regioselective modification is an effective approach to synthesize polysaccharides with different structure features and improved properties. In this study, regioselective sulfation of Artemisia sphaerocephala polysaccharide (SRSASP) was prepared by using triphenylchloromethane (TrCl) as protecting precursor. The decrease in fractal dimension (df) values (1.56-2.04) of SRSASP was observed in size-exclusion chromatography combined with multi angle laser light scattering (SEC-MALLS) analysis. Compared to sample substituted at C-6, SRSASP showed a more expanded conformation of random coil, which was attributed to the breakup of hydrogen bonds and elastic contributions. Circular dichroism (CD), methylene blue (MB) and congo red (CR) spectrophotometric method and atomic force microscopy (AFM) results confirmed the conformational transition and stiffness of the chains after sulfation. SRSASP exhibited enhanced antioxidant activities in the DPPH, superoxide and hydroxyl radical scavenging assay. Sulfation at C-2 or C-3 was favorable for the chelation which might prevent the generation of hydroxyl radicals. It concluded that the degree of substitution and substitution position were the factors influencing biological activities of sulfated polysaccharides. PMID:26572384

  12. Regioselective sulfation of Artemisia sphaerocephala polysaccharide: Solution conformation and antioxidant activities in vitro.

    PubMed

    Wang, Junlong; Yang, Wen; Tang, YinYing; Xu, Qing; Huang, Shengli; Yao, Jian; Zhang, Ji; Lei, Ziqiang

    2016-01-20

    Regioselective modification is an effective approach to synthesize polysaccharides with different structure features and improved properties. In this study, regioselective sulfation of Artemisia sphaerocephala polysaccharide (SRSASP) was prepared by using triphenylchloromethane (TrCl) as protecting precursor. The decrease in fractal dimension (df) values (1.56-2.04) of SRSASP was observed in size-exclusion chromatography combined with multi angle laser light scattering (SEC-MALLS) analysis. Compared to sample substituted at C-6, SRSASP showed a more expanded conformation of random coil, which was attributed to the breakup of hydrogen bonds and elastic contributions. Circular dichroism (CD), methylene blue (MB) and congo red (CR) spectrophotometric method and atomic force microscopy (AFM) results confirmed the conformational transition and stiffness of the chains after sulfation. SRSASP exhibited enhanced antioxidant activities in the DPPH, superoxide and hydroxyl radical scavenging assay. Sulfation at C-2 or C-3 was favorable for the chelation which might prevent the generation of hydroxyl radicals. It concluded that the degree of substitution and substitution position were the factors influencing biological activities of sulfated polysaccharides.

  13. Conformational analysis of a quinolonic ribonucleoside with anti-HSV-1 activity

    NASA Astrophysics Data System (ADS)

    Yoneda, Julliane D.; Velloso, Marcia Helena R.; Leal, Kátia Z.; Azeredo, Rodrigo B. de V.; Sugiura, Makiko; Albuquerque, Magaly G.; Santos, Fernanda da C.; Souza, Maria Cecília B. V. de; Cunha, Anna Claudia; Seidl, Peter R.; Alencastro, Ricardo B. de; Ferreira, Vitor F.

    2011-01-01

    The infections caused by the Herpes Simplex Virus are one of the most common sources of diseases in adults and several natural nucleoside analogues are currently used in the treatment of these infections. In vitro tests of a series of quinolonic ribonucleosides derivatives synthesized by part of our group indicated that some of them have antiviral activity against HSV-1. The conformational analysis of bioactive compounds is extremely important in order to better understand their chemical structures and biological activity. In this work, we have carried out a nuclear relaxation NMR study of 6-Me ribonucleoside derivative in order to determine if the syn or anti conformation is preferential. The NMR analysis permits the determination of inter-atomic distances by using techniques which are based on nuclear relaxation and related phenomena. Those techniques are non-selective longitudinal or spin-lattice relaxation rates and NULL pulse sequence, which allow the determination of distances between pairs of hydrogen atoms. The results of NMR studies were compared with those obtained by molecular modeling.

  14. Exploring the active conformation of cyclohexane carboxylate positive allosteric modulators of the type 4 metabotropic glutamate receptor.

    PubMed

    Rovira, Xavier; Harrak, Youssef; Trapero, Ana; González-Bulnes, Patricia; Malhaire, Fanny; Pin, Jean-Philippe; Goudet, Cyril; Giraldo, Jesús; Llebaria, Amadeu

    2014-12-01

    The active conformation of a family of metabotropic glutamate receptor subtype 4 (mGlu4 ) positive allosteric modulators (PAMs) with the cyclohexane 1,2-dicarboxylic scaffold present in cis-2-(3,5-dichlorophenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041) was investigated by testing structurally similar six-membered ring compounds that have a locked conformation. The norbornane and cyclohexane molecules designed as mGlu4 conformational probes and the enantiomers of the trans diastereomer were computationally characterized and tested in mGlu4 pharmacological assays. The results support a VU0155041 active conformation, with the chair cyclohexane having the aromatic amide substituent in an axial position and the carboxylate in an equatorial position. Moreover, the receptor displays enantiomeric discrimination of the chiral PAMs. The constructed pharmacophore characterized a highly constrained mGlu4 allosteric binding site, thus providing a step forward in structure-based drug design for mGlu4 PAMs.

  15. A Potential Substrate Binding Conformation of β-Lactams and Insight into the Broad Spectrum of NDM-1 Activity

    PubMed Central

    Yuan, Qinghui; He, Lin

    2012-01-01

    New Delhi metallo-β-lactamase 1 (NDM-1) is a key enzyme that the pathogen Klebsiella pneumonia uses to hydrolyze almost all β-lactam antibiotics. It is currently unclear why NDM-1 has a broad spectrum of activity. Docking of the representatives of the β-lactam families into the active site of NDM-1 is reported here. All the β-lactams naturally fit the NDM-1 pocket, implying that NDM-1 can accommodate the substrates without dramatic conformation changes. The docking reveals two major binding modes of the β-lactams, which we tentatively name the S (substrate) and I (inhibitor) conformers. In the S conformers of all the β-lactams, the amide oxygen and the carboxylic group conservatively interact with two zinc ions, while the substitutions on the fused rings show dramatic differences in their conformations and positions. Since the bridging hydroxide ion/water in the S conformer is at the position for the nucleophilic attack, the S conformation may simulate the true binding of a substrate to NDM-1. The I conformer either blocks or displaces the bridging hydroxide ion/water, such as in the case of aztreonam, and is thus inhibitory. The docking also suggests that substitutions on the β-lactam ring are required for β-lactams to bind in the S conformation, and therefore, small β-lactams such as clavulanic acid would be inhibitors of NDM-1. Finally, our docking shows that moxalactam uses its tyrosyl-carboxylic group to compete with the S conformer and would thus be a poor substrate of NDM-1. PMID:22825119

  16. De novo design and in vivo activity of conformationally restrained antimicrobial arylamide foldamers

    PubMed Central

    Choi, Sungwook; Isaacs, Andre; Clements, Dylan; Liu, Dahui; Kim, Hyemin; Scott, Richard W.; Winkler, Jeffrey D.; DeGrado, William F.

    2009-01-01

    The emergence of drug-resistant bacteria has compromised the use of many conventional antibiotics, leading to heightened interest in a variety of antimicrobial peptides. Although these peptides have attractive potential as antibiotics, their size, stability, tissue distribution, and toxicity have hampered attempts to harness these capabilities. To address such issues, we have developed small (molecular mass <1,000 Da) arylamide foldamers that mimic antimicrobial peptides. Hydrogen-bonded restraints in the arylamide template rigidify the conformation via hydrogen bond formation and increase activity toward Staphylococcus aureus and Escherichia coli. The designed foldamers are highly active against S. aureus in an animal model. These results demonstrate the application of foldamer templates as therapeutics. PMID:19359494

  17. Activation of antibody Fc function by antigen-induced conformational changes.

    PubMed Central

    Brown, J C; Koshland, M E

    1975-01-01

    IgM antibody directed against the pheny-beta-lactoside hapten was examined for its capacity to fix complement in the presence of the hapten, monohapten-substituted antigen, and multihapten-substituted antigen. Hapten was found to have no effect; monovalent antigen induced an excellent response which could be inhibited by hapten; and multivalent antigen also induced an excellent response which was related to the number of determinants added and not to the formation of antigen-antibody aggregates. The difference between the activities of hapten and monovalent antigen was reflected in their affinities for the IgM antibody. The monovalent antigen had a lower Ka, indicating that energy from binding was used to activate the Fc complement binding sites. These data show that the expression of IgM Fc function depends on a change in Fc conformation produced by the binding of antigen at the distant Fab combining sites. PMID:1061094

  18. Activation of antibody Fc function by antigen-induced conformational changes.

    PubMed

    Brown, J C; Koshland, M E

    1975-12-01

    IgM antibody directed against the pheny-beta-lactoside hapten was examined for its capacity to fix complement in the presence of the hapten, monohapten-substituted antigen, and multihapten-substituted antigen. Hapten was found to have no effect; monovalent antigen induced an excellent response which could be inhibited by hapten; and multivalent antigen also induced an excellent response which was related to the number of determinants added and not to the formation of antigen-antibody aggregates. The difference between the activities of hapten and monovalent antigen was reflected in their affinities for the IgM antibody. The monovalent antigen had a lower Ka, indicating that energy from binding was used to activate the Fc complement binding sites. These data show that the expression of IgM Fc function depends on a change in Fc conformation produced by the binding of antigen at the distant Fab combining sites.

  19. A sandwich ELISA for the conformation-specific quantification of the activated form of human Bax.

    PubMed

    Teijido, Oscar; Ganesan, Yogesh Tengarai; Llanos, Raul; Peton, Ashley; Urtecho, Jean-Baptiste; Soprani, Adauri; Villamayor, Aimee; Antonsson, Bruno; Manon, Stéphen; Dejean, Laurent

    2016-03-15

    Bcl-2 family proteins are critical regulators of mitochondrial outer membrane permeabilization (MOMP), which represents the point of no return of apoptotic cell death. The exposure of the Bax N-terminus at the mitochondria reflects Bax activation; and this activated configuration of the Bax protein is associated with MOMP. N-terminal exposure can be detected using specific monoclonal and/or polyclonal antibodies, and the onset of activated Bax has extensively been used as an early marker of apoptosis. The protocols of immunoprecipitation and/or immunocytochemistry commonly used to detect activated Bax are long and tedious, and allow semiquantification of the antigen at best. The sandwich ELISA protocol we developed has a 5 ng/mL detection limit and is highly specific for the activated conformation of Bax. This ELISA allows a rapid quantification of activated human Bax in whole cells and isolated mitochondria protein extracts. These properties grant this assay the potential to further clarify the prognostic and diagnostic value of activated Bax in disorders associated with deregulated apoptotic pathways such as degenerative diseases or cancer. PMID:26748144

  20. The N-terminal actin-binding tandem calponin-homology (CH) domain of dystrophin is in a closed conformation in solution and when bound to F-actin.

    PubMed

    Singh, Surinder M; Mallela, Krishna M G

    2012-11-01

    Deficiency of the vital muscle protein dystrophin triggers Duchenne/Becker muscular dystrophy, but the structure-function relationship of dystrophin is poorly understood. To date, molecular structures of three dystrophin domains have been determined, of which the N-terminal actin-binding domain (N-ABD or ABD1) is of particular interest. This domain is composed of two calponin-homology (CH) domains, which form an important class of ABDs in muscle proteins. A previously determined x-ray structure indicates that the dystrophin N-ABD is a domain-swapped dimer, with each monomer adopting an extended, open conformation in which the two CH domains do not interact. This structure is controversial because it contradicts functional studies and known structures of similar ABDs from other muscle proteins. Here, we investigated the solution conformation of the dystrophin N-ABD using a very simple and elegant technique of pyrene excimer fluorescence. Using the wild-type protein, which contains two cysteines, and the corresponding single-cysteine mutants, we show that the protein is a monomer in solution and is in a closed conformation in which the two CH domains seem to interact, as observed from the excimer fluorescence of pyrene-labeled wild-type protein. Excimer fluorescence was also observed in its actin-bound form, indicating that the dystrophin N-ABD binds to F-actin in a closed conformation. Comparison of the dystrophin N-ABD conformation with other ABDs indicates that the tandem CH domains in general may be monomeric in solution and predominantly occur in closed conformation, whereas their actin-bound conformations may differ.

  1. Copper, differently from zinc, affects the conformation, oligomerization state and activity of bradykinin.

    PubMed

    Naletova, Irina; Nicoletti, Vincenzo G; Milardi, Danilo; Pietropaolo, Adriana; Grasso, Giuseppe

    2016-08-01

    The sole role of bradykinin (BK) as an inflammatory mediator is controversial, as recent data also support an anti-inflammatory role for BK in Alzheimer's disease (AD). The involvement of two different receptors (B1R and B2R) could be a key to understand this issue. However, although copper and zinc dyshomeostasis has been demonstrated to be largely involved in the development of AD, a detailed study of the interaction of BK with these two metal ions has never been addressed. In this work, we have applied mass spectrometry, circular dichroism as well as computational methods in order to assess if copper and zinc have the ability to modulate the conformation and oligomerization of BK. In addition, we have correlated the chemical data with the effect of metals on the activity of BK analyzed in cell cultures by biochemical procedures. The biochemical analyses on monocyte/macrophage cell culture (THP-1 Cell Line human) in line with the effect of metals on the conformation of BK showed that the presence of copper can affect the signaling cascade mediated by the BK receptors. The results obtained show a further role of metal ions, particularly copper, in the development and outcome of neuroinflammatory diseases. The possible implications in AD are discussed. PMID:27328010

  2. The amino terminal helix modulates light activated conformational changes in AsLOV2

    PubMed Central

    Zayner, Josiah P.; Antoniou, Chloe; Sosnick, Tobin R.

    2012-01-01

    The mechanism of light-triggered conformational change and signaling in light-oxygen-voltage (LOV) domains remains elusive in spite of extensive investigation and their use in optogenetic studies. The LOV2 domain of Avena Sativa phototropin1 (AsLOV2), a member of the Per-Arnt-Sim (PAS) family, contains an FMN chromophore that forms a covalent bond with a cysteine upon illumination. This event leads to the release of the carboxy terminal Jα helix, the biological output signal. Using mutational analysis, circular dichroism and NMR, we find that the largely ignored amino terminal helix is a control element in AsLOV2’s light-activated conformational change. We further identify a direct amino-to-carboxy terminal “input-output” signaling pathway. These findings provide a framework to rationalize the LOV domain architecture, as well as the signaling mechanisms in both isolated and tandem arrangements of PAS domains. This knowledge can be applied in engineering LOV-based photoswitches, opening up new design strategies and improving existing ones. PMID:22406525

  3. Interactions of a designed peptide with lipopolysaccharide: Bound conformation and anti-endotoxic activity

    SciTech Connect

    Bhunia, Anirban; Chua, Geok Lin; Domadia, Prerna N.; Warshakoon, Hemamali; Cromer, Jens R.; David, Sunil A.; Bhattacharjya, Surajit

    2008-05-09

    Designed peptides that would selectively interact with lipopolysaccharide (LPS) or endotoxin and fold into specific conformations could serve as important scaffolds toward the development of antisepsis compounds. Here, we describe solution structure of a designed amphipathic peptide, H{sub 2}N-YVKLWRMIKFIR-CONH{sub 2} (YW12D) in complex with endotoxin as determined by transferred nuclear Overhauser effect spectroscopy. The conformation of the isolated peptide is highly flexible, but undergoes a dramatic structural stabilization in the presence of LPS. Structure calculations reveal that the peptide presents two amphipathic surfaces in its bound state to LPS whereby each surface is characterized by two positive charges and a number of aromatic and/or aliphatic residues. ITC data suggests that peptide interacts with two molecules of lipid A. In activity assays, YW12D exhibits neutralization of LPS toxicity with very little hemolysis of red blood cells. Structural and functional properties of YW12D would be applicable in designing low molecular weight non-toxic antisepsis molecules.

  4. Detecting the Active Conformation of Calpain with Calpastatin-Based Reagents

    PubMed Central

    Croall, Dorothy E.; Vanhooser, Lisa M.; Cashon, Robert

    2008-01-01

    SUMMARY The specific, calcium dependent, high affinity interaction between calpain and its endogenous inhibitor calpastatin was exploited to selectively detect the calcium-bound, catalytically competent, conformation of calpain in vitro. Modification of calpastatin-domain-1 (Val114-Ser270) or its N-terminal fragment (Val114-Pro202), at selected unique cysteine residues with maleimide–AlexaFluor546 did not compromise calpastatin function (inhibition of calpain) or its binding with calpain. Ca2+-dependent binding between catalytically–dead calpain-2 (Cys105Ala) fused with eGFP and these fluorigenic calpastatin peptides generates fluorescent resonance energy transfer (FRET). The FRET signal documents proximity of calpain-2-C-terminally linked fluorophore to specific sites within calpastatin when the proteins form a complex. These results provide important insights into the calcium dependent interaction between calpain and calpastatin and for holo-calpain-2 in solution experimentally validate some key features of their predicted interactions. These data also provide proof of concept that the calpastatin based reagents may be useful to selectively detect the active conformation of calpain. PMID:18793761

  5. Elucidation of the active conformation of the APS-kinase domain of human PAPS synthetase 1.

    PubMed

    Sekulic, Nikolina; Dietrich, Kristen; Paarmann, Ingo; Ort, Stephan; Konrad, Manfred; Lavie, Arnon

    2007-03-23

    Bifunctional human PAPS synthetase (PAPSS) catalyzes, in a two-step process, the formation of the activated sulfate carrier 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The first reaction involves the formation of the 5'-adenosine phosphosulfate (APS) intermediate from ATP and inorganic sulfate. APS is then further phosphorylated on its 3'-hydroxyl group by an additional ATP molecule to generate PAPS. The former reaction is catalyzed by the ATP-sulfurylase domain and the latter by the APS-kinase domain. Here, we report the structure of the APS-kinase domain of PAPSS isoform 1 (PAPSS1) representing the Michaelis complex with the products ADP-Mg and PAPS. This structure provides a rare glimpse of the active conformation of an enzyme catalyzing phosphoryl transfer without resorting to substrate analogs, inactivating mutations, or catalytically non-competent conditions. Our structure shows the interactions involved in the binding of the magnesium ion and PAPS, thereby revealing residues critical for catalysis. The essential magnesium ion is observed bridging the phosphate groups of the products. This function of the metal ion is made possible by the DGDN-loop changing its conformation from that previously reported, and identifies these loop residues unambiguously as a Walker B motif. Furthermore, the second aspartate residue of this motif is the likely candidate for initiating nucleophilic attack on the ATP gamma-phosphate group by abstracting the proton from the 3'-hydroxyl group of the substrate APS. We report the structure of the APS-kinase domain of human PAPSS1 in complex with two APS molecules, demonstrating the ability of the ATP/ADP-binding site to bind APS. Both structures reveal extended N termini that approach the active site of the neighboring monomer. Together, these results significantly increase our understandings of how catalysis is achieved by APS-kinase.

  6. QSAR study and conformational analysis of 4-arylthiazolylhydrazones derived from 1-indanones with anti-Trypanosoma cruzi activity.

    PubMed

    Noguera, Guido J; Fabian, Lucas E; Lombardo, Elisa; Finkielsztein, Liliana

    2015-10-12

    A set of 4-arylthiazolylhydrazones derived from 1-indanones (TZHs) previously synthesized and assayed against Trypanosoma cruzi, the causative agent of Chagas disease, were explored in terms of conformational analysis. We found that TZHs can adopt four minimum energy conformations: cis (A, B and C) and trans. The possible bioactive conformation was selected by a 3D-QSAR model. Different molecular parameters were calculated to produce QSAR second-generation models. These QSAR results are discussed in conjunction with conformational analysis from molecular modeling studies. The main factor to determine the activity of the compounds was the partial charge at the N(3) atom (qN3). The predictive ability of the QSAR equations proposed was experimentally validated. The QSAR models developed in this study will be helpful to design novel potent TZHs.

  7. Retinobenzoic acids. 4. Conformation of aromatic amides with retinoidal activity. Importance of trans-amide structure for the activity.

    PubMed

    Kagechika, H; Himi, T; Kawachi, E; Shudo, K

    1989-10-01

    N-Methylation of two retinoidal amide compounds, 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]benz oic acid (3, Am80) and 4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)carbonyl]amino]benzoic acid (5, Am580), resulted in the disappearance of their potent differentiation-inducing activity on human promyelocytic leukemia cell line HL-60. Studies with 1H NMR and UV spectroscopy indicated that large conformational differences exist between the active secondary amides and the inactive N-methyl amides. From a comparison of the spectroscopic results of these amides with those of stilbene derivatives, the conformations of the active amides are expected to resemble that of (E)-stilbene, whereas the inactive amides resemble the Z isomer: 3 (Am80) and 5 (Am580) have a trans-amide bond and their whole structures are elongated, while the N-methylated compounds [4 (Am90) and 6 (Am590)] have a cis-amide bond, resulting in the folding of the two benzene rings. These structures in the crystals were related to those in solution by 13C NMR spectroscopic comparison between the two phases (solid and solution).

  8. c-Abl Tyrosine Kinase Adopts Multiple Active Conformational States in Solution.

    PubMed

    Badger, John; Grover, Prerna; Shi, Haibin; Panjarian, Shoghag B; Engen, John R; Smithgall, Thomas E; Makowski, Lee

    2016-06-14

    Protein tyrosine kinases of the Abl family have diverse roles in normal cellular regulation and drive several forms of leukemia as oncogenic fusion proteins. In the crystal structure of the inactive c-Abl kinase core, the SH2 and SH3 domains dock onto the back of the kinase domain, resulting in a compact, assembled state. This inactive conformation is stabilized by the interaction of the myristoylated N-cap with a pocket in the C-lobe of the kinase domain. Mutations that perturb these intramolecular interactions result in kinase activation. Here, we present X-ray scattering solution structures of multidomain c-Abl kinase core proteins modeling diverse active states. Surprisingly, the relative positions of the regulatory N-cap, SH3, and SH2 domains in an active myristic acid binding pocket mutant (A356N) were virtually identical to those of the assembled wild-type kinase core, indicating that Abl kinase activation does not require dramatic reorganization of the downregulated core structure. In contrast, the positions of the SH2 and SH3 domains in a clinically relevant imatinib-resistant gatekeeper mutant (T315I) appear to be reconfigured relative to their positions in the wild-type protein. Our results demonstrate that c-Abl kinase activation can occur either with (T315I) or without (A356N) global allosteric changes in the core, revealing the potential for previously unrecognized signaling diversity. PMID:27166638

  9. c-Abl Tyrosine Kinase Adopts Multiple Active Conformational States in Solution

    PubMed Central

    2016-01-01

    Protein tyrosine kinases of the Abl family have diverse roles in normal cellular regulation and drive several forms of leukemia as oncogenic fusion proteins. In the crystal structure of the inactive c-Abl kinase core, the SH2 and SH3 domains dock onto the back of the kinase domain, resulting in a compact, assembled state. This inactive conformation is stabilized by the interaction of the myristoylated N-cap with a pocket in the C-lobe of the kinase domain. Mutations that perturb these intramolecular interactions result in kinase activation. Here, we present X-ray scattering solution structures of multidomain c-Abl kinase core proteins modeling diverse active states. Surprisingly, the relative positions of the regulatory N-cap, SH3, and SH2 domains in an active myristic acid binding pocket mutant (A356N) were virtually identical to those of the assembled wild-type kinase core, indicating that Abl kinase activation does not require dramatic reorganization of the downregulated core structure. In contrast, the positions of the SH2 and SH3 domains in a clinically relevant imatinib-resistant gatekeeper mutant (T315I) appear to be reconfigured relative to their positions in the wild-type protein. Our results demonstrate that c-Abl kinase activation can occur either with (T315I) or without (A356N) global allosteric changes in the core, revealing the potential for previously unrecognized signaling diversity. PMID:27166638

  10. Lipopeptaibol metabolites of tolypocladium geodes: total synthesis, preferred conformation, and membrane activity.

    PubMed

    Rainaldi, Mario; Moretto, Alessandro; Peggion, Cristina; Formaggio, Fernando; Mammi, Stefano; Peggion, Evaristo; Galvez, José Antonio; Díaz-de-Villegas, Maria Dolores; Cativiela, Carlos; Toniolo, Claudio

    2003-08-01

    We have synthesized by solution methods and characterized the lipopeptaibol metabolite LP237-F8 extracted from the fungus Tolypocladium geodes and five selected analogues with the Etn-->Aib or Etn-->Nva replacement at position 8 and/or a triple Gln-->Glu(OMe) replacement at positions 5, 6, and 9 (Etn=Calpha-ethylnorvaline, Aib=alpha-aminoisobutyric acid, Nva=norvaline). Conformation analysis, performed by FT-IR absorption, NMR, and CD techniques, strongly supports the view that the six terminally blocked decapeptides are highly helical in solution. Helix topology and amphiphilic character are responsible for their remarkable membrane activity. At position 8 the combination of high hydrophobicity and Calpha tetrasubstitution, as in the Etn-containing LP237-F8 metabolite, has a positive effect on membrane interaction.

  11. Effect of copper oxide nanoparticles on the conformation and activity of β-galactosidase.

    PubMed

    Rabbani, Gulam; Khan, Mohd Jahir; Ahmad, Abrar; Maskat, Mohamad Yusof; Khan, Rizwan Hasan

    2014-11-01

    The primary objective of this study is to explore the interaction of β-galactosidase with copper oxide nanoparticles (CuO NPs). Steady-state absorption, fluorescence and circular dichroism (CD) spectroscopic techniques have been employed to unveil the conformational changes of β-galactosidase induced by the binding of CuO NPs. Temperature dependent fluorescence quenching results indicates a static quenching mechanism in the present case. The binding thermodynamic parameters delineate the predominant role of H-bonding and van der Waals forces between β-galactosidase and CuO NPs binding process. The binding was studied by isothermal titration calorimetry (ITC) and the result revealed that the complexation is enthalpy driven, the ΔH°<0, ΔS°<0 indicates the formation of hydrogen bonds between β-galactosidase and CuO NPs occurs. Disruption of the native conformation of the protein upon binding with CuO NPs is reflected through a reduced functionality (in terms of hydrolase activity) of the protein CuO NPs conjugate system in comparison to the native protein and CuO NPs exhibited a competitive mode of inhibition. This also supports the general belief that H-bond formation occurs with NPs is associated with a lesser extent of modification in the native structure. Morphological features and size distributions were investigated using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Additionally the considerable increase in the Rh following the addition of CuO NPs accounts for the unfolding of β-galactosidase. Chemical and thermal unfolding of β-galactosidase, when carried out in the presence of CuO NPs, also indicated a small perturbation in the protein structure. These alterations in functional activity of nanoparticle bound β-galactosidase which will have important consequences should be taken into consideration while using nanoparticles for diagnostic and therapeutic purposes.

  12. Effect of copper oxide nanoparticles on the conformation and activity of β-galactosidase.

    PubMed

    Rabbani, Gulam; Khan, Mohd Jahir; Ahmad, Abrar; Maskat, Mohamad Yusof; Khan, Rizwan Hasan

    2014-11-01

    The primary objective of this study is to explore the interaction of β-galactosidase with copper oxide nanoparticles (CuO NPs). Steady-state absorption, fluorescence and circular dichroism (CD) spectroscopic techniques have been employed to unveil the conformational changes of β-galactosidase induced by the binding of CuO NPs. Temperature dependent fluorescence quenching results indicates a static quenching mechanism in the present case. The binding thermodynamic parameters delineate the predominant role of H-bonding and van der Waals forces between β-galactosidase and CuO NPs binding process. The binding was studied by isothermal titration calorimetry (ITC) and the result revealed that the complexation is enthalpy driven, the ΔH°<0, ΔS°<0 indicates the formation of hydrogen bonds between β-galactosidase and CuO NPs occurs. Disruption of the native conformation of the protein upon binding with CuO NPs is reflected through a reduced functionality (in terms of hydrolase activity) of the protein CuO NPs conjugate system in comparison to the native protein and CuO NPs exhibited a competitive mode of inhibition. This also supports the general belief that H-bond formation occurs with NPs is associated with a lesser extent of modification in the native structure. Morphological features and size distributions were investigated using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Additionally the considerable increase in the Rh following the addition of CuO NPs accounts for the unfolding of β-galactosidase. Chemical and thermal unfolding of β-galactosidase, when carried out in the presence of CuO NPs, also indicated a small perturbation in the protein structure. These alterations in functional activity of nanoparticle bound β-galactosidase which will have important consequences should be taken into consideration while using nanoparticles for diagnostic and therapeutic purposes. PMID:25260221

  13. Conformal thermal therapy using planar ultrasound transducers and adaptive closed-loop MR temperature control: demonstration in gel phantoms and ex vivo tissues.

    PubMed

    Tang, K; Choy, V; Chopra, R; Bronskill, M J

    2007-05-21

    MRI-guided transurethral ultrasound therapy offers a minimally invasive approach for the treatment of localized prostate cancer. Integrating a multi-element planar transducer with active MR temperature feedback can enable three-dimensional conformal thermal therapy of a target region within the prostate gland while sparing surrounding normal tissues. Continuous measurement of the temperature distribution in tissue enables dynamic compensation for unknown changes in blood flow and tissue properties during treatment. The main goal of this study was to evaluate the feasibility of using active temperature feedback on a clinical 1.5 T MR imager for conformal thermal therapy. MR thermometry was performed during heating in both gel phantoms and excised tissue with a transurethral heating applicator, and the rotation rate and power were varied based on the thermal measurements. The capability to produce a region of thermal damage that matched a target boundary was evaluated. The influence of a cooling gradient (to simulate cooling of the rectum or urethra) on the desired pattern of thermal damage was also investigated in gel phantoms. Results showed high correlation between the desired target boundary and the 55 degrees C isotherm generated during heating with an average distance error of 0.9 mm +/- 0.4 mm (n = 6) in turkey breasts, 1.4 mm +/- 0.6 mm (n = 4) in gel phantoms without rectal cooling and 1.4 mm +/- 0.6 mm (n = 3) in gel phantoms with rectal cooling. The results were obtained using a temporal update rate of 5 s, a spatial resolution of 3 x 3 x 10 mm for the control point, and a temperature uncertainty of approximately 1 degrees C. The performance of the control algorithm under these conditions was comparable to that of simulations conducted previously by our group. Overall, the feasibility of generating targeted regions of thermal damage with a transurethral heating applicator and active MR temperature feedback has been demonstrated experimentally. This method

  14. Conformal thermal therapy using planar ultrasound transducers and adaptive closed-loop MR temperature control: demonstration in gel phantoms and ex vivo tissues

    NASA Astrophysics Data System (ADS)

    Tang, K.; Choy, V.; Chopra, R.; Bronskill, M. J.

    2007-05-01

    MRI-guided transurethral ultrasound therapy offers a minimally invasive approach for the treatment of localized prostate cancer. Integrating a multi-element planar transducer with active MR temperature feedback can enable three-dimensional conformal thermal therapy of a target region within the prostate gland while sparing surrounding normal tissues. Continuous measurement of the temperature distribution in tissue enables dynamic compensation for unknown changes in blood flow and tissue properties during treatment. The main goal of this study was to evaluate the feasibility of using active temperature feedback on a clinical 1.5 T MR imager for conformal thermal therapy. MR thermometry was performed during heating in both gel phantoms and excised tissue with a transurethral heating applicator, and the rotation rate and power were varied based on the thermal measurements. The capability to produce a region of thermal damage that matched a target boundary was evaluated. The influence of a cooling gradient (to simulate cooling of the rectum or urethra) on the desired pattern of thermal damage was also investigated in gel phantoms. Results showed high correlation between the desired target boundary and the 55 °C isotherm generated during heating with an average distance error of 0.9 mm ± 0.4 mm (n = 6) in turkey breasts, 1.4 mm ± 0.6 mm (n = 4) in gel phantoms without rectal cooling and 1.4 mm ± 0.6 mm (n = 3) in gel phantoms with rectal cooling. The results were obtained using a temporal update rate of 5 s, a spatial resolution of 3 × 3 × 10 mm for the control point, and a temperature uncertainty of approximately 1 °C. The performance of the control algorithm under these conditions was comparable to that of simulations conducted previously by our group. Overall, the feasibility of generating targeted regions of thermal damage with a transurethral heating applicator and active MR temperature feedback has been demonstrated experimentally. This method of treatment

  15. Fucosylated chondroitin sulfates from the body wall of the sea cucumber Holothuria forskali: conformation, selectin binding, and biological activity.

    PubMed

    Panagos, Charalampos G; Thomson, Derek S; Moss, Claire; Hughes, Adam D; Kelly, Maeve S; Liu, Yan; Chai, Wengang; Venkatasamy, Radhakrishnan; Spina, Domenico; Page, Clive P; Hogwood, John; Woods, Robert J; Mulloy, Barbara; Bavington, Charlie D; Uhrín, Dušan

    2014-10-10

    Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: → 3)GalNAcβ4,6S(1 → 4) [FucαX(1 → 3)]GlcAβ(1 →, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Le(x) blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu(2+)-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention.

  16. Conformational study reveals amino acid residues essential for hemagglutinating and anti-proliferative activities of Clematis montana lectin.

    PubMed

    Lu, Bangmin; Zhang, Bin; Qi, Wei; Zhu, Yanan; Zhao, Yan; Zhou, Nan; Sun, Rong; Bao, Jinku; Wu, Chuanfang

    2014-11-01

    Clematis montana lectin (CML), a novel mannose-binding lectin purified from C. montana Buch.-Ham stem (Ranunculaceae), has been proved to have hemagglutinating activity in rabbit erythrocytes and apoptosis-inducing activity in tumor cells. However, the biochemical properties of CML have not revealed and its structural information still needs to be elucidated. In this study, it was found that CML possessed quite good thermostability and alkaline resistance, and its hemagglutinating activity was bivalent metal cation dependent. In addition, hemagglutination test and fluorescence spectroscopy proved that GuHCl, urea, and sodium dodecyl sulfate could change the conformation of CML and further caused the loss of hemagglutination activity. Moreover, the changes of fluorescence spectrum indicated that the tryptophan (Trp) microenvironment conversion might be related to the conformation and bioactivities of CML. In addition, it was also found that Trp residues, arginine (Arg) residues, and sulfhydryl were important for the hemagglutinating activity of CML, but only Trp was proved to be crucial for the CML conformation. Furthermore, the Trp, Arg, and sulfhydryl-modified CML exhibited 97.17%, 76.99%, and 49.64% loss of its anti-proliferative activity, respectively, which was consistent with the alterations of its hemagglutinating activity. Given these findings, Trp residues on the surface of CML are essential for the active center to form substrate-accessible conformation and suitable environment for carbohydrate binding. PMID:25239139

  17. Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity.

    PubMed

    Markovski, Monica; Bohrhunter, Jessica L; Lupoli, Tania J; Uehara, Tsuyoshi; Walker, Suzanne; Kahne, Daniel E; Bernhardt, Thomas G

    2016-04-26

    To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a of Escherichia coli require the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b-LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed. PMID:27071112

  18. Extended Synaptotagmin Interaction with the Fibroblast Growth Factor Receptor Depends on Receptor Conformation, Not Catalytic Activity.

    PubMed

    Tremblay, Michel G; Herdman, Chelsea; Guillou, François; Mishra, Prakash K; Baril, Joëlle; Bellenfant, Sabrina; Moss, Tom

    2015-06-26

    We previously demonstrated that ESyt2 interacts specifically with the activated FGF receptor and is required for a rapid phase of receptor internalization and for functional signaling via the ERK pathway in early Xenopus embryos. ESyt2 is one of the three-member family of Extended Synaptotagmins that were recently shown to be implicated in the formation of endoplasmic reticulum (ER)-plasma membrane (PM) junctions and in the Ca(2+) dependent regulation of these junctions. Here we show that ESyt2 is directed to the ER by its putative transmembrane domain, that the ESyts hetero- and homodimerize, and that ESyt2 homodimerization in vivo requires a TM adjacent sequence but not the SMP domain. ESyt2 and ESyt3, but not ESyt1, selectively interact in vivo with activated FGFR1. In the case of ESyt2, this interaction requires a short TM adjacent sequence and is independent of receptor autophosphorylation, but dependent on receptor conformation. The data show that ESyt2 recognizes a site in the upper kinase lobe of FGFR1 that is revealed by displacement of the kinase domain activation loop during receptor activation.

  19. Conformational Changes in Orotidine 5’-Monophosphate Decarboxylase: A Structure-Based Explanation for How the 5’-Phosphate Group Activates the Enzyme†

    PubMed Central

    Desai, Bijoy J.; Wood, McKay; Fedorov, Alexander A.; Fedorov, Elena V.; Goryanova, Bogdana; Amyes, Tina L.; Richard, John P.; Almo, Steven C.; Gerlt, John A.

    2012-01-01

    The binding of a ligand to orotidine 5’-monophosphate decarboxylase (OMPDC) is accompanied by a conformational change from an open, inactive conformation (Eo) to a closed, active conformation (Ec). As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions are enforced that destabilize the carboxylate group of the substrate as well as stabilize the intermediate (in the Ec•S‡ complex). Focusing on the OMPDC from Methanothermobacter thermautotrophicus, the “remote” 5’-phosphate group of the substrate activates the enzyme 2.4 × 108-fold; the activation is equivalently described by an intrinsic binding energy (IBE) of 11.4 kcal/mol. We studied residues in the activation that 1) directly contact the 5’-phosphate group; 2) participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from solvent; and 3) form hydrogen-bonding interactions across the interface between the “mobile” and “fixed” half-barrel domains of the (β/α8-barrel structure. Our data support a model in which the IBE provided by the 5’-phosphate group is used to enable interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the Ec•S and Ec•S‡ complexes relative to the Eo•S complex. The conclusion that the IBE of the 5’-phosphate group provides stabilization of both the Ec•S and Ec•S‡ complexes, not just the Ec•S‡ complex, is central to understanding the structural origins of enzymatic catalysis as well as the requirements for the de novo design of enzymes that catalyze novel reactions. PMID:23030629

  20. Active conformation of the erythropoietin receptor: random and cysteine-scanning mutagenesis of the extracellular juxtamembrane and transmembrane domains.

    PubMed

    Lu, Xiaohui; Gross, Alec W; Lodish, Harvey F

    2006-03-17

    In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxtamembrane and transmembrane domains, leading to activation of JAK2 kinase and induction of proliferation and survival signals. To define the active EpoR conformation(s) we screened libraries of EpoRs with random mutations in the transmembrane domain and identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domain residues (Leu(226) and Ile(227)) to cysteine. Following this discovery, we performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at positions 223, 226, or 227 activated EpoR signal transduction pathways and supported proliferation of Ba/F3 cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and the connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Based on our data and the previously determined structure of Epo bound to a dimer of the EpoR extracellular domain, we present a model of the active and inactive conformations of the Epo receptor.

  1. 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.

  2. Functionally relevant diversity of closely related Nitrospira in activated sludge

    PubMed Central

    Gruber-Dorninger, Christiane; Pester, Michael; Kitzinger, Katharina; Savio, Domenico F; Loy, Alexander; Rattei, Thomas; Wagner, Michael; Daims, Holger

    2015-01-01

    Nitrospira are chemolithoautotrophic nitrite-oxidizing bacteria that catalyze the second step of nitrification in most oxic habitats and are important for excess nitrogen removal from sewage in wastewater treatment plants (WWTPs). To date, little is known about their diversity and ecological niche partitioning within complex communities. In this study, the fine-scale community structure and function of Nitrospira was analyzed in two full-scale WWTPs as model ecosystems. In Nitrospira-specific 16S rRNA clone libraries retrieved from each plant, closely related phylogenetic clusters (16S rRNA identities between clusters ranged from 95.8% to 99.6%) within Nitrospira lineages I and II were found. Newly designed probes for fluorescence in situ hybridization (FISH) allowed the specific detection of several of these clusters, whose coexistence in the WWTPs was shown for prolonged periods of several years. In situ ecophysiological analyses based on FISH, relative abundance and spatial arrangement quantification, as well as microautoradiography revealed functional differences of these Nitrospira clusters regarding the preferred nitrite concentration, the utilization of formate as substrate and the spatial coaggregation with ammonia-oxidizing bacteria as symbiotic partners. Amplicon pyrosequencing of the nxrB gene, which encodes subunit beta of nitrite oxidoreductase of Nitrospira, revealed in one of the WWTPs as many as 121 species-level nxrB operational taxonomic units with highly uneven relative abundances in the amplicon library. These results show a previously unrecognized high diversity of Nitrospira in engineered systems, which is at least partially linked to niche differentiation and may have important implications for process stability. PMID:25148481

  3. Functionally relevant diversity of closely related Nitrospira in activated sludge.

    PubMed

    Gruber-Dorninger, Christiane; Pester, Michael; Kitzinger, Katharina; Savio, Domenico F; Loy, Alexander; Rattei, Thomas; Wagner, Michael; Daims, Holger

    2015-03-01

    Nitrospira are chemolithoautotrophic nitrite-oxidizing bacteria that catalyze the second step of nitrification in most oxic habitats and are important for excess nitrogen removal from sewage in wastewater treatment plants (WWTPs). To date, little is known about their diversity and ecological niche partitioning within complex communities. In this study, the fine-scale community structure and function of Nitrospira was analyzed in two full-scale WWTPs as model ecosystems. In Nitrospira-specific 16S rRNA clone libraries retrieved from each plant, closely related phylogenetic clusters (16S rRNA identities between clusters ranged from 95.8% to 99.6%) within Nitrospira lineages I and II were found. Newly designed probes for fluorescence in situ hybridization (FISH) allowed the specific detection of several of these clusters, whose coexistence in the WWTPs was shown for prolonged periods of several years. In situ ecophysiological analyses based on FISH, relative abundance and spatial arrangement quantification, as well as microautoradiography revealed functional differences of these Nitrospira clusters regarding the preferred nitrite concentration, the utilization of formate as substrate and the spatial coaggregation with ammonia-oxidizing bacteria as symbiotic partners. Amplicon pyrosequencing of the nxrB gene, which encodes subunit beta of nitrite oxidoreductase of Nitrospira, revealed in one of the WWTPs as many as 121 species-level nxrB operational taxonomic units with highly uneven relative abundances in the amplicon library. These results show a previously unrecognized high diversity of Nitrospira in engineered systems, which is at least partially linked to niche differentiation and may have important implications for process stability. PMID:25148481

  4. Different conformations of ribosomal DNA in active and inactive chromatin in Xenopus laevis.

    PubMed

    Spadafora, C; Riccardi, P

    1985-12-20

    The chromatin structure of the ribosomal DNA in Xenopus laevis was studied by micrococcal nuclease digestions of blood, liver and embryonic cell nuclei. We have found that BglI-restricted DNA from micrococcal nuclease-digested blood cell nuclei has an increased electrophoretic mobility compared to the undigested control. Micrococcal nuclease digestion of liver cell nuclei causes a very slight shift in mobility, only in the region of the spacer containing the "Bam Islands". In contrast, the mobility of ribosomal DNA in chromatin of embryonic cells, under identical digestion conditions, remains unaffected by the nuclease activity. Denaturing gels or ligase action on the nuclease-treated DNA abolishes the differences in the electrophoretic mobility. Ionic strength and ethidium bromide influence the relative electrophoretic migration of the two DNA fragment populations, suggesting that secondary structure may play an important role in the observed phenomena. In addition, restriction analysis under native electrophoretic conditions of DNA prepared from blood, liver and embryonic cells shows that blood cell DNA restriction fragments always have a faster mobility than the corresponding fragments of liver and embryo cell DNA. We therefore propose that nicking activity by micrococcal nuclease modifies the electrophoretic mobility of an unusual DNA conformation, present in blood cell, and to a lesser extent, in liver cell ribosomal chromatin. A possible function for these structures is discussed. The differences of the ribosomal chromatin structures in adult and embryonic tissues may reflect the potential of the genes to be expressed.

  5. 12 CFR 225.126 - Activities not closely related to banking.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Activities not closely related to banking. 225.126 Section 225.126 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE... Financial Holding Companies Interpretations § 225.126 Activities not closely related to banking. Pursuant...

  6. 12 CFR 225.126 - Activities not closely related to banking.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 3 2011-01-01 2011-01-01 false Activities not closely related to banking. 225.126 Section 225.126 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE... Financial Holding Companies Interpretations § 225.126 Activities not closely related to banking. Pursuant...

  7. Rate of hydrolysis in ATP synthase is fine-tuned by α-subunit motif controlling active site conformation

    PubMed Central

    Beke-Somfai, Tamás; Lincoln, Per; Nordén, Bengt

    2013-01-01

    Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. FoF1 ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F1 performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate. PMID:23345443

  8. Jatrophidin I, a cyclic peptide from Brazilian Jatropha curcas L.: isolation, characterization, conformational studies and biological activity.

    PubMed

    Altei, Wanessa F; Picchi, Douglas G; Abissi, Barbara M; Giesel, Guilherme M; Flausino, Otavio; Reboud-Ravaux, Michèle; Verli, Hugo; Crusca, Edson; Silveira, Edilberto R; Cilli, Eduardo M; Bolzani, Vanderlan S

    2014-11-01

    A cyclic peptide, jatrophidin I, was isolated from the latex of Jatropha curcas L. Its structure was elucidated by extensive 2D NMR spectroscopic analysis, with additional conformational studies performed using Molecular Dynamics/Simulated Annealing (MD/SA). Jatrophidin I had moderate protease inhibition activity when compared with pepstatin A; however, the peptide was inactive in antimalarial, cytotoxic and antioxidant assays.

  9. Rate of hydrolysis in ATP synthase is fine-tuned by α-subunit motif controlling active site conformation.

    PubMed

    Beke-Somfai, Tamás; Lincoln, Per; Nordén, Bengt

    2013-02-01

    Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate. PMID:23345443

  10. Conformation-selective ATP-competitive inhibitors control regulatory interactions and noncatalytic functions of mitogen-activated protein kinases.

    PubMed

    Hari, Sanjay B; Merritt, Ethan A; Maly, Dustin J

    2014-05-22

    Most potent protein kinase inhibitors act by competing with ATP to block the phosphotransferase activity of their targets. However, emerging evidence demonstrates that ATP-competitive inhibitors can affect kinase interactions and functions in ways beyond blocking catalytic activity. Here, we show that stabilizing alternative ATP-binding site conformations of the mitogen-activated protein kinases (MAPKs) p38α and Erk2 with ATP-competitive inhibitors differentially, and in some cases divergently, modulates the abilities of these kinases to interact with upstream activators and deactivating phosphatases. Conformation-selective ligands are also able to modulate Erk2's ability to allosterically activate the MAPK phosphatase DUSP6, highlighting how ATP-competitive ligands can control noncatalytic kinase functions. Overall, these studies underscore the relationship between the ATP-binding and regulatory sites of MAPKs and provide insight into how ATP-competitive ligands can be designed to confer graded control over protein kinase function.

  11. Relating conformation to function in integrin α5β1.

    PubMed

    Su, Yang; Xia, Wei; Li, Jing; Walz, Thomas; Humphries, Martin J; Vestweber, Dietmar; Cabañas, Carlos; Lu, Chafen; Springer, Timothy A

    2016-07-01

    Whether β1 integrin ectodomains visit conformational states similarly to β2 and β3 integrins has not been characterized. Furthermore, despite a wealth of activating and inhibitory antibodies to β1 integrins, the conformational states that these antibodies stabilize, and the relation of these conformations to function, remain incompletely characterized. Using negative-stain electron microscopy, we show that the integrin α5β1 ectodomain adopts extended-closed and extended-open conformations as well as a bent conformation. Antibodies SNAKA51, 8E3, N29, and 9EG7 bind to different domains in the α5 or β1 legs, activate, and stabilize extended ectodomain conformations. Antibodies 12G10 and HUTS-4 bind to the β1 βI domain and hybrid domains, respectively, activate, and stabilize the open headpiece conformation. Antibody TS2/16 binds a similar epitope as 12G10, activates, and appears to stabilize an open βI domain conformation without requiring extension or hybrid domain swing-out. mAb13 and SG/19 bind to the βI domain and βI-hybrid domain interface, respectively, inhibit, and stabilize the closed conformation of the headpiece. The effects of the antibodies on cell adhesion to fibronectin substrates suggest that the extended-open conformation of α5β1 is adhesive and that the extended-closed and bent-closed conformations are nonadhesive. The functional effects and binding sites of antibodies and fibronectin were consistent with their ability in binding to α5β1 on cell surfaces to cross-enhance or inhibit one another by competitive or noncompetitive (allosteric) mechanisms.

  12. Active vibration and balance system for closed cycle thermodynamic machines

    NASA Technical Reports Server (NTRS)

    Qiu, Songgang (Inventor); Augenblick, John E. (Inventor); Peterson, Allen A. (Inventor); White, Maurice A. (Inventor)

    2004-01-01

    An active balance system is provided for counterbalancing vibrations of an axially reciprocating machine. The balance system includes a support member, a flexure assembly, a counterbalance mass, and a linear motor or an actuator. The support member is configured for attachment to the machine. The flexure assembly includes at least one flat spring having connections along a central portion and an outer peripheral portion. One of the central portion and the outer peripheral portion is fixedly mounted to the support member. The counterbalance mass is fixedly carried by the flexure assembly along another of the central portion and the outer peripheral portion. The linear motor has one of a stator and a mover fixedly mounted to the support member and another of the stator and the mover fixedly mounted to the counterbalance mass. The linear motor is operative to axially reciprocate the counterbalance mass. A method is also provided.

  13. Conformable actively multiplexed high-density surface electrode array for brain interfacing

    DOEpatents

    Rogers, John; Kim, Dae-Hyeong; Litt, Brian; Viventi, Jonathan

    2015-01-13

    Provided are methods and devices for interfacing with brain tissue, specifically for monitoring and/or actuation of spatio-temporal electrical waveforms. The device is conformable having a high electrode density and high spatial and temporal resolution. A conformable substrate supports a conformable electronic circuit and a barrier layer. Electrodes are positioned to provide electrical contact with a brain tissue. A controller monitors or actuates the electrodes, thereby interfacing with the brain tissue. In an aspect, methods are provided to monitor or actuate spatio-temporal electrical waveform over large brain surface areas by any of the devices disclosed herein.

  14. The impact of active site protonation on substrate ring conformation in Melanocarpus albomyces cellobiohydrolase Cel7B.

    PubMed

    Schutt, Timothy C; Bharadwaj, Vivek S; Granum, David M; Maupin, C Mark

    2015-07-14

    The ability to utilize biomass as a feedstock for liquid fuel and value-added chemicals is dependent on the efficient and economic utilization of lignin, hemicellulose, and cellulose. In current bioreactors, cellulases are used to convert crystalline and amorphous cellulose to smaller oligomers and eventually glucose by means of cellulase enzymes. A critical component of the enzyme catalyzed hydrolysis reaction is the degree to which the enzyme can facilitate substrate ring deformation from the chair to a more catalytically active conformation (e.g. skewed boat) at the -1 subsite. Presented here is an evaluation of the impact of the protonation state for critical active site residues (i.e. Glu212, Asp214, Glu217, and His228) in Melanocarpus albomyces (Ma) Cellobiohydrolase Cel7B on the substrate's orientation and ring conformation. It is found that the protonation state of the active site can disrupt the intra-enzyme hydrogen bonding network and enhance the sampling of various ring puckering conformations for the substrate ring at the +1 and -1 subsites. In particular it is observed that the protonation state of Asp214 dictates the accessibility of the glycosidic bond to the catalytic acid/base Glu217 by influencing the φ/ψ dihedral angles and the puckering of the ring structure. The protonation-orientation-conformation analysis has revealed an active site that primarily utilizes two highly coupled protonation schemes; one protonation scheme to orient the substrate and generate catalytically favorable substrate geometries and ring puckering conformations and another protonation scheme to hydrolyze the glycosidic bond. In addition to identifying how enzymes utilize protonation state to manipulate substrate geometry, this study identifies possible directions for improving catalytic activity through protein engineering. PMID:26061383

  15. Methods for determining enzymatic activity comprising heating and agitation of closed volumes

    DOEpatents

    Thompson, David Neil; Henriksen, Emily DeCrescenzo; Reed, David William; Jensen, Jill Renee

    2016-03-15

    Methods for determining thermophilic enzymatic activity include heating a substrate solution in a plurality of closed volumes to a predetermined reaction temperature. Without opening the closed volumes, at least one enzyme is added, substantially simultaneously, to the closed volumes. At the predetermined reaction temperature, the closed volumes are agitated and then the activity of the at least one enzyme is determined. The methods are conducive for characterizing enzymes of high-temperature reactions, with insoluble substrates, with substrates and enzymes that do not readily intermix, and with low volumes of substrate and enzyme. Systems for characterizing the enzymes are also disclosed.

  16. Stabilizing effects of G protein on the active conformation of adenosine A1 receptor differ depending on G protein type.

    PubMed

    Tateyama, Michihiro; Kubo, Yoshihiro

    2016-10-01

    G protein coupled receptors (GPCRs) trigger various cellular and physiological responses upon the ligand binding. The ligand binding induces conformational change in GPCRs which allows G protein to interact with the receptor. The interaction of G protein also affects the active conformation of GPCRs. In this study, we have investigated the effects of Gαi1, Gαo and chimeric Gαqi5 on the active conformation of the adenosine A1 receptor, as each Gα showed difference in the interaction with adenosine A1 receptor. The conformational changes in the adenosine A1 receptor were detected as the agonist-induced decreases in efficiency of Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) fused at the two intracellular domains of the adenosine A1 receptor. Amplitudes of the agonist-induced FRET decreases were subtle when the FP-tagged adenosine A1 receptor was expressed alone, whereas they were significantly enhanced when co-expressed with Gαi1Gβ1Gγ22 (Gi1) or Gαqi5Gβ1Gγ22 (Gqi5) but not with GαοGβ1Gγ22 (Go). The enhancement of the agonist-induced FRET decrease in the presence of Gqi5 was significantly larger than that of Gi1. Furthermore, the FRET recovery upon the agonist removal in the presence of Gqi5 was significantly slower than that of Gi1. From these results it was revealed that the agonist-bound active conformation of adenosine A1 receptor is unstable without the binding of G protein and that the stabilizing effects of G protein differ depending on the types of G protein.

  17. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment.

    PubMed

    Fyfe, Cameron D; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W; Cogdell, Richard J; Wall, Daniel M; Burchmore, Richard J S; Byron, Olwyn; Walker, Daniel

    2015-07-01

    Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  18. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    PubMed Central

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-01-01

    Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macro­globulin, this protease-activation mechanism is likely to operate across the diverse members of this group. PMID:26143919

  19. Activation of the aryl hydrocarbon receptor by carbaryl: Computational evidence of the ability of carbaryl to assume a planar conformation.

    PubMed

    Casado, Susana; Alonso, Mercedes; Herradón, Bernardo; Tarazona, José V; Navas, José

    2006-12-01

    It has been accepted that aryl hydrocarbon receptor (AhR) ligands are compounds with two or more aromatic rings in a coplanar conformation. Although general agreement exists that carbaryl is able to activate the AhR, it has been proposed that such activation could occur through alternative pathways without ligand binding. This idea was supported by studies showing a planar conformation of carbaryl as unlikely. The objective of the present work was to clarify the process of AhR activation by carbaryl. In rat H4IIE cells permanently transfected with a luciferase gene under the indirect control of AhR, incubation with carbaryl led to an increase of luminescence. Ligand binding to the AhR was studied by means of a cell-free in vitro system in which the activation of AhR can occur only by ligand binding. In this system, exposure to carbaryl also led to activation of AhR. These results were similar to those obtained with the AhR model ligand beta-naphthoflavone, although this compound exhibited higher potency than carbaryl in both assays. By means of computational modeling (molecular mechanics and quantum chemical calculations), the structural characteristics and electrostatic properties of carbaryl were described in detail, and it was observed that the substituent at C-1 and the naphthyl ring were not coplanar. Assuming that carbaryl would interact with the AhR through a hydrogen bond, this interaction was studied computationally using hydrogen fluoride as a model H-bond donor. Under this situation, the stabilization energy of the carbaryl molecule would permit it to adopt a planar conformation. These results are in accordance with the mechanism traditionally accepted for AhR activation: Binding of ligands in a planar conformation.

  20. Membrane-induced helical conformation of an active candidacidal fragment of salivary histatins.

    PubMed

    Raj, P A; Soni, S D; Levine, M J

    1994-04-01

    The conformational preference of the candidacidal C-terminal 16 residue fragment (9-24; G-Y-K-R-K-F-H-E-K-H-H-S-H-R-G-Y) of salivary histatin 5 was examined in water, methanol, and dimethyl sulfoxide solutions using 500 MHz two-dimensional-NMR. Fourier transform infrared and CD spectroscopy were used to delineate its membrane-bound conformation in lipid vesicles. The peptide backbone and side-chain proton resonance assignments were accomplished by two-dimensional total correlated and nuclear Overhauser effect (NOE) spectra. The coupling constant (JNH-C alpha H) values determined from the double quantum-filtered correlated spectra, temperature coefficients of NH chemical shifts (d delta/dT), 1H/2H exchange rates on amide resonances, and the set of NOE connectivities were used to delineate backbone conformational features. The high JNH-C alpha H values (> or = 7.4 Hz), absence of any characteristic NH-NH (i, i + 1) or C alpha H-C beta H (i, i + 3) NOE connectivities, high d delta/dT values (> or = 0.004), and the fast 1H/2H amide exchange suggest that the histatin peptide favors unfolded random conformations in aqueous solution at pH 3.8. In contrast, the JNH-C alpha H values (< or = 6.5 Hz), slow 1H/2H exchange, low d delta/dT values (< or = 0.003) observed for amide resonances of residues 5-16, and the characteristic NH-NH (i, i + 1), C alpha H-C beta H (i, i + 3) NOE connectivities, provide evidence for the presence of largely alpha-helical conformations in dimethyl sulfoxide, which mimics the polar aprotic membrane environment. In methanolic solutions, 3(10)-helical conformations could exist as a minor population together with the major alpha-helical conformations. Fourier transform infrared spectroscopy and CD data indicate that lipid environments such as dimyristoylphosphatidylcholine vesicles could induce the peptide to fold into predominantly alpha-helical conformation. The results suggest that in dimethyl sulfoxide and dimyristoylphosphatidylcholine vesicles

  1. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    SciTech Connect

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-06-30

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  2. Design and synthesis of conformationally restricted inhibitors of active thrombin activatable fibrinolysis inhibitor (TAFIa).

    PubMed

    Brink, Mikael; Dahlén, Anders; Olsson, Thomas; Polla, Magnus; Svensson, Tor

    2014-04-01

    A series of 4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid and 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid derivatives designed as inhibitors of TAFIa has been prepared via a common hydrogenation-alkylation sequence starting from the appropriate benzimidazole and imidazopyridine system. We present a successful design strategy using a conformational restriction approach resulting in potent and selective inhibitors of TAFIa. The X-ray structure of compound 5 in complex with a H333Y/H335Q double mutant TAFI indicate that the conformational restriction is responsible for the observed potency increase. PMID:24588961

  3. ATPase active-site electrostatic interactions control the global conformation of the 100 kDa SecA translocase.

    PubMed

    Kim, Dorothy M; Zheng, Haiyan; Huang, Yuanpeng J; Montelione, Gaetano T; Hunt, John F

    2013-02-27

    SecA is an intensively studied mechanoenzyme that uses ATP hydrolysis to drive processive extrusion of secreted proteins through a protein-conducting channel in the cytoplasmic membrane of eubacteria. The ATPase motor of SecA is strongly homologous to that in DEAD-box RNA helicases. It remains unclear how local chemical events in its ATPase active site control the overall conformation of an ~100 kDa multidomain enzyme and drive protein transport. In this paper, we use biophysical methods to establish that a single electrostatic charge in the ATPase active site controls the global conformation of SecA. The enzyme undergoes an ATP-modulated endothermic conformational transition (ECT) believed to involve similar structural mechanics to the protein transport reaction. We have characterized the effects of an isosteric glutamate-to-glutamine mutation in the catalytic base, a mutation which mimics the immediate electrostatic consequences of ATP hydrolysis in the active site. Calorimetric studies demonstrate that this mutation facilitates the ECT in Escherichia coli SecA and triggers it completely in Bacillus subtilis SecA. Consistent with the substantial increase in entropy observed in the course of the ECT, hydrogen-deuterium exchange mass spectrometry demonstrates that it increases protein backbone dynamics in domain-domain interfaces at remote locations from the ATPase active site. The catalytic glutamate is one of ~250 charged amino acids in SecA, and yet neutralization of its side chain charge is sufficient to trigger a global order-disorder transition in this 100 kDa enzyme. The intricate network of structural interactions mediating this effect couples local electrostatic changes during ATP hydrolysis to global conformational and dynamic changes in SecA. This network forms the foundation of the allosteric mechanochemistry that efficiently harnesses the chemical energy stored in ATP to drive complex mechanical processes. PMID:23167435

  4. Predicting order of conformational changes during protein conformational transitions using an interpolated elastic network model.

    PubMed

    Tekpinar, Mustafa; Zheng, Wenjun

    2010-08-15

    The decryption of sequence of structural events during protein conformational transitions is essential to a detailed understanding of molecular functions of various biological nanomachines. Coarse-grained models have proven useful by allowing highly efficient simulations of protein conformational dynamics. By combining two coarse-grained elastic network models constructed based on the beginning and end conformations of a transition, we have developed an interpolated elastic network model to generate a transition pathway between the two protein conformations. For validation, we have predicted the order of local and global conformational changes during key ATP-driven transitions in three important biological nanomachines (myosin, F(1) ATPase and chaperonin GroEL). We have found that the local conformational change associated with the closing of active site precedes the global conformational change leading to mechanical motions. Our finding is in good agreement with the distribution of intermediate experimental structures, and it supports the importance of local motions at active site to drive or gate various conformational transitions underlying the workings of a diverse range of biological nanomachines.

  5. Potent and selective activation of abscisic acid receptors in vivo by mutational stabilization of their agonist-bound conformation

    PubMed Central

    Mosquna, Assaf; Peterson, Francis C.; Park, Sang-Youl; Lozano-Juste, Jorge; Volkman, Brian F.; Cutler, Sean R.

    2011-01-01

    Pyrabactin resistance (PYR) 1 and its relatives belong to a family of soluble abscisic acid (ABA) receptors that inhibit type 2C protein phosphatases (PP2C) when in their agonist-stabilized conformation. Given their switch-like properties, we envisioned that mutations that stabilize their agonist-bound conformation could be used to activate signaling in vivo. To identify such mutations, we subjected PYR1 to site-saturation mutagenesis at 39 highly conserved residues that participate in ABA or PP2C contacts. All 741 possible single amino acid substitutions at these sites were tested to identify variants that increase basal PYR1-PP2C interactions, which uncovered activating mutations in 10 residues that preferentially cluster in PYR1's gate loop and C-terminal helix. The mutations cause measurable but incomplete receptor activation in vitro; however, specific triple and quadruple mutant combinations were constructed that promote an agonist-bound conformation, as measured by heteronuclear single quantum coherence NMR, and lead to full receptor activation. Moreover, these mutations retain functionality when introduced into divergent family members, and can therefore be used to dissect individual receptor function in vivo, which has been problematic because of redundancy and family size. Expression of activated PYL2 in Arabidopsis seeds activates ABA signaling by a number of measures: modulation of ABA-regulated gene expression, induction of hyperdormancy, and suppression of ABA deficiency phenotypes in the aba2-1 mutant. Our results set the stage for systematic gain-of-function studies of PYR1 and related ABA receptors and reveal that, despite the large number of receptors, activation of a single receptor is sufficient to activate signaling in planta. PMID:22139369

  6. Conformational Dynamics of Kir3.1/Kir3.2 Channel Activation Via δ-Opioid Receptors

    PubMed Central

    Richard-Lalonde, Melissa; Nagi, Karim; Audet, Nicolas; Sleno, Rory; Amraei, Mohammad; Hogue, Mireille; Balboni, Gianfranco; Schiller, Peter W.; Bouvier, Michel; Hébert, Terence E.; Pineyro, Graciela

    2013-01-01

    This study assessed how conformational information encoded by ligand binding to δ-opioid receptors (DORs) is transmitted to Kir3.1/Kir3.2 channels. Human embryonic kidney 293 cells were transfected with bioluminescence resonance energy transfer (BRET) donor/acceptor pairs that allowed us to evaluate independently reciprocal interactions among signaling partners. These and coimmunoprecipitation studies indicated that DORs, Gβγ, and Kir3 subunits constitutively interacted with one another. GαoA associated with DORs and Gβγ, but despite being part of the complex, no evidence of its direct association with the channel was obtained. DOR activation by different ligands left DOR-Kir3 interactions unmodified but modulated BRET between DOR-GαoA, DOR-Gβγ, GαoA-Gβγ, and Gβγ-Kir3 interfaces. Ligand-induced BRET changes assessing Gβγ-Kir3.1 subunit interaction 1) followed similar kinetics to those monitoring the GαoA-Gβγ interface, 2) displayed the same order of efficacy as those observed at the DOR-Gβγ interface, 3) were sensitive to pertussis toxin, and 4) were predictive of whether a ligand could evoke channel currents. Conformational changes at the Gβγ/Kir3 interface were lost when Kir3.1 subunits were replaced by a mutant lacking essential sites for Gβγ-mediated activation. Thus, conformational information encoded by agonist binding to the receptor is relayed to the channel via structural rearrangements that involve repositioning of Gβγ with respect to DORs, GαoA, and channel subunits. Further, the fact that BRET changes at the Gβγ-Kir3 interface are predictive of a ligand’s ability to induce channel currents points to these conformational biosensors as screening tools for identifying GPCR ligands that induce Kir3 channel activation. PMID:23175530

  7. Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates

    PubMed Central

    Teixeira, Luis Gustavo D.; Malavolta, Luciana; Bersanetti, Patrícia A.; Schreier, Shirley; Carmona, Adriana K.; Nakaie, Clovis R.

    2015-01-01

    Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the

  8. Dimethyl sulfoxide induced structural transformations and non-monotonic concentration dependence of conformational fluctuation around active site of lysozyme

    NASA Astrophysics Data System (ADS)

    Roy, Susmita; Jana, Biman; Bagchi, Biman

    2012-03-01

    Experimental studies have observed significant changes in both structure and function of lysozyme (and other proteins) on addition of a small amount of dimethyl sulfoxide (DMSO) in aqueous solution. Our atomistic molecular dynamic simulations of lysozyme in water-DMSO reveal the following sequence of changes on increasing DMSO concentration. (i) At the initial stage (around 5% DMSO concentration) protein's conformational flexibility gets markedly suppressed. From study of radial distribution functions, we attribute this to the preferential solvation of exposed protein hydrophobic residues by the methyl groups of DMSO. (ii) In the next stage (10-15% DMSO concentration range), lysozome partially unfolds accompanied by an increase both in fluctuation and in exposed protein surface area. (iii) Between 15-20% concentration ranges, both conformational fluctuation and solvent accessible protein surface area suddenly decrease again indicating the formation of an intermediate collapse state. These results are in good agreement with near-UV circular dichroism (CD) and fluorescence studies. We explain this apparently surprising behavior in terms of a structural transformation which involves clustering among the methyl groups of DMSO. (iv) Beyond 20% concentration of DMSO, the protein starts its final sojourn towards the unfolding state with further increase in conformational fluctuation and loss in native contacts. Most importantly, analysis of contact map and fluctuation near the active site reveal that both partial unfolding and conformational fluctuations are centered mostly on the hydrophobic core of active site of lysozyme. Our results could offer a general explanation and universal picture of the anomalous behavior of protein structure-function observed in the presence of cosolvents (DMSO, ethanol, tertiary butyl alcohol, dioxane) at their low concentrations.

  9. An adjustment in NiTi closed coil spring for an extended range of activation.

    PubMed

    Ravipati, Raghu Ram; Sivakumar, Arunachalam; Sudhakar, P; Padmapriya, C V; Bhaskar, Mummudi; Azharuddin, Mohammad

    2014-01-01

    The Nickel Titanium (NiTi) closed coil springs serve as an efficient force delivery system in orthodontic space closure mechanics. The closed coil springs with the eyelets come in various lengths to broaden its force characteristics for an expedient space closure. However, at a certain point of time of progressive space closure, the coil spring can be expanded no further for an adequate force delivery. In such situations, the clinician prefers to replace the existing spring with another short length spring. The present article describes a simple conservative technique for progressively re-activating the same NiTi closed coil spring for complete space closure.

  10. Immobilization of the distal hinge in the labile serpin plasminogen activator inhibitor 1: identification of a transition state with distinct conformational and functional properties.

    PubMed

    De Taeye, Bart; Compernolle, Griet; Dewilde, Maarten; Biesemans, Wouter; Declerck, Paul J

    2003-06-27

    The serpin plasminogen activator inhibitor-1 (PAI-1) plays an important role in the regulation of the fibrinolytic activity in blood. In plasma, PAI-1 circulates mainly in the active conformation. However, PAI-1 spontaneously converts to a latent conformation. This conversion comprises drastic conformational changes in both the distal and the proximal hinge region of the reactive center loop. To study the functional and conformational rearrangements associated solely with the mobility of the proximal hinge, disulfide bonds were introduced to immobilize the distal hinge region. These mutants exhibited specific activities comparable with that of PAI-1-wt. However, the engineered disulfide bond had a major effect on the conformational and associated functional transitions. Strikingly, in contrast to PAI-1-wt, inactivation of these mutants yielded a virtually complete conversion to a substrate-like conformation. Comparison of the digestion pattern (with trypsin and elastase) of the mutants and PAI-1-wt revealed that the inactivated mutants have a conformation differing from that of latent and active PAI-1-wt. Unique trypsin-susceptible cleavage sites arose upon inactivation of these mutants. The localization of these exposed residues provides evidence that a displacement of alphahF has occurred, indicating that the proximal hinge is partly inserted between s3A and s5A. In conclusion, immobilization of the distal hinge region in PAI-1 allowed the identification of an "intermediate" conformation characterized by a partial insertion of the proximal hinge region. We hypothesize that locking PAI-1 in this transition state between active and latent conformations is associated with a displacement of alphahF, subsequently resulting in substrate behavior.

  11. The Effects of Close Companions (and Rotation) on the Magnetic Activity of M Dwarfs

    NASA Astrophysics Data System (ADS)

    Morgan, Dylan P.; West, Andrew A.; Garcés, Ane; Catalán, Silvia; Dhital, Saurav; Fuchs, Miriam; Silvestri, Nicole M.

    2012-10-01

    We present a study of close white dwarf and M dwarf (WD+dM) binary systems and examine the effect that a close companion has on the magnetic field generation in M dwarfs. We use a base sample of 1602 white dwarf main-sequence binaries from Rebassa-Mansergas et al. to develop a set of color cuts in GALEX, SDSS, UKIDSS, and 2MASS color space. Then using the SDSS Data Release 8 spectroscopic database, we construct a sample of 1756 WD+dM high-quality pairs from our color cuts and previous catalogs. We separate the individual WD and dM from each spectrum using an iterative technique that compares the WD and dM components to best-fit templates. Using the absolute height above the Galactic plane as a proxy for age, and the Hα emission line as an indicator for magnetic activity, we investigate the age-activity relation for our sample for spectral types <= M7. Our results show that early-type M dwarfs (<=M4) in close binary systems are more likely to be active and have longer activity lifetimes compared to their field counterparts. However, at a spectral type of M5 (just past the onset of full convection in M dwarfs), the activity fraction and lifetimes of WD+dM binary systems become more comparable to that of the field M dwarfs. One of the implications of having a close binary companion is presumed to be increased stellar rotation through disk disruption, tidal effects, or angular momentum exchange. Thus, we interpret the similarity in activity behavior between late-type dMs in WD+dM pairs and late-type field dMs to be due to a decrease in sensitivity in close binary companions (or stellar rotation), which has implications for the nature of magnetic activity in fully convective stars. Using the WD components of the pairs, we find WD cooling ages to use as an additional constraint on the age-activity relation for our sample. We find that, on average, active early-type dMs tend to be younger and that active late-type dMs span a much broader age regime making them

  12. THE EFFECTS OF CLOSE COMPANIONS (AND ROTATION) ON THE MAGNETIC ACTIVITY OF M DWARFS

    SciTech Connect

    Morgan, Dylan P.; West, Andrew A.; Dhital, Saurav; Fuchs, Miriam; Garces, Ane; Catalan, Silvia; Silvestri, Nicole M.

    2012-10-01

    We present a study of close white dwarf and M dwarf (WD+dM) binary systems and examine the effect that a close companion has on the magnetic field generation in M dwarfs. We use a base sample of 1602 white dwarf main-sequence binaries from Rebassa-Mansergas et al. to develop a set of color cuts in GALEX, SDSS, UKIDSS, and 2MASS color space. Then using the SDSS Data Release 8 spectroscopic database, we construct a sample of 1756 WD+dM high-quality pairs from our color cuts and previous catalogs. We separate the individual WD and dM from each spectrum using an iterative technique that compares the WD and dM components to best-fit templates. Using the absolute height above the Galactic plane as a proxy for age, and the H{alpha} emission line as an indicator for magnetic activity, we investigate the age-activity relation for our sample for spectral types {<=} M7. Our results show that early-type M dwarfs ({<=}M4) in close binary systems are more likely to be active and have longer activity lifetimes compared to their field counterparts. However, at a spectral type of M5 (just past the onset of full convection in M dwarfs), the activity fraction and lifetimes of WD+dM binary systems become more comparable to that of the field M dwarfs. One of the implications of having a close binary companion is presumed to be increased stellar rotation through disk disruption, tidal effects, or angular momentum exchange. Thus, we interpret the similarity in activity behavior between late-type dMs in WD+dM pairs and late-type field dMs to be due to a decrease in sensitivity in close binary companions (or stellar rotation), which has implications for the nature of magnetic activity in fully convective stars. Using the WD components of the pairs, we find WD cooling ages to use as an additional constraint on the age-activity relation for our sample. We find that, on average, active early-type dMs tend to be younger and that active late-type dMs span a much broader age regime making them

  13. Synthesis of selenium-containing Artemisia sphaerocephala polysaccharides: Solution conformation and anti-tumor activities in vitro.

    PubMed

    Wang, Junlong; Li, Qingyao; Bao, Aijuan; Liu, Xiurong; Zeng, Junyuan; Yang, Xiaopin; Yao, Jian; Zhang, Ji; Lei, Ziqiang

    2016-11-01

    It has been reported in our previous work that selenized Artemisia sphaerocephala polysaccharides (SeASPs) with the Se content range of 168-1703μg/g were synthesized by using Na2SeO3/HNO3/BaCl2 system. In the present work, the solution property of SeASP was studied by using size exclusion chromatography combined with multi angle laser light scattering (SEC-MALLS). A decrease in df values indicated that SeASPs with different conformational features that were highly dependent on MW. SeASPs exhibited a more rigid conformation (df value of 1.29-1.52) in low molecular weight range (MW of 1.026-1.426×10(4)g/mol) and compact spherical conformation in high molecular weight range (MW of 2.268-4.363×10(4)g/mol). It could be due to the degradation of polysaccharide chains in HNO3, which was supported in monosaccharide composition analysis. Congo red (CR) spectrophotometric method and atomic force microscopy (AFM) results also confirmed the conformational transition and the evidence on the shape of the rigid chains. In vitro anti-tumor assays, SeASP2 displayed greater anti-proliferative effects against three tumor cell lines (hepatocellular carcinoma HepG-2 cells, lung adenocarcinom A549 cells and cervical squamous carcinoma Hela cells) in a dose-dependent manner. This suggested that selenylation could significantly enhance the anti-tumor activities of polysaccharide derivatives in vitro. PMID:27516251

  14. Small angle neutron scattering reveals pH-dependent conformational changes in Trichoderma reesei cellobiohydrolase I: implications for enzymatic activity.

    PubMed

    Pingali, Sai Venkatesh; O'Neill, Hugh M; McGaughey, Joseph; Urban, Volker S; Rempe, Caroline S; Petridis, Loukas; Smith, Jeremy C; Evans, Barbara R; Heller, William T

    2011-09-16

    Cellobiohydrolase I (Cel7A) of the fungus Trichoderma reesei (now classified as an anamorph of Hypocrea jecorina) hydrolyzes crystalline cellulose to soluble sugars, making it of key interest for producing fermentable sugars from biomass for biofuel production. The activity of the enzyme is pH-dependent, with its highest activity occurring at pH 4-5. To probe the response of the solution structure of Cel7A to changes in pH, we measured small angle neutron scattering of it in a series of solutions having pH values of 7.0, 6.0, 5.3, and 4.2. As the pH decreases from 7.0 to 5.3, the enzyme structure remains well defined, possessing a spatial differentiation between the cellulose binding domain and the catalytic core that only changes subtly. At pH 4.2, the solution conformation of the enzyme changes to a structure that is intermediate between a properly folded enzyme and a denatured, unfolded state, yet the secondary structure of the enzyme is essentially unaltered. The results indicate that at the pH of optimal activity, the catalytic core of the enzyme adopts a structure in which the compact packing typical of a fully folded polypeptide chain is disrupted and suggest that the increased range of structures afforded by this disordered state plays an important role in the increased activity of Cel7A through conformational selection.

  15. Visualization of Activated Platelets by Targeted Magnetic Resonance Imaging Utilizing Conformation-Specific Antibodies against Glycoprotein IIb/IIIa

    PubMed Central

    von zur Muhlen, Constantin; Peter, Karlheinz; Ali, Ziad A.; Schneider, Jürgen E.; McAteer, Martina A.; Neubauer, Stefan; Channon, Keith M.; Bode, Christoph; Choudhury, Robin P.

    2009-01-01

    Ruptured atherosclerotic plaques, lined with activated platelets, constitute an attractive target for magnetic resonance imaging (MRI). This study evaluated whether microparticles of iron oxide (MPIO) targeting ligand-induced binding sites (LIBS) on the activated conformation of glycoprotein IIb/IIIa could be used to image platelets. MPIO (size: 1 μm) were conjugated to anti-LIBS or control single-chain antibody. Following guidewire injury to mouse femoral artery, platelet adhesion was present after 24 h. Mice were perfused with anti-LIBS-MPIO (or control MPIO) via the left ventricle and 11.7-tesla MRI was performed on femoral arteries ex vivo. A 3D gradient echo sequence attained an isotropic resolution of 25 μm. MPIO binding, quantified by MRI, was 4-fold higher with anti-LIBS-MPIO in comparison to control MPIO (p < 0.01). In histological sections, low signal zones on MRI and MPIO correlated strongly (R2 = 0.72; p < 0.001), indicating accurate MR quantification. In conclusion, anti-LIBS-MPIO bind to activated platelets in mouse arteries, providing a basis for the use of function-specific single-chain antibody-MPIO conjugates for molecular MRI, and represent the first molecular imaging of a conformational change in a surface receptor. This presents an opportunity to specifically image activated platelets involved in acute atherothrombosis with MRI. PMID:18515970

  16. Spacer conformation in biologically active molecules. Part 2. Structure and conformation of 4-[2-(diphenylmethylamino)ethyl]-1-(2-methoxyphenyl) piperazine and its diphenylmethoxy analog—potential 5-HT 1A receptor ligands

    NASA Astrophysics Data System (ADS)

    Karolak-Wojciechowska, J.; Fruziński, A.; Czylkowski, R.; Paluchowska, M. H.; Mokrosz, M. J.

    2003-09-01

    As a part of studies on biologically active molecule structures with aliphatic linking chain, the structures of 4-[2-diphenylmethylamino)ethyl]-1-(2-methoxyphenyl)piperazine dihydrochloride ( 1) and 4-[2-diphenylmethoxy)ethyl]-1-(2-methoxyphenyl)piperazine fumarate ( 2) have been reported. In both compounds, four atomic non-all-carbons linking chains (N)C-C-X-C are present. The conformation of that linking spacer depends on the nature of the X-atom. The preferred conformation for chain with XNH has been found to be fully extended while for that with XO—the bend one. It was confirmed by conformational calculations (strain energy distribution and random search) and crystallographic data, including statistics from CCDC.

  17. Modeling of solvent-dependent conformational transitions in Burkholderia cepacia lipase

    PubMed Central

    Trodler, Peter; Schmid, Rolf D; Pleiss, Jürgen

    2009-01-01

    Background The characteristic of most lipases is the interfacial activation at a lipid interface or in non-polar solvents. Interfacial activation is linked to a large conformational change of a lid, from a closed to an open conformation which makes the active site accessible for substrates. While for many lipases crystal structures of the closed and open conformation have been determined, the pathway of the conformational transition and possible bottlenecks are unknown. Therefore, molecular dynamics simulations of a closed homology model and an open crystal structure of Burkholderia cepacia lipase in water and toluene were performed to investigate the influence of solvents on structure, dynamics, and the conformational transition of the lid. Results The conformational transition of B. cepacia lipase was dependent on the solvent. In simulations of closed B. cepacia lipase in water no conformational transition was observed, while in three independent simulations of the closed lipase in toluene the lid gradually opened during the first 10–15 ns. The pathway of conformational transition was accessible and a barrier was identified, where a helix prevented the lid from opening to the completely open conformation. The open structure in toluene was stabilized by the formation of hydrogen bonds. In simulations of open lipase in water, the lid closed slowly during 30 ns nearly reaching its position in the closed crystal structure, while a further lid opening compared to the crystal structure was observed in toluene. While the helical structure of the lid was intact during opening in toluene, it partially unfolded upon closing in water. The closing of the lid in water was also observed, when with eight intermediate structures between the closed and the open conformation as derived from the simulations in toluene were taken as starting structures. A hydrophobic β-hairpin was moving away from the lid in all simulations in water, which was not observed in simulations in

  18. Effects of calcium binding and the hypertrophic cardiomyopathy A8V mutation on the dynamic equilibrium between closed and open conformations of the regulatory N-domain of isolated cardiac troponin C.

    PubMed

    Cordina, Nicole M; Liew, Chu K; Gell, David A; Fajer, Piotr G; Mackay, Joel P; Brown, Louise J

    2013-03-19

    Troponin C (TnC) is the calcium-binding subunit of the troponin complex responsible for initiating striated muscle contraction in response to calcium influx. In the skeletal TnC isoform, calcium binding induces a structural change in the regulatory N-domain of TnC that involves a transition from a closed to open structural state and accompanying exposure of a large hydrophobic patch for troponin I (TnI) to subsequently bind. However, little is understood about how calcium primes the N-domain of the cardiac isoform (cTnC) for interaction with the TnI subunit as the open conformation of the regulatory domain of cTnC has been observed only in the presence of bound TnI. Here we use paramagnetic relaxation enhancement (PRE) to characterize the closed to open transition of isolated cTnC in solution, a process that cannot be observed by traditional nuclear magnetic resonance methods. Our PRE data from four spin-labeled monocysteine constructs of isolated cTnC reveal that calcium binding triggers movement of the N-domain helices toward an open state. Fitting of the PRE data to a closed to open transition model reveals the presence of a small population of cTnC molecules in the absence of calcium that possess an open conformation, the level of which increases substantially upon Ca(2+) binding. These data support a model in which calcium binding creates a dynamic equilibrium between the closed and open structural states to prime cTnC for interaction with its target peptide. We also used PRE data to assess the structural effects of a familial hypertrophic cardiomyopathy point mutation located within the N-domain of cTnC (A8V). The PRE data show that the Ca(2+) switch mechanism is perturbed by the A8V mutation, resulting in a more open N-domain conformation in both the apo and holo states.

  19. An evolutionary vaccination game in the modified activity driven network by considering the closeness

    NASA Astrophysics Data System (ADS)

    Han, Dun; Sun, Mei

    2016-02-01

    In this paper, we explore an evolutionary vaccination game in the modified activity driven network by considering the closeness. We set a closeness parameter p which is used to describe the way of connection between two individuals. The simulation results show that the closeness p may have an active role in weakening both the spreading of epidemic and the vaccination. Besides, when vaccination is not allowed, the final recovered density increases with the value of the ratio of the infection rate to the recovery rate λ / μ. However, when vaccination is allowed the final density of recovered individual first increases and then decreases with the value of λ / μ. Two variables are designed to identify the relation between the individuals' activities and their states. The results draw that both recovered and vaccinated frequency increase with the increase of the individuals' activities. Meanwhile, the immune fee has less impact on the individuals' vaccination than the closeness. While the λ / μ is in a certain range, with the increase of the value of λ / μ, the recovered frequency of the whole crowds reduces. Our results, therefore, reveal the fact that the best of intentions may lead to backfire.

  20. 78 FR 44622 - Agency Information Collection; Activity Under OMB Review; Confidential Close Call Reporting System

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-24

    ..., 2013 (78 FR 27479) and the comment period ended on July 10, 2013. The 60-day notice produced no... Research and Innovative Technology Administration Agency Information Collection; Activity Under OMB Review; Confidential Close Call Reporting System AGENCY: Research & Innovative Technology Administration (RITA),...

  1. ATPase activity and ATP/ADP-induced conformational change in the soluble domain of the bacterial protein translocator HlyB.

    PubMed

    Koronakis, V; Hughes, C; Koronakis, E

    1993-06-01

    The haemolysin exporter HlyB and its homologues are central to the unconventional signal-peptide-independent secretion of toxins, proteases and nodulation proteins by bacteria. HlyB is a member of the ATP-binding cassette (ABC) or traffic ATPase superfamily, and resembles closely in structure and function mammalian exporters such as the multidrug-resistance P-glycoprotein, combining both integral membrane and cytosolic domains. Overproduction of the HlyB cytoplasmic domain as a C-terminal peptide fused to glutathione S-transferase allowed the direct affinity purification and concentration of 30-50 mg ml-1 of soluble protein (GST-Bctp) in an apparently dimeric form possessing both transferase and ATPase activity. GST-Bctp bound to ADP-agarose and was eluted specifically by ATP and ADP, affinity behaviour which was confirmed in both the full-length HlyB and the unfused HlyB cytoplasmic domain synthesized in vitro. The stoichiometry of binding to MgATP and MgADP was close to equimolar and both ligands induced substantial conformational change in the protein. Mg(2+)-dependent ATPase activity of GST-Bctp (Vmax 1 mumol min-1 mg-1, Km 0.2 mM) was comparable with the activity of the bacterial importer MalK and human P-glycoprotein reconstituted into proteoliposomes, and over an order of magnitude higher than in vitro measurements of disaggregated MalK purified from inclusion bodies. Activity was unaffected by inhibitors of F- and V-type ATPases, non-hydrolysable ATP analogues, or translocation substrate, but was severely inhibited by inhibitors of E1E2 (P-type) ATPases, and the acidic phospholipid phosphatidyl glycerol. PMID:8361361

  2. Lecithin cholesterol acyltransferase (LCAT) activity in the presence of Apo-AI-derived peptides exposed to disorder-order conformational transitions.

    PubMed

    Aguilar-Espinosa, S L; Mendoza-Espinosa, P; Delgado-Coello, B; Mas-Oliva, J

    2013-10-25

    Although the association of Apo AI with HDLs has been proposed to activate LCAT activity, the detailed molecular mechanisms involved in the process are not known. Therefore, in this study we have investigated how conformational changes in several exposed regions of Apo-AI might cause LCAT activation and for this purpose, designed a strategy to investigate three Apo AI-derived peptides. Since these peptides present the ability to adopt several secondary structure conformations, they were used to determine whether LCAT activity could be modulated in the presence of a particular conformation. Circular dichroism experiments showed that Apo AI-derived peptides in PBS displayed a disordered arrangement, with a strong tendency to adopt β-sheet and random conformational structures as a function of concentration. However, in the presence of Lyso-C12PC, maximal percentages of α-helical structures were observed. Performed in human plasma, time-course experiments of LCAT activity under control conditions reached the highest level of (3)H-cholesteryl esters after 2.5 h incubation. In the presence of Apo AI-derived peptides, a significant increase in the production of (3)H-cholesteryl esters was observed. The present study provides an important insight into the potential interactions between LCAT and lipoproteins and also suggests that peptides, initially present in a disordered conformation, are able to sense the lipid environment provided by lipoproteins of plasma and following a disorder-to-order transition, change their conformation to an ordered α-helix. PMID:24513206

  3. Lecithin cholesterol acyltransferase (LCAT) activity in the presence of Apo-AI-derived peptides exposed to disorder-order conformational transitions.

    PubMed

    Aguilar-Espinosa, S L; Mendoza-Espinosa, P; Delgado-Coello, B; Mas-Oliva, J

    2013-11-15

    Although the association of Apo AI with HDLs has been proposed to activate LCAT activity, the detailed molecular mechanisms involved in the process are not known. Therefore, in this study we have investigated how conformational changes in several exposed regions of Apo-AI might cause LCAT activation and for this purpose, designed a strategy to investigate three Apo AI-derived peptides. Since these peptides present the ability to adopt several secondary structure conformations, they were used to determine whether LCAT activity could be modulated in the presence of a particular conformation. Circular dichroism experiments showed that Apo AI-derived peptides in PBS displayed a disordered arrangement, with a strong tendency to adopt β-sheet and random conformational structures as a function of concentration. However, in the presence of Lyso-C12PC, maximal percentages of α-helical structures were observed. Performed in human plasma, time-course experiments of LCAT activity under control conditions reached the highest level of (3)H-cholesteryl esters after 2.5 h incubation. In the presence of Apo AI-derived peptides, a significant increase in the production of (3)H-cholesteryl esters was observed. The present study provides an important insight into the potential interactions between LCAT and lipoproteins and also suggests that peptides, initially present in a disordered conformation, are able to sense the lipid environment provided by lipoproteins of plasma and following a disorder-to-order transition, change their conformation to an ordered α-helix. PMID:24383078

  4. Type I beta-turn conformation is important for biological activity of the melanocyte-stimulating hormone analogues.

    PubMed

    Li, S Z; Lee, J H; Lee, W; Yoon, C J; Baik, J H; Lim, S K

    1999-10-01

    In order to define which structure of alpha-melanocyte-stimulating hormone (MSH) analogues plays a critical role for ligand-receptor interaction and selectivity, we analysed receptor-binding and cAMP-generating activity in Chinese hamster ovary cell lines stably transfected with rMC3R and hMC4R, as well as the NMR structures of chemically synthesized alpha-MSH analogues. Compared with [Ahx4]alpha-MSH, the linear MTII designated as alpha-MSH-ND revealed a preference for the MC4R, whereas its IC50 and EC50 values were comparable to those of MTII reported previously. Truncation of Ahx4 and Asp5 of alpha-MSH-ND remarkably decreased the receptor-binding and cAMP-generating activity. Meanwhile, maximum cAMP-generating activity was observed at a higher concentration (10(-5) M) of alpha-MSH-ND(6-10), and MC4R preference was changed into MC3R preference. In contrast, [Gln6]alpha-MSH-ND(6-10) lost its cAMP-generating activity almost completely, even though it bound to both receptors. Whereas the solution conformation of alpha-MSH-ND revealed a stable type I beta-turn structure, [Gln6]alpha-MSH-ND(6-10) revealed a tight gamma-turn composed of Gln6-D-Phe7-Arg8. Replacement of the His6 residue of alpha-MSH-ND by Gln, Asn, Arg or Lys decreased not only the receptor binding, but also the cAMP-generating activity in both the MC3R and the MC4R. The structure of [Gln6]alpha-MSH-ND exhibited a stable type I' beta-turn comprising Asp5, Gln6, D-Phe7 and Arg8. [Lys6]alpha-MSH-ND showed a greatly reduced binding affinity and cAMP-generating activity with the loss of MC4R selectivity. In NMR studies, [Lys6]alpha-MSH-ND also demonstrated a gamma-turn conformation around Lys6-DPhe7-Arg8. From the above results, we conclude that a type I beta-turn conformation comprising the residues Asp5-His6-(D-Phe7)-Arg8 was important for receptor binding and activation, as well as the selectivity of MSH analogues.

  5. The Crystal Structure of a Cardiovirus RNA-Dependent RNA Polymerase Reveals an Unusual Conformation of the Polymerase Active Site

    PubMed Central

    Vives-Adrian, Laia; Lujan, Celia; Oliva, Baldo; van der Linden, Lonneke; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; van Kuppeveld, Frank J. M.

    2014-01-01

    ABSTRACT Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-π interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A. IMPORTANCE The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated

  6. Structure-Activity study of retinoid agonists bearing substituted dicarba-closo-dodecaborane. Relation between retinoidal activity and conformation of two aromatic nuclei.

    PubMed

    Endo, Y; Iijima, T; Yaguchi, K; Kawachi, E; Inoue, N; Kagechika, H; Kubo, A; Itai, A

    2001-05-21

    We have investigated the structure activity relationships of the potent retinoid agonist, 4-[4-(2-propyl-1,2-dicarba-closo-dodecaboran-l-yl)phenylamino]benzoic acid (BR403), which we have previously reported. Substitution of a methyl group on the aromatic nucleus or a methyl group on the nitrogen atom, or replacement of the amino group with ether, methylene, carboxyl or 1,1-ethylene greatly decreased the activity. The relatively planar conformation at the phenyl-N-phenyl moiety seems to play a critical role in the appearance of the biological activity.

  7. The Effects of Close Companions on the Magnetic Activity of M Dwarfs

    NASA Astrophysics Data System (ADS)

    Morgan, D. P.; West, A. A.; Silvestri, N. M.

    2011-12-01

    We present a study of close white dwarf and M dwarf (WD+dM) binary systems that examines the effects that close companions have on magnetic field generation in M dwarfs. We used a base sample of 1602 white dwarf -- main seqeuence (WDMS) binaries from Rebassa-Mansergas et al. to determine a set of color cuts in u, g, r, i, and z. Then using the Sloan Digital Sky Survey (SDSS) Data Release 7 (DR7) we constructed a sample of 2292 WD+dM pairs. We separated the dM and WD from each combined spectrum using an iterative technique that compared the WD and dM components to best-fit templates. Using the absolute height above the Galactic Plane as a proxy for age, and the Hα emission line as an indicator for magnetic activity, we investigated the age-activity relation for our sample; spectral types ≤M5. Our results show that early-type M dwarfs in close binary systems have increased magnetic activity in both magnitude and duration compared to their field counterparts. However, we begin to see a transition at spectral type M5 (just past the onset of full convection in M dwarfs), where the magnitude and duration in close WD+dM binary systems become more comparable to that of the field M dwarfs.

  8. Self-objectification, feminist activism and conformity to feminine norms among female vegetarians, semi-vegetarians, and non-vegetarians.

    PubMed

    Brinkman, Britney G; Khan, Aliya; Edner, Benjamin; Rosén, Lee A

    2014-01-01

    Recent research has suggested that vegetarians may be at an increased risk for developing disordered eating or body image issues when compared to non-vegetarians. However, the results of such studies are mixed, and no research has explored potential connections between vegetarianism and self-objectification. In the current study, the authors examine factors that predicted body surveillance, body shame, and appearance control beliefs; three aspects of self-objectification. Surveys were completed by 386 women from the United States who were categorized as vegetarian, semi-vegetarian, or non-vegetarian. The three groups differed regarding dietary motivations, levels of feminist activism, and body shame, but did not differ on their conformity to feminine norms. While conformity to feminine norms predicted body surveillance and body shame levels among all three groups of women, feminist activism predicted appearance control beliefs among non-vegetarians only. These findings suggest that it is important for researchers and clinicians to distinguish among these three groups when examining the relationship between vegetarianism and self-objectification.

  9. Self-objectification, feminist activism and conformity to feminine norms among female vegetarians, semi-vegetarians, and non-vegetarians.

    PubMed

    Brinkman, Britney G; Khan, Aliya; Edner, Benjamin; Rosén, Lee A

    2014-01-01

    Recent research has suggested that vegetarians may be at an increased risk for developing disordered eating or body image issues when compared to non-vegetarians. However, the results of such studies are mixed, and no research has explored potential connections between vegetarianism and self-objectification. In the current study, the authors examine factors that predicted body surveillance, body shame, and appearance control beliefs; three aspects of self-objectification. Surveys were completed by 386 women from the United States who were categorized as vegetarian, semi-vegetarian, or non-vegetarian. The three groups differed regarding dietary motivations, levels of feminist activism, and body shame, but did not differ on their conformity to feminine norms. While conformity to feminine norms predicted body surveillance and body shame levels among all three groups of women, feminist activism predicted appearance control beliefs among non-vegetarians only. These findings suggest that it is important for researchers and clinicians to distinguish among these three groups when examining the relationship between vegetarianism and self-objectification. PMID:24411771

  10. Ser¹¹⁹ phosphorylation modulates the activity and conformation of PRRXL1, a homeodomain transcription factor.

    PubMed

    Soares-dos-Reis, Ricardo; Pessoa, Ana S; Matos, Mariana R; Falcão, Miguel; Mendes, Vera M; Manadas, Bruno; Monteiro, Filipe A; Lima, Deolinda; Reguenga, Carlos

    2014-05-01

    PRRXL1 [paired related homeobox-like 1; also known as DRG11 (dorsal root ganglia 11)] is a paired-like homeodomain transcription factor expressed in DRG and dSC (dorsal spinal cord) nociceptive neurons. PRRXL1 is crucial for the establishment and maintenance of nociceptive circuitry, as Prrxl1(-/-) mice present neuronal loss, reduced pain sensitivity and failure to thrive. In the present study, we show that PRRXL1 is highly phosphorylated in vivo, and that its multiple band pattern on electrophoretic analysis is the result of different phosphorylation states. PRRXL1 phosphorylation appears to be differentially regulated along the dSC and DRG development and it is mapped to two functional domains. One region comprises amino acids 107-143, whereas the other one encompasses amino acids 227-263 and displays repressor activity. Using an immunoprecipitation-MS approach, two phosphorylation sites were identified, Ser¹¹⁹ and Ser²³⁸. Phosphorylation at Ser¹¹⁹ is shown to be determinant for PRRXL1 conformation and transcriptional activity. Ser¹¹⁹ phosphorylation is thus proposed as a mechanism for regulating PRRXL1 function and conformation during nociceptive system development.

  11. Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to Drug Discovery.

    PubMed

    Hu, Jianping; Feng, Zhiwei; Ma, Shifan; Zhang, Yu; Tong, Qin; Alqarni, Mohammed Hamed; Gou, Xiaojun; Xie, Xiang-Qun

    2016-06-27

    Cannabinoid receptor 2 (CB2), a G protein-coupled receptor (GPCR), is a promising target for the treatment of neuropathic pain, osteoporosis, immune system, cancer, and drug abuse. The lack of an experimental three-dimensional CB2 structure has hindered not only the development of studies of conformational differences between the inactive and active CB2 but also the rational discovery of novel functional compounds targeting CB2. In this work, we constructed models of both inactive and active CB2 by homology modeling. Then we conducted two comparative 100 ns molecular dynamics (MD) simulations on the two systems-the active CB2 bound with both the agonist and G protein and the inactive CB2 bound with inverse agonist-to analyze the conformational difference of CB2 proteins and the key residues involved in molecular recognition. Our results showed that the inactive CB2 and the inverse agonist remained stable during the MD simulation. However, during the MD simulations, we observed dynamical details about the breakdown of the "ionic lock" between R131(3.50) and D240(6.30) as well as the outward/inward movements of transmembrane domains of the active CB2 that bind with G proteins and agonist (TM5, TM6, and TM7). All of these results are congruent with the experimental data and recent reports. Moreover, our results indicate that W258(6.48) in TM6 and residues in TM4 (V164(4.56)-L169(4.61)) contribute greatly to the binding of the agonist on the basis of the binding energy decomposition, while residues S180-F183 in extracellular loop 2 (ECL2) may be of importance in recognition of the inverse agonist. Furthermore, pharmacophore modeling and virtual screening were carried out for the inactive and active CB2 models in parallel. Among all 10 hits, two compounds exhibited novel scaffolds and can be used as novel chemical probes for future studies of CB2. Importantly, our studies show that the hits obtained from the inactive CB2 model mainly act as inverse agonist(s) or neutral

  12. Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to Drug Discovery.

    PubMed

    Hu, Jianping; Feng, Zhiwei; Ma, Shifan; Zhang, Yu; Tong, Qin; Alqarni, Mohammed Hamed; Gou, Xiaojun; Xie, Xiang-Qun

    2016-06-27

    Cannabinoid receptor 2 (CB2), a G protein-coupled receptor (GPCR), is a promising target for the treatment of neuropathic pain, osteoporosis, immune system, cancer, and drug abuse. The lack of an experimental three-dimensional CB2 structure has hindered not only the development of studies of conformational differences between the inactive and active CB2 but also the rational discovery of novel functional compounds targeting CB2. In this work, we constructed models of both inactive and active CB2 by homology modeling. Then we conducted two comparative 100 ns molecular dynamics (MD) simulations on the two systems-the active CB2 bound with both the agonist and G protein and the inactive CB2 bound with inverse agonist-to analyze the conformational difference of CB2 proteins and the key residues involved in molecular recognition. Our results showed that the inactive CB2 and the inverse agonist remained stable during the MD simulation. However, during the MD simulations, we observed dynamical details about the breakdown of the "ionic lock" between R131(3.50) and D240(6.30) as well as the outward/inward movements of transmembrane domains of the active CB2 that bind with G proteins and agonist (TM5, TM6, and TM7). All of these results are congruent with the experimental data and recent reports. Moreover, our results indicate that W258(6.48) in TM6 and residues in TM4 (V164(4.56)-L169(4.61)) contribute greatly to the binding of the agonist on the basis of the binding energy decomposition, while residues S180-F183 in extracellular loop 2 (ECL2) may be of importance in recognition of the inverse agonist. Furthermore, pharmacophore modeling and virtual screening were carried out for the inactive and active CB2 models in parallel. Among all 10 hits, two compounds exhibited novel scaffolds and can be used as novel chemical probes for future studies of CB2. Importantly, our studies show that the hits obtained from the inactive CB2 model mainly act as inverse agonist(s) or neutral

  13. A closed-loop model of the respiratory system: focus on hypercapnia and active expiration.

    PubMed

    Molkov, Yaroslav I; Shevtsova, Natalia A; Park, Choongseok; Ben-Tal, Alona; Smith, Jeffrey C; Rubin, Jonathan E; Rybak, Ilya A

    2014-01-01

    Breathing is a vital process providing the exchange of gases between the lungs and atmosphere. During quiet breathing, pumping air from the lungs is mostly performed by contraction of the diaphragm during inspiration, and muscle contraction during expiration does not play a significant role in ventilation. In contrast, during intense exercise or severe hypercapnia forced or active expiration occurs in which the abdominal "expiratory" muscles become actively involved in breathing. The mechanisms of this transition remain unknown. To study these mechanisms, we developed a computational model of the closed-loop respiratory system that describes the brainstem respiratory network controlling the pulmonary subsystem representing lung biomechanics and gas (O2 and CO2) exchange and transport. The lung subsystem provides two types of feedback to the neural subsystem: a mechanical one from pulmonary stretch receptors and a chemical one from central chemoreceptors. The neural component of the model simulates the respiratory network that includes several interacting respiratory neuron types within the Bötzinger and pre-Bötzinger complexes, as well as the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) representing the central chemoreception module targeted by chemical feedback. The RTN/pFRG compartment contains an independent neural generator that is activated at an increased CO2 level and controls the abdominal motor output. The lung volume is controlled by two pumps, a major one driven by the diaphragm and an additional one activated by abdominal muscles and involved in active expiration. The model represents the first attempt to model the transition from quiet breathing to breathing with active expiration. The model suggests that the closed-loop respiratory control system switches to active expiration via a quantal acceleration of expiratory activity, when increases in breathing rate and phrenic amplitude no longer provide sufficient ventilation. The model

  14. Conformable, flexible, large-area networks of pressure and thermal sensors with organic transistor active matrixes

    NASA Astrophysics Data System (ADS)

    Someya, Takao; Kato, Yusaku; Sekitani, Tsuyoshi; Iba, Shingo; Noguchi, Yoshiaki; Murase, Yousuke; Kawaguchi, Hiroshi; Sakurai, Takayasu

    2005-08-01

    Skin-like sensitivity, or the capability to recognize tactile information, will be an essential feature of future generations of robots, enabling them to operate in unstructured environments. Recently developed large-area pressure sensors made with organic transistors have been proposed for electronic artificial skin (E-skin) applications. These sensors are bendable down to a 2-mm radius, a size that is sufficiently small for the fabrication of human-sized robot fingers. Natural human skin, however, is far more complex than the transistor-based imitations demonstrated so far. It performs other functions, including thermal sensing. Furthermore, without conformability, the application of E-skin on three-dimensional surfaces is impossible. In this work, we have successfully developed conformable, flexible, large-area networks of thermal and pressure sensors based on an organic semiconductor. A plastic film with organic transistor-based electronic circuits is processed to form a net-shaped structure, which allows the E-skin films to be extended by 25%. The net-shaped pressure sensor matrix was attached to the surface of an egg, and pressure images were successfully obtained in this configuration. Then, a similar network of thermal sensors was developed with organic semiconductors. Next, the possible implementation of both pressure and thermal sensors on the surfaces is presented, and, by means of laminated sensor networks, the distributions of pressure and temperature are simultaneously obtained. Author contributions: T. Someya designed research; T. Someya, Y.K., T. Sekitani, S.I., Y.N., Y.M., H.K., and T. Sakurai performed research; and T. Someya wrote the paper.This paper was submitted directly (Track II) to the PNAS office.Freely available online through the PNAS open access option.Abbreviations: E-skin, electronic artificial skin; IDS, source-drain current; PTCDI, 3,4,9,10-perylene-tetracarboxylic-diimide; parylene, polychloro-para-xylylene; CuPc, copper

  15. Dynamic protein conformations preferentially drive energy transfer along the active chain of the photosystem II reaction centre

    NASA Astrophysics Data System (ADS)

    Zhang, Lu; Silva, Daniel-Adriano; Zhang, Houdao; Yue, Alexander; Yan, Yijing; Huang, Xuhui

    2014-06-01

    One longstanding puzzle concerning photosystem II, a core component of photosynthesis, is that only one of the two symmetric branches in its reaction centre is active in electron transfer. To investigate the effect of the photosystem II environment on the preferential selection of the energy transfer pathway (a prerequisite for electron transfer), we have constructed an exciton model via extensive molecular dynamics simulations and quantum mechanics/molecular mechanics calculations based on a recent X-ray structure. Our results suggest that it is essential to take into account an ensemble of protein conformations to accurately compute the site energies. We identify the cofactor CLA606 of active chain as the most probable site for the energy excitation. We further pinpoint a number of charged protein residues that collectively lower the CLA606 site energy. Our work provides insights into the understanding of molecular mechanisms of the core machinery of the green-plant photosynthesis.

  16. Dynamic protein conformations preferentially drive energy transfer along the active chain of the photosystem II reaction centre.

    PubMed

    Zhang, Lu; Silva, Daniel-Adriano; Zhang, Houdao; Yue, Alexander; Yan, YiJing; Huang, Xuhui

    2014-01-01

    One longstanding puzzle concerning photosystem II, a core component of photosynthesis, is that only one of the two symmetric branches in its reaction centre is active in electron transfer. To investigate the effect of the photosystem II environment on the preferential selection of the energy transfer pathway (a prerequisite for electron transfer), we have constructed an exciton model via extensive molecular dynamics simulations and quantum mechanics/molecular mechanics calculations based on a recent X-ray structure. Our results suggest that it is essential to take into account an ensemble of protein conformations to accurately compute the site energies. We identify the cofactor CLA606 of active chain as the most probable site for the energy excitation. We further pinpoint a number of charged protein residues that collectively lower the CLA606 site energy. Our work provides insights into the understanding of molecular mechanisms of the core machinery of the green-plant photosynthesis. PMID:24954746

  17. Influence of sodium alginate pretreated by ultrasound on papain properties: Activity, structure, conformation and molecular weight and distribution.

    PubMed

    Feng, Liping; Cao, Yanping; Xu, Duoxia; You, Sasa; Han, Fu

    2016-09-01

    The aim of the study was to investigate the impact of sodium alginate (ALG) pretreated by ultrasound on the enzyme activity, structure, conformation and molecular weight and distribution of papain. ALG solutions were pretreated with ultrasound at varying power (0.05, 0.15, 0.25, 0.35, 0.45W/cm(2)), 135kHz, 50°C for 20min. The maximum relative activity of papain increased by 10.53% when mixed with ALG pretreated by ultrasound at 0.25W/cm(2), compared with the untreated ALG. The influence of ultrasound pretreated ALG on the conformation and secondary structure of papain were assessed by fluorescence spectroscopy and circular dichroism spectroscopy. The fluorescence spectra revealed that ultrasound pretreated ALG increased the number of tryptophan on papain surface, especially at 0.25W/cm(2). It indicated that ultrasound pretreatment induced molecular unfolding, causing the exposure of more hydrophobic groups and regions from inside to the outside of the papain molecules. Furthermore, ultrasound pretreated ALG resulted in minor changes in the secondary structure of the papain. The content of α-helix was slightly increased after ultrasound pretreatment and no significant change was observed at different ultrasound powers. ALG pretreated by ultrasound enhanced the stability of the secondary structure of papain, especially at 0.25W/cm(2). The free sulfhydryl (SH) content of papain was slightly increased and then decreased with the increase of ultrasonic power. The maximum content of free SH was observed at 0.25W/cm(2), under which the content of the free SH increased by 6.36% compared with the untreated ALG. Dynamic light scattering showed that the effect of ultrasound treatment was mainly the homogenization of the ALG particles in the mixed dispersion. The gel permeation chromatography coupled with the multi-angle laser light scattering photometer analysis showed that the molecular weight (Mw) of papain/ALG was decreased and then increased with the ultrasonic

  18. pH-Dependent Conformational Changes in the HCV NS3 Protein Modulate Its ATPase and Helicase Activities

    PubMed Central

    Ventura, Gustavo Tavares; da Costa, Emmerson Corrêa Brasil; Capaccia, Anne Miranda; Mohana-Borges, Ronaldo

    2014-01-01

    The hepatitis C virus (HCV) infects 170 to 200 million people worldwide and is, therefore, a major health problem. The lack of efficient treatments that specifically target the viral proteins or RNA and its high chronicity rate make hepatitis C the cause of many deaths and hepatic transplants annually. The NS3 protein is considered an important target for the development of anti-HCV drugs because it is composed of two domains (a serine protease in the N-terminal portion and an RNA helicase/NTPase in the C-terminal portion), which are essential for viral replication and proliferation. We expressed and purified both the NS3 helicase domain (NS3hel) and the full-length NS3 protein (NS3FL) and characterized pH-dependent structural changes associated with the increase in their ATPase and helicase activities at acidic pH. Using intrinsic fluorescence experiments, we have observed that NS3hel was less stable at pH 6.4 than at pH 7.2. Moreover, binding curves using an extrinsic fluorescent probe (bis-ANS) and ATPase assays performed under different pH conditions demonstrated that the hydrophobic clefts of NS3 are significantly more exposed to the aqueous medium at acidic pH. Using fluorescence spectroscopy and anisotropy assays, we have also observed more protein interaction with DNA upon pH acidification, which suggests that the hydrophobic clefts exposure on NS3 might be related to a loss of stability that could lead it to adopt a more open conformation. This conformational change at acidic pH would stimulate both its ATPase and helicase activities, as well as its ability to bind DNA. Taken together, our results indicate that the NS3 protein adopts a more open conformation due to acidification from pH 7.2 to 6.4, resulting in a more active form at a pH that is found near Golgi-derived membranes. This increased activity could better allow NS3 to carry out its functions during HCV replication. PMID:25551442

  19. Vibrational and electronic optical activity of the chiral disulphide group: implications for disulphide bridge conformation.

    PubMed

    Bednárová, Lucie; Bour, Petr; Malon, Petr

    2010-05-15

    Using dihydrogendisulphide (H(2)S(2)), dimethyl- ((CH(3))(2)S(2)), and diethyldisulphide ((CH(3)CH(2))(2)S(2))as model molecules, theoretical ECD, VCD, and ROA spectra of nonplanar disulphides were calculated by DFT methods. Most of the calculated electronic and vibrational chiroptical features suffer an equivocal relation between calculatedsigns of ECD, VCD, or ROA and the sense of disulphide nonplanarity as noted earlier for low-lying ECD bands. This is a consequence of local C(2) symmetry of a disulphide group causing most electronic and vibrational transitions to occur as pairs falling to alternative A, B symmetry species, which become degenerate and switch their succession (and consequently the observed chiroptical sign pattern) at the energetically most favorable perpendicular conformation. According to present calculations, the key to resolving this ambiguity may involve the S-S stretching vibrational mode at approximately 500 cm(-1). The relation of signs of the relevant VCD and ROA features to sense of disulphide chirality seems simpler and less ambiguous. The right-handed arrangement of the S-S group (0 < chi(S-S) < 180 degrees) results in mostly negative VCD signals. Although relation to ROA still suffers some ambiguity, it gets clearer along the series H(2)S(2)-(CH(3))(2)S(2)-(CH(3)CH(2))(2)S(2). ROA is also attractive for the analysis of disulphide-containing peptides and proteins, because applying it to aqueous solutions is not problematic.

  20. Evidence that 16S RNA from E. coli can assume two different biologically active conformations.

    PubMed Central

    Hochkeppel, H K; Craven, G R

    1976-01-01

    We have recently shown that 16S RNA can be extracted from 30S ribosomes by an acetic acid-urea precipitation procedure which yields RNA capable of binding 13 individual ribosomal proteins. This is in contrast to phenol extracted 16S RNA which can specifically associate with only 7 proteins2-7. In the experiments reported here, we demonstrate that the difference in protein binding capacities is due to a relatiely more "open" configuration possessed by the acetic acid-urea 16S RNA. Under identical conditions, acetic acid-urea 16S RNA is more susceptible to limited T1-RNase digestion than is phenol-16S RNA. In addition, acetic acid-urea RNA shows a relatively slower electrophoretic mobility. The observable difference in conformation between the two types of RNA is lost by storage at-70 degrees C. This loss is accompanied by a reduction in protein binding capacity of the acetic acid-urea 16S RNA. Images PMID:787927

  1. Activated conformations of the ras-gene-encoded p21 protein. 1. An energy-refined structure for the normal p21 protein complexed with GDP.

    PubMed

    Dykes, D C; Brandt-Rauf, P; Luster, S M; Chung, D; Friedman, F K; Pincus, M R

    1992-06-01

    A complete three-dimensional structure for the ras-gene-encoded p21 protein with Gly 12 and Gln 61, bound to GDP, has been constructed in four stages using the available alpha-carbon coordinates as deposited in the Brookhaven National Laboratories Protein Data Bank. No all-atom structure has been made available despite the fact that the first crystallographic structure for the p21 protein was reported almost four years ago. In the p21 protein, if amino acid substitutions are made at any one of a number of different positions in the amino acid sequence, the protein becomes permanently activated and causes malignant transformation of normal cells or, in some cell lines, differentiation and maturation. For example, all amino acids except Gly and Pro at position 12 result in an oncogenic protein; all amino acids except Gln, Glu and Pro at position 61 likewise cause malignant transformation of cells. We have constructed our all-atom structure of the non-oncogenic protein from the x-ray structure in order to determine how oncogenic amino acid substitutions affect the three-dimensional structure of this protein. In Stage 1 we generated a poly-alanine backbone (except at Gly and Pro residues) through the alpha-carbon structure, requiring the individual Ala, Pro or Gly residues to conform to standard amino acid geometry and to form trans-planar peptide bonds. Since no alpha-carbon coordinates for residues 60-65 have been determined, these residues were modeled by generating them in the extended conformation and then subjecting them to molecular dynamics using the computer application DISCOVER and energy minimization using DISCOVER and the ECEPP (Empirical Conformational Energies for Peptides Program). In Stage 2, the positions of residues that are homologous to corresponding residues of bacterial elongation factor Tu (EF-Tu) to which p21 bears an overall 40% sequence homology, were determined from their corresponding positions in a high-resolution structure of EF-Tu. Non

  2. Exploiting conformational dynamics in drug discovery: design of C-terminal inhibitors of Hsp90 with improved activities

    PubMed Central

    Moroni, Elisabetta; Zhao, Huiping; Blagg, Brian S.J.; Colombo, Giorgio

    2014-01-01

    The interaction that occurs between molecules is a dynamic process that impacts both structural and conformational properties of the ligand and the ligand binding site. Herein, we investigate the dynamic cross-talk between a protein and the ligand as a source for new opportunities in ligand design. Analysis of the formation/disappearance of protein pockets produced in response to a first-generation inhibitor assisted in the identification of functional groups that could be introduced onto scaffolds to facilitate optimal binding, which allowed for increased binding with previously uncharacterized regions. MD simulations were used to elucidate primary changes that occur in the Hsp90 C-terminal binding pocket in the presence of first-generation ligands. This data was then used to design ligands that adapt to these receptor conformations, which provides access to an energy landscape that is not visible in a static model. The newly synthesized compounds demonstrated anti-proliferative activity at ~150 nanomolar concentration. The method identified herein may be used to design chemical probes that provide additional information on structural variations of Hsp90 C-terminal binding site. PMID:24397468

  3. Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent N-Hydroxylase

    PubMed Central

    2015-01-01

    The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N5-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes. PMID:25184411

  4. Monoclonal antibodies that bind the renal Na/sup +//glucose symport system. 2. Stabilization of an active conformation

    SciTech Connect

    Wu, J.S.R.; Lever, J.E.

    1987-09-08

    Conformation-dependent fluorescein isothiocyanate (FITC) labeling of the pig renal Na/sup +//glucose symporter was investigated with specific monoclonal antibodies (MAb's). When renal brush border membranes were pretreated with phenyl isothiocyanate (PITC), washed, and then treated at neutral pH with FITC in the presence of transporter substrates Na/sup +/ and glucose, most of the incorporated fluorescence was associated with a single peak after resolution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular mass of the FITC-labeled species ranged from 79 to 92 kDa. Labeling of this peak was specifically reduced by 70% if Na/sup +/ and glucose were omitted. Na/sup +/ could not be replaced by K/sup +/, Rb/sup +/, or Li/sup +/. FITC labeling of this peak was also stimulated after incubation of membranes with MAb's known to influence high-affinity phlorizin binding, and stimulation was synergistically increased when MAb's were added in the presence of Na/sup +/ and glucose. Substrate-induced or MAb-induced labeling correlated with inactivation of Na/sup +/-dependent phlorizin binding. MAb's recognized an antigen of 75 kDa in the native membranes whereas substrate-induced FITC labeling was accompanied by loss of antigen recognition and protection from proteolysis. These findings are consistent with a model in which MAb's stabilize a Na/sup +/-induced active conformer of the Na/sup +//glucose symport system.

  5. A convergent ring-closing metathesis approach to carbohydrate-based macrolides with potential antibiotic activity.

    PubMed

    Blom, Petra; Ruttens, Bart; Van Hoof, Steven; Hubrecht, Idzi; Van der Eycken, Johan; Sas, Benedikt; Van hemel, Johan; Vandenkerckhove, Jan

    2005-11-25

    [reaction: see text] An efficient convergent approach has been developed for the construction of novel, non-natural, carbohydrate-based macrolides. The key step in the synthesis is the formation of the macrocyclic ring via a ring-closing metathesis reaction. The obtained macrolide analogues have been screened for biological activity against gram-positive and gram-negative bacteria, including resistant strains, yeasts, and molds.

  6. Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes

    PubMed Central

    Goñi, Guillermina M.; Epifano, Carolina; Boskovic, Jasminka; Camacho-Artacho, Marta; Zhou, Jing; Bronowska, Agnieszka; Martín, M. Teresa; Eck, Michael J.; Kremer, Leonor; Gräter, Frauke; Gervasio, Francesco Luigi; Perez-Moreno, Mirna; Lietha, Daniel

    2014-01-01

    Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase (NRTK) with key roles in integrating growth and cell matrix adhesion signals, and FAK is a major driver of invasion and metastasis in cancer. Cell adhesion via integrin receptors is well known to trigger FAK signaling, and many of the players involved are known; however, mechanistically, FAK activation is not understood. Here, using a multidisciplinary approach, including biochemical, biophysical, structural, computational, and cell biology approaches, we provide a detailed view of a multistep activation mechanism of FAK initiated by phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Interestingly, the mechanism differs from canonical NRTK activation and is tailored to the dual catalytic and scaffolding function of FAK. We find PI(4,5)P2 induces clustering of FAK on the lipid bilayer by binding a basic region in the regulatory 4.1, ezrin, radixin, moesin homology (FERM) domain. In these clusters, PI(4,5)P2 induces a partially open FAK conformation where the autophosphorylation site is exposed, facilitating efficient autophosphorylation and subsequent Src recruitment. However, PI(4,5)P2 does not release autoinhibitory interactions; rather, Src phosphorylation of the activation loop in FAK results in release of the FERM/kinase tether and full catalytic activation. We propose that PI(4,5)P2 and its generation in focal adhesions by the enzyme phosphatidylinositol 4-phosphate 5-kinase type Iγ are important in linking integrin signaling to FAK activation. PMID:25049397

  7. Active Site Detection by Spatial Conformity and Electrostatic Analysis—Unravelling a Proteolytic Function in Shrimp Alkaline Phosphatase

    PubMed Central

    Chakraborty, Sandeep; Minda, Renu; Salaye, Lipika; Bhattacharjee, Swapan K.; Rao, Basuthkar J.

    2011-01-01

    Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural information that supplement sequence conservation based analyses. Development of tools that compute electrostatic potentials has further improved our ability to better characterize the active site residues in proteins. We have described a computational methodology for detecting active sites based on structural and electrostatic conformity - CataLytic Active Site Prediction (CLASP). In our pipelined model, physical 3D signature of any particular enzymatic function as defined by its active sites is used to obtain spatially congruent matches. While previous work has revealed that catalytic residues have large pKa deviations from standard values, we show that for a given enzymatic activity, electrostatic potential difference (PD) between analogous residue pairs in an active site taken from different proteins of the same family are similar. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are rejected. We first present the results of active site prediction by CLASP for two enzymatic activities - β-lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA) are also presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP), one of the well known promiscuous enzymes, for additional activities. Such a search has led us to predict a hitherto unknown function of shrimp alkaline phosphatase (SAP), where the protein acts as a protease. Finally, we present experimental evidence of the prediction by CLASP by showing that SAP indeed has protease activity in vitro. PMID

  8. The Effect of Close Companions on the Magnetic Activity of M Dwarfs

    NASA Astrophysics Data System (ADS)

    Morgan, Dylan; West, A. A.; Silvestri, N. M.

    2011-05-01

    We used close white dwarf and M dwarf (WD+dM) binary systems as a method to understand the effect that close companions have on magnetic field generation in M dwarfs. We used a base sample of 1602 white dwarf - main sequence (WDMS) binaries from Rebassa-Mansergas et al. (2010) to aid in determining a set of color cuts using GALEX, SDSS, 2MASS, and UKIDSS colors. Using the Sloan Digital Sky Survey (SDSS) Data Release 8 (DR8) we constructed a sample of 1800 WD+dM pairs. We separated the dM and WD from each combined spectrum using an iterative technique that compares the WD and dM components to best-fit templates. Using the absolute height above the Galactic plane as a proxy for age and the Hα emission line as an indicator for magnetic activity, we investigated the age-activity relation for our sample. Our results show that M dwarfs in close binary systems have increased magnetic activity in both magnitude and duration compared to their field counterparts.

  9. The Activity of Escherichia coli Chaperone SurA Is Regulated by Conformational Changes Involving a Parvulin Domain

    PubMed Central

    Soltes, Garner R.; Schwalm, Jaclyn; Ricci, Dante P.

    2016-01-01

    ABSTRACT The periplasmic chaperone SurA is critical for the biogenesis of outer membrane proteins (OMPs) and, thus, the maintenance of membrane integrity in Escherichia coli. The activity of this modular chaperone has been attributed to a core chaperone module, with only minor importance assigned to the two SurA peptidyl-prolyl isomerase (PPIase) domains. In this work, we used synthetic phenotypes and covalent tethering to demonstrate that the activity of SurA is regulated by its PPIase domains and, furthermore, that its activity is correlated with the conformational state of the chaperone. When combined with mutations in the β-barrel assembly machine (BAM), SurA mutations resulting in deletion of the second parvulin domain (P2) inhibit OMP assembly, suggesting that P2 is involved in the regulation of SurA. The first parvulin domain (P1) potentiates this autoinhibition, as mutations that covalently tether the P1 domain to the core chaperone module severely impair OMP assembly. Furthermore, these inhibitory mutations negate the suppression of and biochemically stabilize the protein specified by a well-characterized gain-of-function mutation in P1, demonstrating that SurA cycles between distinct conformational and functional states during the OMP assembly process. IMPORTANCE This work reveals the reversible autoinhibition of the SurA chaperone imposed by a heretofore underappreciated parvulin domain. Many β-barrel-associated outer membrane (OM) virulence factors, including the P-pilus and type I fimbriae, rely on SurA for proper assembly; thus, a mechanistic understanding of SurA function and inhibition may facilitate antibiotic intervention against Gram-negative pathogens, such as uropathogenic Escherichia coli, E. coli O157:H7, Shigella, and Salmonella. In addition, SurA is important for the assembly of critical OM biogenesis factors, such as the lipopolysaccharide (LPS) transport machine, suggesting that specific targeting of SurA may provide a useful means to

  10. The membrane anchor of the transcriptional activator SREBP is characterized by intrinsic conformational flexibility

    PubMed Central

    Linser, Rasmus; Salvi, Nicola; Briones, Rodolfo; Rovó, Petra; de Groot, Bert L.; Wagner, Gerhard

    2015-01-01

    Regulated intramembrane proteolysis (RIP) is a conserved mechanism crucial for numerous cellular processes, including signaling, transcriptional regulation, axon guidance, cell adhesion, cellular stress responses, and transmembrane protein fragment degradation. Importantly, it is relevant in various diseases including Alzheimer’s disease, cardiovascular diseases, and cancers. Even though a number of structures of different intramembrane proteases have been solved recently, fundamental questions concerning mechanistic underpinnings of RIP and therapeutic interventions remain. In particular, this includes substrate recognition, what properties render a given substrate amenable for RIP, and how the lipid environment affects the substrate cleavage. Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors are critical regulators of genes involved in cholesterol/lipid homeostasis. After site-1 protease cleavage of the inactive SREBP transmembrane precursor protein, RIP of the anchor intermediate by site-2 protease generates the mature transcription factor. In this work, we have investigated the labile anchor intermediate of SREBP-1 using NMR spectroscopy. Surprisingly, NMR chemical shifts, site-resolved solvent exposure, and relaxation studies show that the cleavage site of the lipid-signaling protein intermediate bears rigid α-helical topology. An evolutionary conserved motif, by contrast, interrupts the secondary structure ∼9–10 residues C-terminal of the scissile bond and acts as an inducer of conformational flexibility within the carboxyl-terminal transmembrane region. These results are consistent with molecular dynamics simulations. Topology, stability, and site-resolved dynamics data suggest that the cleavage of the α-helical substrate in the case of RIP may be associated with a hinge motion triggered by the molecular environment. PMID:26392539

  11. Structural Determinants for the Selective Anti-HIV-1 Activity of the All-β Alternative Conformer of XCL1

    PubMed Central

    Guzzo, Christina; Fox, Jamie C.; Miao, Huiyi; Volkman, Brian F.

    2015-01-01

    ABSTRACT HIV-1 replication is regulated in vivo by a complex network of cytokines and chemokines. XCL1/lymphotactin, a unique metamorphic chemokine, was recently identified as a broad-spectrum endogenous HIV-1 inhibitor that blocks viral entry via direct interaction with the gp120 envelope glycoprotein. HIV-1 inhibition by XCL1 requires access to the alternative all-β conformation, which interacts with glycosaminoglycans (GAGs) but not with the specific XCL1 receptor, XCR1. To investigate the structural determinants of the HIV-inhibitory function of XCL1, we performed a detailed structure-function analysis of a stabilized all-β variant, XCL1 W55D. Individual alanine substitutions of two basic residues within the 40s' loop, K42 and R43, abrogated the ability of XCL1 to bind to the viral envelope and block HIV-1 infection; moreover, a loss of HIV-inhibitory function, albeit less marked, was seen upon individual mutation of three additional basic residues: R18, R35, and K46. In contrast, mutation of K42 to arginine did not cause any loss of function, suggesting that the interaction with gp120 is primarily electrostatic in nature. Strikingly, four of these five residues cluster to form a large (∼350 Å2) positively charged surface in the all-β XCL1 conformation, whereas they are dissociated in the classic chemokine fold, which is inactive against HIV-1, providing a structural basis for the selective antiviral activity of the alternatively folded XCL1. Furthermore, we observed that changes to the N-terminal domain, which is proximal to the cluster of putative HIV-1 gp120-interacting residues, also affect the antiviral activity of XCL1. Interestingly, the complement of residues involved in HIV-1 blockade is partially overlapping, but distinct from those involved in the GAG-binding function of XCL1. These data identify key structural determinants of anti-HIV activity in XCL1, providing new templates for the development of HIV-1 entry inhibitors. IMPORTANCE The host

  12. Conformational entropic maps of functional coupling domains in GPCR activation: A case study with beta2 adrenergic receptor

    NASA Astrophysics Data System (ADS)

    Liu, Fan; Abrol, Ravinder; Goddard, William, III; Dougherty, Dennis

    2014-03-01

    Entropic effect in GPCR activation is poorly understood. Based on the recent solved structures, researchers in the GPCR structural biology field have proposed several ``local activating switches'' that consisted of a few number of conserved residues, but have long ignored the collective dynamical effect (conformational entropy) of a domain comprised of an ensemble of residues. A new paradigm has been proposed recently that a GPCR can be viewed as a composition of several functional coupling domains, each of which undergoes order-to-disorder or disorder-to-order transitions upon activation. Here we identified and studied these functional coupling domains by comparing the local entropy changes of each residue between the inactive and active states of the β2 adrenergic receptor from computational simulation. We found that agonist and G-protein binding increases the heterogeneity of the entropy distribution in the receptor. This new activation paradigm and computational entropy analysis scheme provides novel ways to design functionally modified mutant and identify new allosteric sites for GPCRs. The authors thank NIH and Sanofi for funding this project.

  13. Enzymic and immunochemical properties of lysozyme. X. Conformation, enzymic activity and immunochemistry of lysozyme reduced at two carboxyl groups.

    PubMed

    Atassi, M Z; Suliman, A M; Habeeb, A F

    1975-10-20

    Reduction of lysozyme by diborane, followed by air oxidation of the reduced disulfides and chromatography on CM-cellulose, yielded a homogeneous derivative. In the derivative, the carboxyl groups of aspartic acid 119 and the end-chain leucine residue were reduced to their corresponding alcohols. Correct re-forming of the disulfide bonds was demonstrated by peptide mapping of the tryptic hydrolysates of the derivative and lysozyme without breaking the disulfide bonds, followed by identification of the disulfide-containing peptides. Correct disulfide pairing in the two-disulfide peptide in the tryptic hydrolysate was established from its immunochemical behavior. Preparations of the two-disulfide fragment from lysozyme and derivative had equal inhibitory activities (26 or 32%) of the reaction of lysozyme with two homologous antisera. In ORD measurements, lysozyme and the derivative had equal rotatory powers at neutral pH. However, the bo value for the derivative decreased by about 10%. Below pH 6.4 and above pH 8.0, the derivative was less rotatory than native lysozyme. In CD measurements at neutral pH, the negative ellipticity bands at 220 and 208 nm showed little or no decrease in the derivative relative to the native protein. Although conformational differences between the derivative and its parent protein were almost undetectable by ORD and CD measurements, they were readily detected by chemical monitoring of the conformation. In the derivative, both accessibility to tryptic hydrolysis and reducibility of the disulfide bonds increased markedly. The enzymic activity of the derivative was decreased but retained the same pH optimum. With antisera to lysozyme or antisera to the derivative, lysozyme and its derivative possessed equal antigenic reactivities. The immunochemical findings further confirm the correct refolding of the disulfides. Also, they indicate that aspartic acid 119 and the C-terminal leucine residue are not part of an antigenic reactive region in

  14. When your friends make you cringe: social closeness modulates vicarious embarrassment-related neural activity.

    PubMed

    Müller-Pinzler, Laura; Rademacher, Lena; Paulus, Frieder M; Krach, Sören

    2016-03-01

    Social closeness is a potent moderator of vicarious affect and specifically vicarious embarrassment. The neural pathways of how social closeness to another person affects our experience of vicarious embarrassment for the other's public flaws, failures and norm violations are yet unknown. To bridge this gap, we examined the neural response of participants while witnessing threats to either a friend's or a stranger's social integrity. The results show consistent responses of the anterior insula (AI) and anterior cingulate cortex (ACC), shared circuits of the aversive quality of affect, as well as the medial prefrontal cortex and temporal pole, central structures of the mentalizing network. However, the ACC/AI network activation was increased during vicarious embarrassment in response to a friend's failures. At the same time, the precuneus, a brain region associated with self-related thoughts, showed a specific activation and an increase in functional connectivity with the shared circuits in the frontal lobe while observing friends. This might indicate a neural systems mechanism for greater affective sharing and self-involvement while people interact with close others that are relevant to oneself.

  15. Structural basis of conformational transitions in the active site and 80′s loop in the FK506-binding protein FKBP12

    PubMed Central

    Mustafi, Sourajit M.; Brecher, Matthew; Zhang, Jing; Li, Hongmin; Lemaster, David M.; Hernández, Griselda

    2014-01-01

    The extensive set of NMR doublings exhibited by the immunophilin FKBP12 (FK506-binding protein 12) arose from a slow transition to the cis-peptide configuration at Gly89 near the tip of the 80′s loop, the site for numerous protein-recognition interactions for both FKBP12 and other FKBP domain proteins. The 80′s loop also exhibited linebroadening, indicative of microsecond to millisecond conformational dynamics, but only in the trans-peptide state. The G89A variant shifted the trans–cis peptide equilibrium from 88:12 to 33:67, whereas a proline residue substitution induced fully the cis-peptide configuration. The 80′s loop conformation in the G89P crystal structure at 1.50 Å resolution differed from wild-type FKBP12 primarily at residues 88, 89 and 90, and it closely resembled that reported for FKBP52. Structure-based chemical-shift predictions indicated that the microsecond to millisecond dynamics in the 80′s loop probably arose from a concerted main chain (ψ88 and ϕ89) torsion angle transition. The indole side chain of Trp59 at the base of the active-site cleft was reoriented ~90o and the adjacent backbone was shifted in the G89P crystal structure. NOE analysis of wild-type FKBP12 demonstrated that this indole populates the perpendicular orientation at 20%. The 15N relaxation analysis was consistent with the indole reorientation occurring in the nanosecond timeframe. Recollection of the G89P crystal data at 1.20 Å resolution revealed a weaker wild-type-like orientation for the indole ring. Differences in the residues that underlie the Trp59 indole ring and altered interactions linking the 50′s loop to the active site suggested that reorientation of this ring may be disfavoured in the other six members of the FKBP domain family that bear this active-site tryptophan residue. PMID:24405377

  16. Actively Closing the Gap? Social Class, Organized Activities, and Academic Achievement in High School

    ERIC Educational Resources Information Center

    Morris, David S.

    2015-01-01

    Participation in Organized Activities (OA) is associated with positive behavioral and developmental outcomes in children. However, less is known about how particular aspects of participation affect the academic achievement of high school students from different social class positions. Using the Education Longitudinal Study of 2002, this study…

  17. The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation.

    PubMed

    Kanade, Santosh R; Paul, Beena; Rao, A G Appu; Gowda, Lalitha R

    2006-05-01

    Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase)--a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen--and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1+/-2 to 75.9+/-0.6 A (1 A=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack. PMID:16393141

  18. Novel human D-amino acid oxidase inhibitors stabilize an active-site lid-open conformation

    PubMed Central

    Terry-Lorenzo, Ryan T.; Chun, Lawrence E.; Brown, Scott P.; Heffernan, Michele L. R.; Fang, Q. Kevin; Orsini, Michael A.; Pollegioni, Loredano; Hardy, Larry W.; Spear, Kerry L.; Large, Thomas H.

    2014-01-01

    The NMDAR (N-methyl-D-aspartate receptor) is a central regulator of synaptic plasticity and learning and memory. hDAAO (human D-amino acid oxidase) indirectly reduces NMDAR activity by degrading the NMDAR co-agonist D-serine. Since NMDAR hypofunction is thought to be a foundational defect in schizophrenia, hDAAO inhibitors have potential as treatments for schizophrenia and other nervous system disorders. Here, we sought to identify novel chemicals that inhibit hDAAO activity. We used computational tools to design a focused, purchasable library of compounds. After screening this library for hDAAO inhibition, we identified the structurally novel compound, ‘compound 2’ [3-(7-hydroxy-2-oxo-4-phenyl-2H-chromen-6-yl)propanoic acid], which displayed low nM hDAAO inhibitory potency (Ki=7 nM). Although the library was expected to enrich for compounds that were competitive for both D-serine and FAD, compound 2 actually was FAD uncompetitive, much like canonical hDAAO inhibitors such as benzoic acid. Compound 2 and an analog were independently co-crystalized with hDAAO. These compounds stabilized a novel conformation of hDAAO in which the active-site lid was in an open position. These results confirm previous hypotheses regarding active-site lid flexibility of mammalian D-amino acid oxidases and could assist in the design of the next generation of hDAAO inhibitors. PMID:25001371

  19. β-galactosidase at the membrane-water interface: a case of an active enzyme with non-native conformation.

    PubMed

    Sánchez, Julieta M; Nolan, Verónica; Perillo, María A

    2013-08-01

    Previously we demonstrated that Escherichia coli beta-galactosidase (β-Gal) binds to zwitterionic lipid membranes improving its catalytic activity. To understand the activation mechanism from the protein perspective, here the thermal dependence of the catalytic activity was evaluated in conjunction with parameters derived from spectroscopy and calorimetry, in the presence and absence of egg-yolk phosphatidylcholine vesicles. In solution, the native state of β-Gal exhibits a loose conformation according to the λmax of fluorescence emission, which is in the upper end of the emission range for most proteins. A non-two state thermal unfolding mechanism was derived from DSC experiments and supported by the sequential unfolding temperatures exhibited by fluorescence (55°C) and CD (60°C) spectroscopies. Quenching of β-Gal's intrinsic fluorescence, provided evidence for a novel and even looser folding for the lipid-bound protein. However, DSC data showed that the thermal unfolding in the presence of lipids occurred with a significant decrease in ΔH compared to what happened in solution, suggesting that only the population of non-bound protein molecules were involved in this process. Concluding, upon binding to a lipid-water interface β-Gal becomes trapped in a partially unfolded state, more active than that of the native protein in solution.

  20. Conformations of tissue plasminogen activator (tPA) orchestrate neuronal survival by a crosstalk between EGFR and NMDAR

    PubMed Central

    Bertrand, T; Lesept, F; Chevilley, A; Lenoir, S; Aimable, M; Briens, A; Hommet, Y; Bardou, I; Parcq, J; Vivien, D

    2015-01-01

    Tissue-type plasminogen activator (tPA) is a pleiotropic serine protease of the central nervous system (CNS) with reported neurotrophic and neurotoxic functions. Produced and released under its single chain form (sc), the sc-tPA can be cleaved by plasmin or kallikrein in a two chain form, tc-tPA. Although both sc-tPA and tc-tPA display a similar fibrinolytic activity, we postulated here that these two conformations of tPA (sc-tPA and tc-tPA) could differentially control the effects of tPA on neuronal survival. Using primary cultures of mouse cortical neurons, our present study reveals that sc-tPA is the only one capable to promote N-methyl-D-aspartate receptor (NMDAR)-induced calcium influx and subsequent excitotoxicity. In contrast, both sc-tPA and tc-tPA are capable to activate epidermal growth factor receptors (EGFRs), a mechanism mediating the antiapoptotic effects of tPA. Interestingly, we revealed a tPA dependent crosstalk between EGFR and NMDAR in which a tPA-dependent activation of EGFRs leads to downregulation of NMDAR signaling and to subsequent neurotrophic effects. PMID:26469972

  1. Linear Closed-form Solution and Finite-element Analysis of an Active Tensegrity Unit

    NASA Astrophysics Data System (ADS)

    Kmeť, Stanislav; Platko, Peter

    2012-11-01

    Results of the linear closed form solution of an active or adaptive tensegrity unit, as well as its numerical analysis using finite element method are presented in the paper. The shape of the unit is an octahedral cell with a square base and it is formed by thirteen members (four bottom and four top cables, four edge struts and one central strut). The central strut is designed as an actuator that allows for an adjustment of the shape of the unit which leads to changes of tensile forces in the cables. Due to the diagonal symmetry of the 3D tensegrity unit the closed-form analysis is based on the 2D solution of the equivalent planar biconvex cable system with one central strut under a vertical point load.

  2. Enhancement of Chaperone Activity of Plant-Specific Thioredoxin through γ-Ray Mediated Conformational Change.

    PubMed

    Lee, Seung Sik; Jung, Hyun Suk; Park, Soo-Kwon; Lee, Eun Mi; Singh, Sudhir; Lee, Yuno; Lee, Kyun Oh; Lee, Sang Yeol; Chung, Byung Yeoup

    2015-11-13

    AtTDX, a thioredoxin-like plant-specific protein present in Arabidopsis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW) complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins.

  3. Enhancement of Chaperone Activity of Plant-Specific Thioredoxin through γ-Ray Mediated Conformational Change.

    PubMed

    Lee, Seung Sik; Jung, Hyun Suk; Park, Soo-Kwon; Lee, Eun Mi; Singh, Sudhir; Lee, Yuno; Lee, Kyun Oh; Lee, Sang Yeol; Chung, Byung Yeoup

    2015-01-01

    AtTDX, a thioredoxin-like plant-specific protein present in Arabidopsis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW) complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins. PMID:26580605

  4. Enhancement of Chaperone Activity of Plant-Specific Thioredoxin through γ-Ray Mediated Conformational Change

    PubMed Central

    Lee, Seung Sik; Jung, Hyun Suk; Park, Soo-Kwon; Lee, Eun Mi; Singh, Sudhir; Lee, Yuno; Lee, Kyun Oh; Lee, Sang Yeol; Chung, Byung Yeoup

    2015-01-01

    AtTDX, a thioredoxin-like plant-specific protein present in Arabidospis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW) complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins. PMID:26580605

  5. Mapping the conformational space accessible to catechol-O-methyltransferase.

    PubMed

    Ehler, Andreas; Benz, Jörg; Schlatter, Daniel; Rudolph, Markus G

    2014-08-01

    Methylation catalysed by catechol-O-methyltransferase (COMT) is the main pathway of catechol neurotransmitter deactivation in the prefrontal cortex. Low levels of this class of neurotransmitters are held to be causative of diseases such as schizophrenia, depression and Parkinson's disease. Inhibition of COMT may increase neurotransmitter levels, thus offering a route for treatment. Structure-based drug design hitherto seems to be based on the closed enzyme conformation. Here, a set of apo, semi-holo, holo and Michaelis form crystal structures are described that define the conformational space available to COMT and that include likely intermediates along the catalytic pathway. Domain swaps and sizeable loop movements around the active site testify to the flexibility of this enzyme, rendering COMT a difficult drug target. The low affinity of the co-substrate S-adenosylmethionine and the large conformational changes involved during catalysis highlight significant energetic investment to achieve the closed conformation. Since each conformation of COMT is a bona fide target for inhibitors, other states than the closed conformation may be promising to address. Crystallographic data for an alternative avenue of COMT inhibition, i.e. locking of the apo state by an inhibitor, are presented. The set of COMT structures may prove to be useful for the development of novel classes of inhibitors.

  6. Mapping the conformational space accessible to catechol-O-methyltransferase.

    PubMed

    Ehler, Andreas; Benz, Jörg; Schlatter, Daniel; Rudolph, Markus G

    2014-08-01

    Methylation catalysed by catechol-O-methyltransferase (COMT) is the main pathway of catechol neurotransmitter deactivation in the prefrontal cortex. Low levels of this class of neurotransmitters are held to be causative of diseases such as schizophrenia, depression and Parkinson's disease. Inhibition of COMT may increase neurotransmitter levels, thus offering a route for treatment. Structure-based drug design hitherto seems to be based on the closed enzyme conformation. Here, a set of apo, semi-holo, holo and Michaelis form crystal structures are described that define the conformational space available to COMT and that include likely intermediates along the catalytic pathway. Domain swaps and sizeable loop movements around the active site testify to the flexibility of this enzyme, rendering COMT a difficult drug target. The low affinity of the co-substrate S-adenosylmethionine and the large conformational changes involved during catalysis highlight significant energetic investment to achieve the closed conformation. Since each conformation of COMT is a bona fide target for inhibitors, other states than the closed conformation may be promising to address. Crystallographic data for an alternative avenue of COMT inhibition, i.e. locking of the apo state by an inhibitor, are presented. The set of COMT structures may prove to be useful for the development of novel classes of inhibitors. PMID:25084335

  7. Characterization and restoration of performance of {open_quotes}aged{close_quotes} radioiodine removing activated carbons

    SciTech Connect

    Freeman, W.P.

    1997-08-01

    The degradation of radioiodine removal performance for impregnated activated carbons because of ageing is well established. However, the causes for this degradation remain unclear. One theory is that this reduction in performance from the ageing process results from an oxidation of the surface of the carbon. Radioiodine removing activated carbons that failed radioiodine removal tests showed an oxidized surface that had become hydrophilic compared with new carbons. We attempted to restore the performance of these {open_quotes}failed{close_quotes} carbons with a combination of thermal and chemical treatment. The results of these investigations are presented and discussed with the view of extending the life of radioiodine removing activated carbons. 4 refs., 2 tabs.

  8. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

    PubMed

    Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

  9. Design and synthesis of conformationally constrained analogues of cis-cinnamic acid and evaluation of their plant growth inhibitory activity.

    PubMed

    Nishikawa, Keisuke; Fukuda, Hiroshi; Abe, Masato; Nakanishi, Kazunari; Tazawa, Yuta; Yamaguchi, Chihiro; Hiradate, Syuntaro; Fujii, Yoshiharu; Okuda, Katsuhiro; Shindo, Mitsuru

    2013-12-01

    1-O-cis-Cinnamoyl-β-D-glucopyranose is known to be one of the most potent allelochemical candidates and was isolated from Spiraea thunbergii Sieb by Hiradate et al. (2004), who suggested that it derived its strong inhibitory activity from cis-cinnamic acid, which is crucial for phytotoxicity. In this study, key structural features and substituent effects of cis-cinnamic acid (cis-CA) on lettuce root growth inhibition was investigated. These structure-activity relationship studies indicated the importance of the spatial relationship of the aromatic ring and carboxylic acid moieties. In this context, conformationally constrained cis-CA analogues, in which the aromatic ring and cis-olefin were connected by a carbon bridge, were designed, synthesized, and evaluated as plant growth inhibitors. The results of the present study demonstrated that the inhibitory activities of the five-membered and six-membered bridged compounds were enhanced, up to 0.27 μM, and were ten times higher than cis-CA, while the potency of the other compounds was reduced.

  10. Conformational Changes in a Hyperthermostable Glycoside Hydrolase: Enzymatic Activity Is a Consequence of the Loop Dynamics and Protonation Balance

    PubMed Central

    de Oliveira, Leandro C.; da Silva, Viviam M.; Colussi, Francieli; Cabral, Aline D.; de Oliveira Neto, Mario; Squina, Fabio M.; Garcia, Wanius

    2015-01-01

    Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features. PMID:25723179

  11. Detailed conformation dynamics and activation process of wild type c-Abl and T315I mutant

    NASA Astrophysics Data System (ADS)

    Yang, Li-Jun; Zhao, Wen-Hua; Liu, Qian

    2014-10-01

    Bcr-Abl is an important target for therapy against chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). The synergistic effect between myristyl pocket and the ATP pocket has been found. But its detailed information based on molecular level still has not been achieved. In this study, conventional molecular dynamics (CMD) and target molecular dynamics (TMD) simulations were performed to explore the effect of T315I mutation on dynamics and activation process of Abl containing the N-terminal cap (Ncap). The CMD simulation results reveal the increasing flexibility of ATP pocket in kinase domain (KD) after T315I mutation which confirms the disability of ATP-pocket inhibitors to the Abl-T315I mutant. On the contrary, the T315I mutation decreased the flexibility of remote helix αI which suggests the synergistic effect between them. The mobility of farther regions containing Ncap, SH3, SH2 and SH2-KD linker were not affected by T315I mutation. The TMD simulation results show that the activation process of wild type Abl and Abl-T315I mutant experienced global conformation change. Their differences were elucidated by the activation motion of subsegments including A-loop, P-loop and Ncap. Besides, the T315I mutation caused decreasing energy barrier and increasing intermediate number in activation process, which results easier activation process. The TMD and CMD results indicate that a drug targeting only the ATP pocket is not enough to inhibit the Abl-T315I mutant. An effective way to inhibit the abnormal activity of Abl-T315I mutant is to combine the ATP-pocket inhibitors with inhibitors binding at non-ATP pockets mainly related to Ncap, SH2-KD linker and myristyl pocket.

  12. A 1.3-Å resolution crystal structure of the HIV-1 trans-activation response region RNA stem reveals a metal ion-dependent bulge conformation

    PubMed Central

    Ippolito, Joseph A.; Steitz, Thomas A.

    1998-01-01

    The crystal structure of an HIV-1 trans-activation response region (TAR) RNA fragment containing the binding site for the trans-activation protein Tat has been determined to 1.3-Å resolution. In this crystal structure, the characteristic UCU bulge of TAR adopts a conformation that is stabilized by three divalent calcium ions and differs from those determined previously by solution NMR. One metal ion, crucial to the loop conformation, binds directly to three phosphates in the loop region. The structure emphasizes the influence of metal ion binding on RNA structure and, given the abundance of divalent metal ion in the cell, raises the question of whether metal ions play a role in the conformation of TAR RNA and the interaction of TAR with Tat and cyclin T in vivo. PMID:9707559

  13. The conformation and CETP inhibitory activity of [10]-dehydrogingerdione isolated from Zingiber officinale.

    PubMed

    Choi, Soon-Yong; Park, Gil-Soon; Lee, Sung Yoon; Kim, Ji Yeon; Kim, Young Kook

    2011-05-01

    In the course of searching for cholesteryl ester transfer protein (CETP) inhibitors from natural sources, a new type of CETP inhibitor, [10]-dehydrogingerdione (1), was isolated from the extract of rhizomes of Zingiber officinale Roscoe. By NMR spectroscopic analysis of its (1)HNMR, (13)C-NMR, and (1)H-(1)H COSY, HMBC, HMQC and NOESY, more precise structure, compared with its originally proposed structures, of [10]-dehydrogingerdione has been elucidated. This active compound inhibited human plasma CETP with IC(50) values of 35 μM.

  14. The conformation and CETP inhibitory activity of [10]-dehydrogingerdione isolated from Zingiber officinale.

    PubMed

    Choi, Soon-Yong; Park, Gil-Soon; Lee, Sung Yoon; Kim, Ji Yeon; Kim, Young Kook

    2011-05-01

    In the course of searching for cholesteryl ester transfer protein (CETP) inhibitors from natural sources, a new type of CETP inhibitor, [10]-dehydrogingerdione (1), was isolated from the extract of rhizomes of Zingiber officinale Roscoe. By NMR spectroscopic analysis of its (1)HNMR, (13)C-NMR, and (1)H-(1)H COSY, HMBC, HMQC and NOESY, more precise structure, compared with its originally proposed structures, of [10]-dehydrogingerdione has been elucidated. This active compound inhibited human plasma CETP with IC(50) values of 35 μM. PMID:21656357

  15. Role of Methoxypolyethylene Glycol on the Hydration, Activity, Conformation and Dynamic Properties of a Lipase in a Dry Film

    PubMed Central

    Secundo, Francesco; Barletta, Gabriel; Mazzola, Giorgio

    2009-01-01

    A combined approach based on the use of ATR-FT/IR and steady-state fluorescence spectroscopy allowed to shed light on the effects of the additive methoxypolyethylene glycol (MePEG) on the hydration, conformation and dynamic properties of lipase from Burkholderia cepacia dehydrated to form a film. Spectroscopic data show that the additive has little effect on the structure of the protein; however, H/D exchange kinetic and fluorescence anisotropy suggest a more flexible enzyme molecule when in the presence of MePEG. By infrared spectroscopy, we estimated that, after conditioning the films at water activity of 1, the water content in the lipase dehydrated with MePEG is 5.4-and 4.7-fold higher than in the absence of the additive and the additive alone, respectively. Additionally, our infrared data suggest that MePEG acts by hindering intermolecular protein–protein interactions and contributing to increase the accessibility and flexibility of the lipase in the dehydrated solid film. These factors also explain the enhancement of the enzyme catalytic activity (i.e., up to 3.7-fold in neat organic solvent) when in the presence of MePEG. The method and results presented might better address the use of additives for the preparation of enzymes employed in non-aqueous media or of proteins used in a dry form in different fields of biotechnology. PMID:18727030

  16. Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation

    PubMed Central

    Jinek, Martin; Jiang, Fuguo; Taylor, David W.; Sternberg, Samuel H.; Kaya, Emine; Ma, Enbo; Anders, Carolin; Hauer, Michael; Zhou, Kaihong; Lin, Steven; Kaplan, Matias; Iavarone, Anthony T.; Charpentier, Emmanuelle; Nogales, Eva; Doudna, Jennifer A.

    2014-01-01

    Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA–induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation. PMID:24505130

  17. Evidence That GH115 α-Glucuronidase Activity, Which Is Required to Degrade Plant Biomass, Is Dependent on Conformational Flexibility*

    PubMed Central

    Rogowski, Artur; Baslé, Arnaud; Farinas, Cristiane S.; Solovyova, Alexandra; Mortimer, Jennifer C.; Dupree, Paul; Gilbert, Harry J.; Bolam, David N.

    2014-01-01

    The microbial degradation of the plant cell wall is an important biological process that is highly relevant to environmentally significant industries such as the bioenergy and biorefining sectors. A major component of the wall is glucuronoxylan, a β1,4-linked xylose polysaccharide that is decorated with α-linked glucuronic and/or methylglucuronic acid (GlcA/MeGlcA). Recently three members of a glycoside hydrolase family, GH115, were shown to hydrolyze MeGlcA side chains from the internal regions of xylan, an activity that has not previously been described. Here we show that a dominant member of the human microbiota, Bacteroides ovatus, contains a GH115 enzyme, BoAgu115A, which displays glucuronoxylan α-(4-O-methyl)-glucuronidase activity. The enzyme is significantly more active against substrates in which the xylose decorated with GlcA/MeGlcA is flanked by one or more xylose residues. The crystal structure of BoAgu115A revealed a four-domain protein in which the active site, comprising a pocket that abuts a cleft-like structure, is housed in the second domain that adopts a TIM barrel-fold. The third domain, a five-helical bundle, and the C-terminal β-sandwich domain make inter-chain contacts leading to protein dimerization. Informed by the structure of the enzyme in complex with GlcA in its open ring form, in conjunction with mutagenesis studies, the potential substrate binding and catalytically significant amino acids were identified. Based on the catalytic importance of residues located on a highly flexible loop, the enzyme is required to undergo a substantial conformational change to form a productive Michaelis complex with glucuronoxylan. PMID:24214982

  18. The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation

    PubMed Central

    Kanade, Santosh R.; Paul, Beena; Rao, A. G. Appu; Gowda, Lalitha R.

    2006-01-01

    Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase) – a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen – and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1±2 to 75.9±0.6 Å (1 Å=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack. PMID:16393141

  19. Role of the backbone conformation at position 7 in the structure and activity of marinostatin, an ester-linked serine protease inhibitor.

    PubMed

    Taichi, Misako; Yamazaki, Toshimasa; Nishiuchi, Yuji

    2012-09-01

    Rational design of inhibitors: The cis-amide backbone at position 7 in the serine protease inhibitor marinostatin was replaced with an E or Z olefin. The E olefin analogue was not active, but the Z analogue was. The cis conformation might play a critical role in organizing a canonical structure for binding to proteases.

  20. Structure, chain conformation, and immunomodulatory activity of the polysaccharide purified from Bacillus Calmette Guerin formulation.

    PubMed

    Liu, Wei; Wang, Hong; Yu, Juping; Liu, Yameng; Lu, Weisheng; Chai, Yin; Liu, Chao; Pan, Chun; Yao, Wenbing; Gao, Xiangdong

    2016-10-01

    A polysaccharide, coded as BDP, purified from the injection powder of Bacillus Calmette Guerin (BCG) polysaccharide and nucleic acid, was composed mainly of α-D-(1→4)-linked glucan with (1→6)-linked branches and trace amounts of fucose and mannose from the results of FT-IR, HPAEC-PAD and NMR spectrum. The Mw, Mn, Mz, and [Formula: see text] were determined to be 1.320×10(5)g/mol, 1.012×10(5)g/mol, 2.139×10(5)g/mol, and 21.8±3.2%nm by using HPSEC-MALLS, respectively. The ν value from [Formula: see text] was calculated to be 0.52±0.01, which firstly clarified that BDP existed as random coils in 0.9% NaCl aqueous solution. AFM and SEM combined with Congo-red test also revealed that the polysaccharide was irregular globular like or curly structure. Furthermore, in vitro tests on RAW264.7 murine macrophages cells revealed that BDP exhibited significant immunomodulatory activity. PMID:27312624

  1. Conformational changes in the amino-terminal helix of the G protein alpha(i1) following dissociation from Gbetagamma subunit and activation.

    PubMed

    Medkova, Martina; Preininger, Anita M; Yu, Nan-Jun; Hubbell, Wayne L; Hamm, Heidi E

    2002-08-01

    G protein alpha subunits mediate activation of signaling pathways through G protein-coupled receptors (GPCR) by virtue of GTP-dependent conformational rearrangements. It is known that regions of disorder in crystal structures can be indicative of conformational flexibility within a molecule, and there are several such regions in G protein alpha subunits. The amino-terminal 29 residues of Galpha are alpha-helical only in the heterotrimer, where they contact the side of Gbeta, but little is known about the conformation of this region in the active GTP bound state. To address the role of the Galpha amino-terminus in G-protein activation and to investigate whether this region undergoes activation-dependent conformational changes, a site-directed cysteine mutagenesis study was carried out. Engineered Galpha(i1) proteins were created by first removing six native reactive cysteines to yield a mutant Galpha(i1)-C3S-C66A-C214S-C305S-C325A-C351I that no longer reacts with cysteine-directed labels. Several cysteine substitutions along the amino-terminal region were then introduced. All mutant proteins were shown to be folded properly and functional. An environmentally sensitive probe, Lucifer yellow, linked to these sites showed a fluorescence change upon interaction with Gbetagamma and with activation by AlF(4)(-). Other fluorescent probes of varying charge, size, and hydrophobicity linked to amino-terminal residues also revealed changes upon activation with bulkier probes reporting larger changes. Site-directed spin-labeling studies showed that the N-terminus of the Galpha subunit is dynamically disordered in the GDP bound state, but adopts a structure consistent with an alpha-helix upon interaction with Gbetagamma. Interaction of the resulting spin-labeled Galphabetagamma with photoactivated rhodopsin, followed by rhodopsin-catalyzed GTPgammaS binding, caused the amino-terminal domain of Galpha to revert to a dynamically disordered state similar to that of the GDP

  2. Chemical Synthesis of Arabidopsis CLV3 Glycopeptide Reveals the Impact of Hydroxyproline Arabinosylation on Peptide Conformation and Activity

    PubMed Central

    Shinohara, Hidefumi; Matsubayashi, Yoshikatsu

    2013-01-01

    Arabinosylation of hydroxyproline (Hyp) is a post-translational modification often found in secreted peptide signals in plants. The physiological importance of this modification was highlighted by the finding that CLAVATA3 (CLV3), a key peptide signal for regulating the fate of stem cells in the shoot apical meristem in Arabidopsis, contains three l-arabinose residues linked via linear β-1,2-linkages. However, understanding the functions and properties of arabinosylated peptides has been hindered by difficulties in synthesizing the complex arabinose chain. Here we report the stereoselective total synthesis of β-1,2-linked triarabinosylated CLV3 peptide ([Ara3]CLV3). Chemically synthesized [Ara3]CLV3 restricted stem cell activity more effectively than did unmodified CLV3 peptide. Comparison of mono-, di- and triarabinosylated CLV3 glycopeptides revealed that the biological activity increased progressively as the arabinose chain length increased. Thus, the arabinose chain length of CLV3 is important for its biological activity. Nuclear magnetic resonance spectroscopy and nuclear Overhauser effect-based structure calculations further revealed the structural impact of the arabinose chain on peptide conformation. The arabinose chain of [Ara3]CLV3 extends toward the C-terminal end of the peptide, and its non-reducing end is positioned proximal to the peptide backbone. Consequently, the arabinose chain causes distinct distortion in the C-terminal half of the peptide in a highly directional manner. The established synthetic route of [Ara3]CLV3 will greatly contribute to our understanding of the biology and biochemistry of arabinosylated peptide signals in plants. PMID:23256149

  3. Chemical synthesis of Arabidopsis CLV3 glycopeptide reveals the impact of hydroxyproline arabinosylation on peptide conformation and activity.

    PubMed

    Shinohara, Hidefumi; Matsubayashi, Yoshikatsu

    2013-03-01

    Arabinosylation of hydroxyproline (Hyp) is a post-translational modification often found in secreted peptide signals in plants. The physiological importance of this modification was highlighted by the finding that CLAVATA3 (CLV3), a key peptide signal for regulating the fate of stem cells in the shoot apical meristem in Arabidopsis, contains three l-arabinose residues linked via linear β-1,2-linkages. However, understanding the functions and properties of arabinosylated peptides has been hindered by difficulties in synthesizing the complex arabinose chain. Here we report the stereoselective total synthesis of β-1,2-linked triarabinosylated CLV3 peptide ([Ara3]CLV3). Chemically synthesized [Ara3]CLV3 restricted stem cell activity more effectively than did unmodified CLV3 peptide. Comparison of mono-, di- and triarabinosylated CLV3 glycopeptides revealed that the biological activity increased progressively as the arabinose chain length increased. Thus, the arabinose chain length of CLV3 is important for its biological activity. Nuclear magnetic resonance spectroscopy and nuclear Overhauser effect-based structure calculations further revealed the structural impact of the arabinose chain on peptide conformation. The arabinose chain of [Ara3]CLV3 extends toward the C-terminal end of the peptide, and its non-reducing end is positioned proximal to the peptide backbone. Consequently, the arabinose chain causes distinct distortion in the C-terminal half of the peptide in a highly directional manner. The established synthetic route of [Ara3]CLV3 will greatly contribute to our understanding of the biology and biochemistry of arabinosylated peptide signals in plants.

  4. Closed Loop Active Flow Separation Detection and Control in a Multistage Compressor

    NASA Technical Reports Server (NTRS)

    Bright, Michelle M.; Culley, Dennis E.; Braunscheidel, Edward P.; Welch, Gerard E.

    2005-01-01

    Active closed loop flow control was successfully demonstrated on a full annulus of stator vanes in a low speed axial compressor. Two independent methods of detecting separated flow conditions on the vane suction surface were developed. The first technique detects changes in static pressure along the vane suction surface, while the second method monitors variation in the potential field of the downstream rotor. Both methods may feasibly be used in future engines employing embedded flow control technology. In response to the detection of separated conditions, injection along the suction surface of each vane was used. Injected mass flow on the suction surface of stator vanes is known to reduce separation and the resulting limitation on static pressure rise due to lowered diffusion in the vane passage. A control algorithm was developed which provided a proportional response of the injected mass flow to the degree of separation, thereby minimizing the performance penalty on the compressor system.

  5. Resonant activation in a colored multiplicative thermal noise driven closed system

    SciTech Connect

    Ray, Somrita; Bag, Bidhan Chandra; Mondal, Debasish

    2014-05-28

    In this paper, we have demonstrated that resonant activation (RA) is possible even in a thermodynamically closed system where the particle experiences a random force and a spatio-temporal frictional coefficient from the thermal bath. For this stochastic process, we have observed a hallmark of RA phenomena in terms of a turnover behavior of the barrier-crossing rate as a function of noise correlation time at a fixed noise variance. Variance can be fixed either by changing temperature or damping strength as a function of noise correlation time. Our another observation is that the barrier crossing rate passes through a maximum with increase in coupling strength of the multiplicative noise. If the damping strength is appreciably large, then the maximum may disappear. Finally, we compare simulation results with the analytical calculation. It shows that there is a good agreement between analytical and numerical results.

  6. Evaluating the performance of close-range 3D active vision systems for industrial design applications

    NASA Astrophysics Data System (ADS)

    Beraldin, J.-Angelo; Gaiani, Marco

    2004-12-01

    In recent years, active three-dimensional (3D) active vision systems or range cameras for short have come out of research laboratories to find niche markets in application fields as diverse as industrial design, automotive manufacturing, geomatics, space exploration and cultural heritage to name a few. Many publications address different issues link to 3D sensing and processing but currently these technologies pose a number of challenges to many recent users, i.e., "what are they, how good are they and how do they compare?". The need to understand, test and integrate those range cameras with other technologies, e.g. photogrammetry, CAD, etc. is driven by the quest for optimal resolution, accuracy, speed and cost. Before investing, users want to be certain that a given range camera satisfy their operational requirements. The understanding of the basic theory and best practices associated with those cameras are in fact fundamental to fulfilling the requirements listed above in an optimal way. This paper addresses the evaluation of active 3D range cameras as part of a study to better understand and select one or a number of them to fulfill the needs of industrial design applications. In particular, object material and surface features effect, calibration and performance evaluation are discussed. Results are given for six different range cameras for close range applications.

  7. Evaluating the performance of close-range 3D active vision systems for industrial design applications

    NASA Astrophysics Data System (ADS)

    Beraldin, J.-Angelo; Gaiani, Marco

    2005-01-01

    In recent years, active three-dimensional (3D) active vision systems or range cameras for short have come out of research laboratories to find niche markets in application fields as diverse as industrial design, automotive manufacturing, geomatics, space exploration and cultural heritage to name a few. Many publications address different issues link to 3D sensing and processing but currently these technologies pose a number of challenges to many recent users, i.e., "what are they, how good are they and how do they compare?". The need to understand, test and integrate those range cameras with other technologies, e.g. photogrammetry, CAD, etc. is driven by the quest for optimal resolution, accuracy, speed and cost. Before investing, users want to be certain that a given range camera satisfy their operational requirements. The understanding of the basic theory and best practices associated with those cameras are in fact fundamental to fulfilling the requirements listed above in an optimal way. This paper addresses the evaluation of active 3D range cameras as part of a study to better understand and select one or a number of them to fulfill the needs of industrial design applications. In particular, object material and surface features effect, calibration and performance evaluation are discussed. Results are given for six different range cameras for close range applications.

  8. Conformational change of Dishevelled plays a key regulatory role in the Wnt signaling pathways

    PubMed Central

    Lee, Ho-Jin; Shi, De-Li; Zheng, Jie J

    2015-01-01

    The intracellular signaling molecule Dishevelled (Dvl) mediates canonical and non-canonical Wnt signaling via its PDZ domain. Different pathways diverge at this point by a mechanism that remains unclear. Here we show that the peptide-binding pocket of the Dvl PDZ domain can be occupied by Dvl's own highly conserved C-terminus, inducing a closed conformation. In Xenopus, Wnt-regulated convergent extension (CE) is readily affected by Dvl mutants unable to form the closed conformation than by wild-type Dvl. We also demonstrate that while Dvl cooperates with other Wnt pathway elements to activate canonical Wnt signaling, the open conformation of Dvl more effectively activates Jun N-terminal kinase (JNK). These results suggest that together with other players in the Wnt signaling pathway, the conformational change of Dvl regulates Wnt stimulated JNK activity in the non-canonical Wnt signaling. DOI: http://dx.doi.org/10.7554/eLife.08142.001 PMID:26297804

  9. Conformable seal

    DOEpatents

    Neef, W.S.; Lambert, D.R.

    1982-08-10

    Sealing apparatus and method, comprising first and second surfaces or membranes, at least one of which surfaces is deformable, placed in proximity to one another. Urging means cause these surfaces to contact one another in a manner such that the deformable surface deforms to conform to the geometry of the other surface, thereby creating a seal. The seal is capable of undergoing multiple cycles of sealing and unsealing.

  10. Conformational HER-2/neu B-cell epitope peptide vaccine designed to incorporate two native disulfide bonds enhances tumor cell binding and antitumor activities.

    PubMed

    Dakappagari, Naveen K; Lute, Kenneth D; Rawale, Sharad; Steele, Joan T; Allen, Stephanie D; Phillips, Gary; Reilly, R Todd; Kaumaya, Pravin T P

    2005-01-01

    Cancer vaccines designed to elicit an antibody response that target antigenic sites on a tumor antigen must closely mimic the three-dimensional structure of the corresponding region on the antigen. We have designed a complex immunogen derived from the extracellular domain of human HER-2/neu-(626-649) that represents a three-dimensional epitope. We have successfully introduced two disulfide bonds into this sequence, thereby recapitulating the natural disulfide pairings observed in the native protein. To evaluate the immunogenicity of the doubly cyclized disulfide-linked peptide versus the free uncyclized peptide we examined the induction of antibody responses in both inbred and outbred mice strains, with both constructs eliciting high titered antibodies. The disulfide-paired specific antibodies exhibited enhanced cross-reactivity to HER-2/neu expressed on BT-474 cell line as determined by flow cytometry. The antitumor activities of the disulfidepaired specific antibodies did not improve the in vitro growth inhibition of human breast cancer cells overexpressing HER-2, but showed superior antitumor responses in the context of ADCC and interferon-gamma induction. Inbred mice (FVB/n) vaccinated with the disulfide-paired epitope exhibited a statistically significant reduction in the development of exogenously administered tumors in vivo compared with mice receiving either the free uncyclized or the promiscuous T-cell epitope (MVF) control peptide (p = 0.001). This study demonstrates the feasibility and importance of designing conformational epitopes that mimic the tertiary structure of the native protein for eliciting biologically relevant anti-tumor antibodies. Such approaches are a prerequisite to the design of effective peptide vaccines.

  11. Zeta Inhibitory Peptide Disrupts Electrostatic Interactions That Maintain Atypical Protein Kinase C in Its Active Conformation on the Scaffold p62*

    PubMed Central

    Tsai, Li-Chun Lisa; Xie, Lei; Dore, Kim; Xie, Li; Del Rio, Jason C.; King, Charles C.; Martinez-Ariza, Guillermo; Hulme, Christopher; Malinow, Roberto; Bourne, Philip E.; Newton, Alexandra C.

    2015-01-01

    Atypical protein kinase C (aPKC) enzymes signal on protein scaffolds, yet how they are maintained in an active conformation on scaffolds is unclear. A myristoylated peptide based on the autoinhibitory pseudosubstrate fragment of the atypical PKCζ, zeta inhibitory peptide (ZIP), has been extensively used to inhibit aPKC activity; however, we have previously shown that ZIP does not inhibit the catalytic activity of aPKC isozymes in cells (Wu-Zhang, A. X., Schramm, C. L., Nabavi, S., Malinow, R., and Newton, A. C. (2012) J. Biol. Chem. 287, 12879–12885). Here we sought to identify a bona fide target of ZIP and, in so doing, unveiled a novel mechanism by which aPKCs are maintained in an active conformation on a protein scaffold. Specifically, we used protein-protein interaction network analysis, structural modeling, and protein-protein docking to predict that ZIP binds an acidic surface on the Phox and Bem1 (PB1) domain of p62, an interaction validated by peptide array analysis. Using a genetically encoded reporter for PKC activity fused to the p62 scaffold, we show that ZIP inhibits the activity of wild-type aPKC, but not a construct lacking the pseudosubstrate. These data support a model in which the pseudosubstrate of aPKCs is tethered to the acidic surface on p62, locking aPKC in an open, signaling-competent conformation. ZIP competes for binding to the acidic surface, resulting in displacement of the pseudosubstrate of aPKC and re-engagement in the substrate-binding cavity. This study not only identifies a cellular target for ZIP, but also unveils a novel mechanism by which scaffolded aPKC is maintained in an active conformation. PMID:26187466

  12. Zeta Inhibitory Peptide Disrupts Electrostatic Interactions That Maintain Atypical Protein Kinase C in Its Active Conformation on the Scaffold p62.

    PubMed

    Tsai, Li-Chun Lisa; Xie, Lei; Dore, Kim; Xie, Li; Del Rio, Jason C; King, Charles C; Martinez-Ariza, Guillermo; Hulme, Christopher; Malinow, Roberto; Bourne, Philip E; Newton, Alexandra C

    2015-09-01

    Atypical protein kinase C (aPKC) enzymes signal on protein scaffolds, yet how they are maintained in an active conformation on scaffolds is unclear. A myristoylated peptide based on the autoinhibitory pseudosubstrate fragment of the atypical PKCζ, zeta inhibitory peptide (ZIP), has been extensively used to inhibit aPKC activity; however, we have previously shown that ZIP does not inhibit the catalytic activity of aPKC isozymes in cells (Wu-Zhang, A. X., Schramm, C. L., Nabavi, S., Malinow, R., and Newton, A. C. (2012) J. Biol. Chem. 287, 12879-12885). Here we sought to identify a bona fide target of ZIP and, in so doing, unveiled a novel mechanism by which aPKCs are maintained in an active conformation on a protein scaffold. Specifically, we used protein-protein interaction network analysis, structural modeling, and protein-protein docking to predict that ZIP binds an acidic surface on the Phox and Bem1 (PB1) domain of p62, an interaction validated by peptide array analysis. Using a genetically encoded reporter for PKC activity fused to the p62 scaffold, we show that ZIP inhibits the activity of wild-type aPKC, but not a construct lacking the pseudosubstrate. These data support a model in which the pseudosubstrate of aPKCs is tethered to the acidic surface on p62, locking aPKC in an open, signaling-competent conformation. ZIP competes for binding to the acidic surface, resulting in displacement of the pseudosubstrate of aPKC and re-engagement in the substrate-binding cavity. This study not only identifies a cellular target for ZIP, but also unveils a novel mechanism by which scaffolded aPKC is maintained in an active conformation.

  13. A CORRELATION BETWEEN HOST STAR ACTIVITY AND PLANET MASS FOR CLOSE-IN EXTRASOLAR PLANETS?

    SciTech Connect

    Poppenhaeger, K.; Schmitt, J. H. M. M.

    2011-07-01

    The activity levels of stars are influenced by several stellar properties, such as stellar rotation, spectral type, and the presence of stellar companions. Analogous to binaries, planetary companions are also thought to be able to cause higher activity levels in their host stars, although at lower levels. Especially in X-rays, such influences are hard to detect because coronae of cool stars exhibit a considerable amount of intrinsic variability. Recently, a correlation between the mass of close-in exoplanets and their host star's X-ray luminosity has been detected, based on archival X-ray data from the ROSAT All-Sky Survey. This finding has been interpreted as evidence for star-planet interactions. We show in our analysis that this correlation is caused by selection effects due to the flux limit of the X-ray data used and due to the intrinsic planet detectability of the radial velocity method, and thus does not trace possible planet-induced effects. We also show that the correlation is not present in a corresponding complete sample derived from combined XMM-Newton and ROSAT data.

  14. New Atglistatin closely related analogues: Synthesis and structure-activity relationship towards adipose triglyceride lipase inhibition.

    PubMed

    Roy, Pierre-Philippe; D'Souza, Kenneth; Cuperlovic-Culf, Miroslava; Kienesberger, Petra C; Touaibia, Mohamed

    2016-08-01

    Adipose Triglyceride Lipase (ATGL) performs the first and rate-limiting step in lipolysis by hydrolyzing triacylglycerols stored in lipid droplets to diacylglycerols. By mediating lipolysis in adipose and non-adipose tissues, ATGL is a major regulator of overall energy metabolism and plasma lipid levels. Since chronically high levels of plasma lipids are linked to metabolic disorders including insulin resistance and type 2 diabetes, ATGL is an interesting therapeutic target. In the present study, fourteen closely related analogues of Atglistatin (1), a newly discovered ATGL inhibitor, were synthesized, and their ATGL inhibitory activity was evaluated. The effect of these analogues on lipolysis in 3T3-L1 adipocytes clearly shows that inhibition of the enzyme by Atglistatin (1) is due to the presence of the carbamate and N,N-dimethyl moieties on the biaryl central core at meta and para position, respectively. Mono carbamate-substituted analogue C2, in which the carbamate group was in the meta position as in Atglistatin (1), showed slight inhibition. Low dipole moment of Atglistatin (1) compared to the synthesized analogues possibly explains the lower inhibitory activities.

  15. The Extremely Low Activity Comet 209P/LINEAR During Its Extraordinary Close Approach in 2014

    NASA Astrophysics Data System (ADS)

    Schleicher, David G.; knight, Matthew m.

    2016-10-01

    We present results from our observing campaign of Comet 209P/LINEAR during its exceptionally close approach to Earth during 2014 May, the third smallest perigee of any comet in two centuries. These circumstances permitted us to pursue several studies of this intrinsically faint object, including measurements of gas and dust production rates, searching for coma morphology, and direct detection of the nucleus to measure its properties. Indeed, we successfully measured the lowest water production rates of an intact comet in over 35 years and a corresponding smallest active area, ∼0.007 km2. When combined with the nucleus size found from radar, this also yields the smallest active fraction for any comet, ∼0.024%. In all, this strongly suggests that 209P/LINEAR is on its way to becoming an inert object. The nucleus was detected but could not easily be disentangled from the inner coma due to seeing variations and changing spatial scales. Even so, we were able to measure a double-peaked lightcurve consistent with the shorter of two viable rotational periods found by Hergenrother. Radial profiles of the dust coma are quite steep, similar to that observed for some other very anemic comets, and suggest that vaporizing icy grains are present.

  16. Mutation analysis of PobR and PcaU, closely related transcriptional activators in acinetobacter.

    PubMed

    Kok, R G; D'Argenio, D A; Ornston, L N

    1998-10-01

    Acinetobacter PobR and PcaU are transcriptional activators that closely resemble each other in primary structure, DNA-binding sites, metabolic modulators, and physiological function. PobR responds to the inducer-metabolite p-hydroxybenzoate and activates transcription of pobA, the structural gene for the enzyme that converts p-hydroxybenzoate to protocatechuate. This compound, differing from p-hydroxybenzoate only in that it contains an additional oxygen atom, binds to PcaU and thereby specifically activates transcription of the full set of genes for protocatechuate catabolism. Particular experimental attention has been paid to PobR and PcaU from Acinetobacter strain ADP1, which exhibits exceptional competence for natural transformation. This trait allowed selection of mutant strains in which pobR function had been impaired by nucleotide substitutions introduced by PCR replication errors. Contrary to expectation, the spectrum of amino acids whose substitution led to loss of function in PobR shows no marked similarity to the spectrum of amino acids conserved by the demand for continued function during evolutionary divergence of PobR, PcaU, and related proteins. Surface plasmon resonance was used to determine the ability of mutant PobR proteins to bind to DNA in the pobA-pobR intergenic region. Deleterious mutations that strongly affect DNA binding all cluster in and around the PobR region that contains a helix-turn-helix motif, whereas mutations causing defects in the central portion of the PobR primary sequence do not seem to have a significant effect on operator binding. PCR-generated mutations allowing PobR to mimic PcaU function invariably caused a T57A amino acid substitution, making the helix-turn-helix sequence of PobR more like that of PcaU. The mutant PobR depended on p-hydroxybenzoate for its activity, but this dependence could be relieved by any of six amino acid substitutions in the center of the PobR primary sequence. Independent mutations allowing Pca

  17. [Conformal radiotherapy: principles and classification].

    PubMed

    Rosenwald, J C; Gaboriaud, G; Pontvert, D

    1999-01-01

    'Conformal radiotherapy' is the name fixed by usage and given to a new form of radiotherapy resulting from the technological improvements observed during, the last ten years. While this terminology is now widely used, no precise definition can be found in the literature. Conformal radiotherapy refers to an approach in which the dose distribution is more closely 'conformed' or adapted to the actual shape of the target volume. However, the achievement of a consensus on a more specific definition is hampered by various difficulties, namely in characterizing the degree of 'conformality'. We have therefore suggested a classification scheme be established on the basis of the tools and the procedures actually used for all steps of the process, i.e., from prescription to treatment completion. Our classification consists of four levels: schematically, at level 0, there is no conformation (rectangular fields); at level 1, a simple conformation takes place, on the basis of conventional 2D imaging; at level 2, a 3D reconstruction of the structures is used for a more accurate conformation; and level 3 includes research and advanced dynamic techniques. We have used our personal experience, contacts with colleagues and data from the literature to analyze all the steps of the planning process, and to define the tools and procedures relevant to a given level. The corresponding tables have been discussed and approved at the European level within the Dynarad concerted action. It is proposed that the term 'conformal radiotherapy' be restricted to procedures where all steps are at least at level 2.

  18. Coordination and conformational isomers in mononuclear iron complexes with pertinence to the [FeFe] hydrogenase active site.

    PubMed

    Orthaber, Andreas; Karnahl, Michael; Tschierlei, Stefanie; Streich, Daniel; Stein, Matthias; Ott, Sascha

    2014-03-21

    A series of six mononuclear iron complexes of the type [Fe(X-bdt)(P(R)2N(Ph)2)(CO)] (P(R)2N(Ph)2 = 1,5-diaza-3,7-diphosphaoctane, bdt = benzenedithiolate with X = H, Cl2 or Me and R = Ph, Bn, Cyc or tert-Bu) was prepared. This new class of penta-coordinate iron complexes contains a free coordination site and a pendant base as essential structural features of the [FeFe]-hydrogenase active site. The bidentate nature of the P(R)2N(Ph)2 ligands was found to be crucial for the preferential formation of coordinatively unsaturated penta-coordinate complexes, which is supported by first principle calculations. IR-spectroscopic data suggest the presence of coordination isomers around the metal center, as well as multiple possible conformers of the P(R)2N(Ph)2 ligand. This finding is further corroborated by X-ray crystallographic and computational studies. (31)P{(1)H}-NMR- and IR-spectroscopic as well as electrochemical measurements show that the electronic properties of the complexes are strongly, and independently, influenced by the P-substituents at the P(R)2N(Ph)2 ligand as well as by modifications of the bdt bridge. These results illustrate the advantages of this modular platform, which allows independent and selective tuning through site specific modifications. Potential catalytic intermediates, namely singly reduced and protonated complexes, have been further investigated by spectroscopic methods and exhibit remarkable stability. Finally, their general capacity for electro-catalytic reduction of protons to molecular hydrogen was verified.

  19. One-dimensional TRFLP-SSCP is an effective DNA fingerprinting strategy for soil Archaea that is able to simultaneously differentiate broad taxonomic clades based on terminal fragment length polymorphisms and closely related sequences based on single stranded conformation polymorphisms.

    PubMed

    Swanson, Colby A; Sliwinski, Marek K

    2013-09-01

    DNA fingerprinting methods provide a means to rapidly compare microbial assemblages from environmental samples without the need to first cultivate species in the laboratory. The profiles generated by these techniques are able to identify statistically significant temporal and spatial patterns, correlations to environmental gradients, and biological variability to estimate the number of replicates for clone libraries or next generation sequencing (NGS) surveys. Here we describe an improved DNA fingerprinting technique that combines terminal restriction fragment length polymorphisms (TRFLP) and single stranded conformation polymorphisms (SSCP) so that both can be used to profile a sample simultaneously rather than requiring two sequential steps as in traditional two-dimensional (2-D) gel electrophoresis. For the purpose of profiling Archaeal 16S rRNA genes from soil, the dynamic range of this combined 1-D TRFLP-SSCP approach was superior to TRFLP and SSCP. 1-D TRFLP-SSCP was able to distinguish broad taxonomic clades with genetic distances greater than 10%, such as Euryarchaeota and the Thaumarchaeal clades g_Ca. Nitrososphaera (formerly 1.1b) and o_NRP-J (formerly 1.1c) better than SSCP. In addition, 1-D TRFLP-SSCP was able to simultaneously distinguish closely related clades within a genus such as s_SCA1145 and s_SCA1170 better than TRFLP. We also tested the utility of 1-D TRFLP-SSCP fingerprinting of environmental assemblages by comparing this method to the generation of a 16S rRNA clone library of soil Archaea from a restored Tallgrass prairie. This study shows 1-D TRFLP-SSCP fingerprinting provides a rapid and phylogenetically informative screen of Archaeal 16S rRNA genes in soil samples.

  20. Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

    SciTech Connect

    Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.; Hession, Catherine A.; Miatkowski, Konrad; Wildes, Craig P.; Cullen, Patrick F.; Hong, Victor; Hopkins, Brian T.; Mertsching, Elisabeth; Jenkins, Tracy J.; Romanowski, Michael J.; Baker, Darren P.; Silvian, Laura F.

    2010-11-15

    Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 {angstrom} resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 {angstrom} resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

  1. Transferred nuclear Overhauser effect analyses of membrane-bound enkephalin analogues by sup 1 H nuclear magnetic resonance: Correlation between activities and membrane-bound conformations

    SciTech Connect

    Milon, Alain; Miyazawa, Tatsuo; Higashijima, Tsutomu )

    1990-01-09

    Leu-enkephalin, (D-Ala{sup 2})Leu-enkephalin, and (D-Ala{sup 2})Leu-enkephalinamide (agonists) and (L-Ala{sup 2})Leu-enkephalin (inactive analogue) bind to lipid bilayer consisting of phosphatidylcholine and phosphatidylserine. The conformations that these compounds assume, once bound to perdeuterated phospholipid bilayer, have been shown to be unique, as shown by the transferred nuclear Overhauser effect (TRNOE) of {sup 1}H NMR spectroscopy. In addition, their location in the bilayer was analyzed by TRNOE in the presence of spin-labeled phospholipids. These analyses showed a clear relationship between the activity and the peptide-membrane interaction. The three active peptides, when bound to membranes, adopt the same conformation, characterized by a type II{prime} {beta}-turn around Gly{sup 3}-Phe and a {gamma}-turn around Gly{sup 2} (or D-Ala{sup 2}). The inactive analogue, (L-Ala{sup 2})Leu-enkephalin, displayed a completely different TRNOE pattern corresponding to a different conformation in the membrane-bound state. The tyrosine residue of the active compounds is not inserted into the interior of membrane, but it is inserted into the bilayer for the L-Ala{sup 2} analogue. According to these results, (L-Ala{sup 2})Leu-enkephalin may be explained to be inactive because the mode of binding to the membranes is different from that of active compounds.

  2. Synthesis, conformational analysis, and biological activities of cyclic enkephalins and model cyclic peptides containing thioamides as amide bond replacements

    SciTech Connect

    Sherman, D.B.

    1988-01-01

    This thesis describes the first applications of the thioamide surrogate ({psi}(CSNH)) in model cyclic peptides and cyclic enkephalins. The solution phase synthesis and conformational analysis of two model cyclic endothiopentapeptides, cyclo(D-Phe-Pro{psi} (CSNH)Gly-Pro-Gly) (I) and cyclo(D-Phe-Pro-Gly-Pro{psi}(CSNH)Gly) (II), are reported. The conformations of I and II were analyzed using several 1 and 2D NMR techniques, such as INDOR, temperature and concentration dependence, {sup 1}H-{sup 1}H and {sup 1}H-{sup 13}C COSY, ROESY, and magnetization transfer. Compound I displayed the same general conformation as its parent in CDCl{sub 3}, but the {gamma}-turn hydrogen bond appeared to be weaker, while the {beta}-turn hydrogen bond appeared to be stronger. In DMSO-d{sub 6}, this molecule existed in two conformations in a ratio of 2:1. The major conformer appeared to be the same as that in CDCl{sub 3}, while the second contained at least one cis X-Pro bond, most likely at the Gly{sup 1}-Pro{sup 2} position. Compound II displayed the same conformation as its parent in both solvents. The {gamma}-turn hydrogen bond again appeared to be weaker in CDCl{sub 3}, but comparable with the parent in DMSO-d{sub 6}. Molecular modeling studies of I and II indicated the Pro {psi} angle increased by {approx}6{degree} when a thiocarbonyl was present, thereby reducing steric interactions with the Pro {beta} methylene.

  3. A Distant Echo of Milky Way Central Activity Closes the Galaxy’s Baryon Census

    NASA Astrophysics Data System (ADS)

    Nicastro, F.; Senatore, F.; Krongold, Y.; Mathur, S.; Elvis, M.

    2016-09-01

    We report on the presence of large amounts of million-degree gas in the Milky Way’s interstellar and circum-galactic medium. This gas (1) permeates both the Galactic plane and the halo, (2) extends to distances larger than 60–200 kpc from the center, and (3) its mass is sufficient to close the Galaxy’s baryon census. Moreover, we show that a vast, ∼6 kpc radius, spherically symmetric central region of the Milky Way above and below the 0.16 kpc thick plane has either been emptied of hot gas or the density of this gas within the cavity has a peculiar profile, increasing from the center up to a radius of ∼6 kpc, and then decreasing with a typical halo density profile. This, and several other converging pieces of evidence, suggest that the current surface of the cavity, at 6 kpc from the Galaxy’s center, traces the distant echo of a period of strong nuclear activity of our supermassive black hole, occurring about 6 Myr ago.

  4. A Distant Echo of Milky Way Central Activity Closes the Galaxy’s Baryon Census

    NASA Astrophysics Data System (ADS)

    Nicastro, F.; Senatore, F.; Krongold, Y.; Mathur, S.; Elvis, M.

    2016-09-01

    We report on the presence of large amounts of million-degree gas in the Milky Way’s interstellar and circum-galactic medium. This gas (1) permeates both the Galactic plane and the halo, (2) extends to distances larger than 60-200 kpc from the center, and (3) its mass is sufficient to close the Galaxy’s baryon census. Moreover, we show that a vast, ˜6 kpc radius, spherically symmetric central region of the Milky Way above and below the 0.16 kpc thick plane has either been emptied of hot gas or the density of this gas within the cavity has a peculiar profile, increasing from the center up to a radius of ˜6 kpc, and then decreasing with a typical halo density profile. This, and several other converging pieces of evidence, suggest that the current surface of the cavity, at 6 kpc from the Galaxy’s center, traces the distant echo of a period of strong nuclear activity of our supermassive black hole, occurring about 6 Myr ago.

  5. Active Ammonia Oxidizers in an Acidic Soil Are Phylogenetically Closely Related to Neutrophilic Archaeon

    PubMed Central

    Wang, Baozhan; Zheng, Yan; Huang, Rong; Zhou, Xue; Wang, Dongmei; He, Yuanqiu

    2014-01-01

    All cultivated ammonia-oxidizing archaea (AOA) within the Nitrososphaera cluster (former soil group 1.1b) are neutrophilic. Molecular surveys also indicate the existence of Nitrososphaera-like phylotypes in acidic soil, but their ecological roles are poorly understood. In this study, we present molecular evidence for the chemolithoautotrophic growth of Nitrososphaera-like AOA in an acidic soil with pH 4.92 using DNA-based stable isotope probing (SIP). Soil microcosm incubations demonstrated that nitrification was stimulated by urea fertilization and accompanied by a significant increase in the abundance of AOA rather than ammonia-oxidizing bacteria (AOB). Real-time PCR analysis of amoA genes as a function of the buoyant density of the DNA gradient following the ultracentrifugation of the total DNA extracted from SIP microcosms indicated a substantial growth of soil AOA during nitrification. Pyrosequencing of the total 16S rRNA genes in the “heavy” DNA fractions suggested that archaeal communities were labeled to a much greater extent than soil AOB. Acetylene inhibition further showed that 13CO2 assimilation by nitrifying communities depended solely on ammonia oxidation activity, suggesting a chemolithoautotrophic lifestyle. Phylogenetic analysis of both 13C-labeled amoA and 16S rRNA genes revealed that most of the active AOA were phylogenetically closely related to the neutrophilic strains Nitrososphaera viennensis EN76 and JG1 within the Nitrososphaera cluster. Our results provide strong evidence for the adaptive growth of Nitrososphaera-like AOA in acidic soil, suggesting a greater metabolic versatility of soil AOA than previously appreciated. PMID:24375137

  6. Active ammonia oxidizers in an acidic soil are phylogenetically closely related to neutrophilic archaeon.

    PubMed

    Wang, Baozhan; Zheng, Yan; Huang, Rong; Zhou, Xue; Wang, Dongmei; He, Yuanqiu; Jia, Zhongjun

    2014-03-01

    All cultivated ammonia-oxidizing archaea (AOA) within the Nitrososphaera cluster (former soil group 1.1b) are neutrophilic. Molecular surveys also indicate the existence of Nitrososphaera-like phylotypes in acidic soil, but their ecological roles are poorly understood. In this study, we present molecular evidence for the chemolithoautotrophic growth of Nitrososphaera-like AOA in an acidic soil with pH 4.92 using DNA-based stable isotope probing (SIP). Soil microcosm incubations demonstrated that nitrification was stimulated by urea fertilization and accompanied by a significant increase in the abundance of AOA rather than ammonia-oxidizing bacteria (AOB). Real-time PCR analysis of amoA genes as a function of the buoyant density of the DNA gradient following the ultracentrifugation of the total DNA extracted from SIP microcosms indicated a substantial growth of soil AOA during nitrification. Pyrosequencing of the total 16S rRNA genes in the "heavy" DNA fractions suggested that archaeal communities were labeled to a much greater extent than soil AOB. Acetylene inhibition further showed that (13)CO2 assimilation by nitrifying communities depended solely on ammonia oxidation activity, suggesting a chemolithoautotrophic lifestyle. Phylogenetic analysis of both (13)C-labeled amoA and 16S rRNA genes revealed that most of the active AOA were phylogenetically closely related to the neutrophilic strains Nitrososphaera viennensis EN76 and JG1 within the Nitrososphaera cluster. Our results provide strong evidence for the adaptive growth of Nitrososphaera-like AOA in acidic soil, suggesting a greater metabolic versatility of soil AOA than previously appreciated.

  7. Illuminating the Mechanistic Roles of Enzyme Conformational Dynamics

    SciTech Connect

    Hanson, Jeffrey A.; Dunderstadt, Karl; Watkins, Lucas P.; Bhattacharyya, Sucharita; Brokaw, Jason B.; Chu, Jhih-wei; Yang, Haw

    2007-11-13

    Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme’s lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK’s domain movements on its catalytic time scale. To quantitatively measure the enzyme’s entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly, the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzyme’s conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzyme’s rate-limiting step.

  8. The Tertiary Origin of the Allosteric Activation of E. coli Glucosamine-6-Phosphate Deaminase Studied by Sol-Gel Nanoencapsulation of Its T Conformer

    PubMed Central

    Zonszein, Sergio; Álvarez-Añorve, Laura I.; Vázquez-Núñez, Roberto J.; Calcagno, Mario L.

    2014-01-01

    The role of tertiary conformational changes associated to ligand binding was explored using the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase from Escherichia coli (EcGNPDA) as an experimental model. This is an enzyme of amino sugar catabolism that deaminates GlcN6P, giving fructose 6-phosphate and ammonia, and is allosterically activated by N-acetylglucosamine 6-phosphate (GlcNAc6P). We resorted to the nanoencapsulation of this enzyme in wet silica sol-gels for studying the role of intrasubunit local mobility in its allosteric activation under the suppression of quaternary transition. The gel-trapped enzyme lost its characteristic homotropic cooperativity while keeping its catalytic properties and the allosteric activation by GlcNAc6P. The nanoencapsulation keeps the enzyme in the T quaternary conformation, making possible the study of its allosteric activation under a condition that is not possible to attain in a soluble phase. The involved local transition was slowed down by nanoencapsulation, thus easing the fluorometric analysis of its relaxation kinetics, which revealed an induced-fit mechanism. The absence of cooperativity produced allosterically activated transitory states displaying velocity against substrate concentration curves with apparent negative cooperativity, due to the simultaneous presence of subunits with different substrate affinities. Reaction kinetics experiments performed at different tertiary conformational relaxation times also reveal the sequential nature of the allosteric activation. We assumed as a minimal model the existence of two tertiary states, t and r, of low and high affinity, respectively, for the substrate and the activator. By fitting the velocity-substrate curves as a linear combination of two hyperbolic functions with Kt and Kr as KM values, we obtained comparable values to those reported for the quaternary conformers in solution fitted to MWC model. These results are discussed in the background of the known

  9. WASP-121 b: a hot Jupiter close to tidal disruption transiting an active F star

    NASA Astrophysics Data System (ADS)

    Delrez, L.; Santerne, A.; Almenara, J.-M.; Anderson, D. R.; Collier-Cameron, A.; Díaz, R. F.; Gillon, M.; Hellier, C.; Jehin, E.; Lendl, M.; Maxted, P. F. L.; Neveu-VanMalle, M.; Pepe, F.; Pollacco, D.; Queloz, D.; Ségransan, D.; Smalley, B.; Smith, A. M. S.; Triaud, A. H. M. J.; Udry, S.; Van Grootel, V.; West, R. G.

    2016-06-01

    We present the discovery by the WASP-South survey of WASP-121 b, a new remarkable short-period transiting hot Jupiter. The planet has a mass of 1.183_{-0.062}^{+0.064} MJup, a radius of 1.865 ± 0.044 RJup, and transits every 1.274 9255_{-0.000 0025}^{+0.000 0020} days an active F6-type main-sequence star (V = 10.4, 1.353_{-0.079}^{+0.080} M⊙, 1.458 ± 0.030 R⊙, Teff = 6460 ± 140 K). A notable property of WASP-121 b is that its orbital semimajor axis is only ˜1.15 times larger than its Roche limit, which suggests that the planet is close to tidal disruption. Furthermore, its large size and extreme irradiation (˜7.1 109 erg s-1 cm-2) make it an excellent target for atmospheric studies via secondary eclipse observations. Using the TRAnsiting Planets and PlanetesImals Small Telescope, we indeed detect its emission in the z'-band at better than ˜4σ, the measured occultation depth being 603 ± 130 ppm. Finally, from a measurement of the Rossiter-McLaughlin effect with the CORALIE spectrograph, we infer a sky-projected spin-orbit angle of 257.8°_{-5.5°}^{+5.3°}. This result may suggest a significant misalignment between the spin axis of the host star and the orbital plane of the planet. If confirmed, this high misalignment would favour a migration of the planet involving strong dynamical events with a third body.

  10. Three active galactic nuclei close to the effective Eddington limit for dusty gas

    NASA Astrophysics Data System (ADS)

    Vasudevan, R. V.; Fabian, A. C.; Mushotzky, R. F.; Meléndez, M.; Winter, L. M.; Trippe, M. L.

    2013-06-01

    The effective Eddington limit for dusty gas surrounding active galactic nucleus (AGN) is lower than the canonical Eddington limit for hydrogen gas. Previous results from the Swift/Burst Alert Telescope 9-month catalogue suggested that in the overwhelming majority of local AGN, the dusty absorbing gas is below this effective Eddington limit, implying that radiation pressure is insufficient to blow away the absorbing clouds. We present an analysis of three objects from that sample which were found to be close to the effective Eddington limit (NGC 454, 2MASX J03565655-4041453 and XSS J05054-2348), using newly obtained XMM-Newton data. We use the X-ray data to better constrain the absorbing column density, and supplement them with XMM Optical Monitor data, infrared Spitzer and Herschel data were available to construct a broad-band spectral energy distribution to estimate refined bolometric luminosities and Eddington ratios for these three objects. The new XMM-Newton observations show all three objects moving away from the region expected for short-lived absorption in the NH-λEdd plane into the `long-lived absorption' region. We find our conclusions robust to different methods for estimating the bolometric luminosity and Eddington ratio. Interestingly, 2MASX J03565655-4041453 and XSS J05054-2348 now exhibit complex X-ray spectra, at variance with previous analyses of their Swift/X-Ray Telescope data. We find evidence for absorption variability in NGC 454 and 2MASX J03565655-4041453, perhaps implying that although the radiation pressure from the central engine is insufficient to cause clearly detectable outflows, it may cause absorption variations over longer time-scales. However, more robust black hole mass estimates would improve the accuracy of the Eddington ratio estimates for these objects.

  11. Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

    SciTech Connect

    Wubben, T.; Mesecar, A.D.

    2014-10-02

    Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 {angstrom} resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observed in the MtPPAT-CoA complex.

  12. Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

    SciTech Connect

    Santos, Camila Ramos; Meza, Andreia Navarro; Hoffmam, Zaira Bruna; Silva, Junio Cota; Alvarez, Thabata Maria; Ruller, Roberto; Giesel, Guilherme Menegon; Verli, Hugo; Squina, Fabio Marcio; Prade, Rolf Alexander; Murakami, Mario Tyago

    2010-12-10

    Research highlights: {yields} The hyperthermostable xylanase 10B from Thermotoga petrophila RKU-1 produces exclusively xylobiose at the optimum temperature. {yields} Circular dichroism spectroscopy suggests a coupling effect of temperature-induced structural changes with its enzymatic behavior. {yields} Crystallographic and molecular dynamics studies indicate that conformational changes in the product release area modulate the enzyme action mode. -- Abstract: Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20 {sup o}C, and exclusively xylobiose at 90 {sup o}C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.

  13. The sugar ring conformation of 4'-ethynyl-2-fluoro-2'-deoxyadenosine and its recognition by the polymerase active site of HIV reverse transcriptase.

    PubMed

    Kirby, K A; Singh, K; Michailidis, E; Marchand, B; Kodama, E N; Ashida, N; Mitsuya, H; Parniak, M A; Sarafianos, S G

    2011-01-01

    4' Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is the most potent inhibitor of HIV reverse transcriptase (RT). We have recently named EFdA a Translocation Defective RT Inhibitor (TDRTI) because after its incorporation in the nucleic acid it blocks DNA polymerization, primarily by preventing translocation of RT on the template/primer that has EFdA at the 3'-primer end (T/PEFdA). The sugar ring conformation of EFdA may also influence RT inhibition by a) affecting the binding of EFdA triphosphate (EFdATP) at the RT active site and/or b) by preventing proper positioning of the 3'-OH of EFdA in T/PEFdA that is required for efficient DNA synthesis. Specifically, the North (C2'-exo/C3'-endo), but not the South (C2'-endo/C3'-exo) nucleotide sugar ring conformation is required for efficient binding at the primer-binding and polymerase active sites of RT. In this study we use nuclear magnetic resonance (NMR) spectroscopy experiments to determine the sugar ring conformation of EFdA. We find that unlike adenosine nucleosides unsubstituted at the 4'-position, the sugar ring of EFdA is primarily in the North conformation. This difference in sugar ring puckering likely contributes to the more efficient incorporation of EFdATP by RT than dATP. In addition, it suggests that the 3'-OH of EFdA in T/PEFdA is not likely to prevent incorporation of additional nucleotides and thus it does not contribute to the mechanism of RT inhibition. This study provides the first insights into how structural attributes of EFdA affect its antiviral potency through interactions with its RT target. PMID:21366961

  14. THE SUGAR RING CONFORMATION OF 4’-ETHYNYL-2-FLUORO-2’-DEOXYADENOSINE AND ITS RECOGNITION BY THE POLYMERASE ACTIVE SITE OF HIV REVERSE TRANSCRIPTASE*

    PubMed Central

    Kirby, K.A.; Singh, K.; Michailidis, E.; Marchand, B.; Kodama, E.N.; Ashida, N.; Mitsuya, H.; Parniak, M.A.; Sarafianos, S.G.

    2011-01-01

    4’-Ethynyl-2-fluoro-2’-deoxyadenosine (EFdA) is the most potent inhibitor of HIV reverse transcriptase (RT). We have recently named EFdA a Translocation Defective RT Inhibitor (TDRTI) because after its incorporation in the nucleic acid it blocks DNA polymerization, primarily by preventing translocation of RT on the template/primer that has EFdA at the 3’-primer end (T/PEFdA). The sugar ring conformation of EFdA may also influence RT inhibition by a) affecting the binding of EFdA triphosphate (EFdATP) at the RT active site and/or b) by preventing proper positioning of the 3’-OH of EFdA in T/PEFdA that is required for efficient DNA synthesis. Specifically, the North (C2’-exo/C3’-endo), but not the South (C2’-endo/C3’-exo) nucleotide sugar ring conformation is required for efficient binding at the primer-binding and polymerase active sites of RT. In this study we use nuclear magnetic resonance (NMR) spectroscopy experiments to determine the sugar ring conformation of EFdA. We find that unlike adenosine nucleosides unsubstituted at the 4’-position, the sugar ring of EFdA is primarily in the North conformation. This difference in sugar ring puckering likely contributes to the more efficient incorporation of EFdATP by RT than dATP. In addition, it suggests that the 3’-OH of EFdA in T/PEFdA is not likely to prevent incorporation of additional nucleotides and thus it does not contribute to the mechanism of RT inhibition. This study provides the first insights into how structural attributes of EFdA affect its antiviral potency through interactions with its RT target. PMID:21366961

  15. The conformational activation of antithrombin. A 2.85-A structure of a fluorescein derivative reveals an electrostatic link between the hinge and heparin binding regions.

    PubMed

    Huntington, J A; McCoy, A; Belzar, K J; Pei, X Y; Gettins, P G; Carrell, R W

    2000-05-19

    Antithrombin is unique among the serpins in that it circulates in a native conformation that is kinetically inactive toward its target proteinase, factor Xa. Activation occurs upon binding of a specific pentasaccharide sequence found in heparin that results in a rearrangement of the reactive center loop removing constraints on the active center P1 residue. We determined the crystal structure of an activated antithrombin variant, N135Q S380C-fluorescein (P14-fluorescein), in order to see how full activation is achieved in the absence of heparin and how the structural effects of the substitution in the hinge region are translated to the heparin binding region. The crystal structure resembles native antithrombin except in the hinge and heparin binding regions. The absence of global conformational change allows for identification of specific interactions, centered on Glu(381) (P13), that are responsible for maintenance of the solution equilibrium between the native and activated forms and establishes the existence of an electrostatic link between the hinge region and the heparin binding region. A revised model for the mechanism of the allosteric activation of antithrombin is proposed.

  16. Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion.

    PubMed

    Hohlbein, Johannes; Aigrain, Louise; Craggs, Timothy D; Bermek, Oya; Potapova, Olga; Shoolizadeh, Pouya; Grindley, Nigel D F; Joyce, Catherine M; Kapanidis, Achillefs N

    2013-01-01

    The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.

  17. Mapping the Free Energy Landscape of PKA Inhibition and Activation: A Double-Conformational Selection Model for the Tandem cAMP-Binding Domains of PKA RIα.

    PubMed

    Akimoto, Madoka; McNicholl, Eric Tyler; Ramkissoon, Avinash; Moleschi, Kody; Taylor, Susan S; Melacini, Giuseppe

    2015-01-01

    Protein Kinase A (PKA) is the major receptor for the cyclic adenosine monophosphate (cAMP) secondary messenger in eukaryotes. cAMP binds to two tandem cAMP-binding domains (CBD-A and -B) within the regulatory subunit of PKA (R), unleashing the activity of the catalytic subunit (C). While CBD-A in RIα is required for PKA inhibition and activation, CBD-B functions as a "gatekeeper" domain that modulates the control exerted by CBD-A. Preliminary evidence suggests that CBD-B dynamics are critical for its gatekeeper function. To test this hypothesis, here we investigate by Nuclear Magnetic Resonance (NMR) the two-domain construct RIα (91-379) in its apo, cAMP2, and C-bound forms. Our comparative NMR analyses lead to a double conformational selection model in which each apo CBD dynamically samples both active and inactive states independently of the adjacent CBD within a nearly degenerate free energy landscape. Such degeneracy is critical to explain the sensitivity of CBD-B to weak interactions with C and its high affinity for cAMP. Binding of cAMP eliminates this degeneracy, as it selectively stabilizes the active conformation within each CBD and inter-CBD contacts, which require both cAMP and W260. The latter is contributed by CBD-B and mediates capping of the cAMP bound to CBD-A. The inter-CBD interface is dispensable for intra-CBD conformational selection, but is indispensable for full activation of PKA as it occludes C-subunit recognition sites within CBD-A. In addition, the two structurally homologous cAMP-bound CBDs exhibit marked differences in their residual dynamics profiles, supporting the notion that conservation of structure does not necessarily imply conservation of dynamics.

  18. Short-time dynamics of pH-dependent conformation and substrate binding in the active site of beta-glucosidases: A computational study.

    PubMed

    Flannelly, David F; Aoki, Thalia G; Aristilde, Ludmilla

    2015-09-01

    The complete degradation of cellulose to glucose is essential to carbon turnover in terrestrial ecosystems and to engineered biofuel production. A rate-limiting step in this pathway is catalyzed by beta-glucosidase (BG) enzymes, which convert cellulobiose into two glucose molecules. The activity of these enzymes has been shown to vary with solution pH. However, it is not well understood how pH influences the enzyme conformation required for catalytic action on the substrate. A structural understanding of this pH effect is important for predicting shifts in BG activity in bioreactors and environmental matrices, in addition to informing targeted protein engineering. Here we applied molecular dynamics simulations to explore conformational and substrate binding dynamics in two well-characterized BGs of bacterial (Clostridium cellulovorans) and fungal (Trichoderma reesei) origins as a function of pH. The enzymes were simulated in an explicit solvated environment, with NaCl as electrolytes, at their prominent ionization states obtained at pH 5, 6, 7, and 7.5. Our findings indicated that pH-dependent changes in the ionization states of non-catalytic residues localized outside of the immediate active site led to pH-dependent disruption of the active site conformation. This disruption interferes with favorable H-bonding interactions with catalytic residues required to initiate catalysis on the substrate. We also identified specific non-catalytic residues that are involved in stabilizing the substrate at the optimal pH for enzyme activity. The simulations further revealed the dynamics of water-bridging interactions both outside and inside the substrate binding cleft during structural changes in the enzyme-substrate complex. These findings provide new structural insights into the pH-dependent substrate binding specificity in BGs. PMID:26160737

  19. Short-time dynamics of pH-dependent conformation and substrate binding in the active site of beta-glucosidases: A computational study.

    PubMed

    Flannelly, David F; Aoki, Thalia G; Aristilde, Ludmilla

    2015-09-01

    The complete degradation of cellulose to glucose is essential to carbon turnover in terrestrial ecosystems and to engineered biofuel production. A rate-limiting step in this pathway is catalyzed by beta-glucosidase (BG) enzymes, which convert cellulobiose into two glucose molecules. The activity of these enzymes has been shown to vary with solution pH. However, it is not well understood how pH influences the enzyme conformation required for catalytic action on the substrate. A structural understanding of this pH effect is important for predicting shifts in BG activity in bioreactors and environmental matrices, in addition to informing targeted protein engineering. Here we applied molecular dynamics simulations to explore conformational and substrate binding dynamics in two well-characterized BGs of bacterial (Clostridium cellulovorans) and fungal (Trichoderma reesei) origins as a function of pH. The enzymes were simulated in an explicit solvated environment, with NaCl as electrolytes, at their prominent ionization states obtained at pH 5, 6, 7, and 7.5. Our findings indicated that pH-dependent changes in the ionization states of non-catalytic residues localized outside of the immediate active site led to pH-dependent disruption of the active site conformation. This disruption interferes with favorable H-bonding interactions with catalytic residues required to initiate catalysis on the substrate. We also identified specific non-catalytic residues that are involved in stabilizing the substrate at the optimal pH for enzyme activity. The simulations further revealed the dynamics of water-bridging interactions both outside and inside the substrate binding cleft during structural changes in the enzyme-substrate complex. These findings provide new structural insights into the pH-dependent substrate binding specificity in BGs.

  20. Mapping the Free Energy Landscape of PKA Inhibition and Activation: A Double-Conformational Selection Model for the Tandem cAMP-Binding Domains of PKA RIα

    PubMed Central

    Akimoto, Madoka; McNicholl, Eric Tyler; Ramkissoon, Avinash; Moleschi, Kody; Taylor, Susan S.; Melacini, Giuseppe

    2015-01-01

    Protein Kinase A (PKA) is the major receptor for the cyclic adenosine monophosphate (cAMP) secondary messenger in eukaryotes. cAMP binds to two tandem cAMP-binding domains (CBD-A and -B) within the regulatory subunit of PKA (R), unleashing the activity of the catalytic subunit (C). While CBD-A in RIα is required for PKA inhibition and activation, CBD-B functions as a “gatekeeper” domain that modulates the control exerted by CBD-A. Preliminary evidence suggests that CBD-B dynamics are critical for its gatekeeper function. To test this hypothesis, here we investigate by Nuclear Magnetic Resonance (NMR) the two-domain construct RIα (91–379) in its apo, cAMP2, and C-bound forms. Our comparative NMR analyses lead to a double conformational selection model in which each apo CBD dynamically samples both active and inactive states independently of the adjacent CBD within a nearly degenerate free energy landscape. Such degeneracy is critical to explain the sensitivity of CBD-B to weak interactions with C and its high affinity for cAMP. Binding of cAMP eliminates this degeneracy, as it selectively stabilizes the active conformation within each CBD and inter-CBD contacts, which require both cAMP and W260. The latter is contributed by CBD-B and mediates capping of the cAMP bound to CBD-A. The inter-CBD interface is dispensable for intra-CBD conformational selection, but is indispensable for full activation of PKA as it occludes C-subunit recognition sites within CBD-A. In addition, the two structurally homologous cAMP-bound CBDs exhibit marked differences in their residual dynamics profiles, supporting the notion that conservation of structure does not necessarily imply conservation of dynamics. PMID:26618408

  1. Effects of magnesium chloride on smooth muscle actomyosin adenosine-5'-triphosphatase activity, myosin conformation, and tension development in glycerinated smooth muscle fibers.

    PubMed

    Ikebe, M; Barsotti, R J; Hinkins, S; Hartshorne, D J

    1984-10-01

    The contractile system of smooth muscle exhibits distinctive responses to varying Mg2+ concentrations in that maximum adenosine-5'-triphosphatase (ATPase) activity of actomyosin requires relatively high concentrations of Mg2+ and also that tension in skinned smooth muscle fibers can be induced in the absence of Ca2+ by high Mg2+ concentrations. We have examined the effects of MgCl2 on actomyosin ATPase activity and on tension development in skinned gizzard fibers and suggest that the MgCl2-induced changes may be correlated to shifts in myosin conformation. At low concentrations of free Mg2+ (less than or equal to 1 mM) the actin-activated ATPase activity of phosphorylated turkey gizzard myosin is reduced and is increased as the Mg2+ concentration is raised. The increase in Mg2+ (over a range of 1-10 mM added MgCl2) induces the conversion of 10S phosphorylated myosin to the 6S form, and it was found that the proportion of myosin as 10S is inversely related to the level of actin-activated ATPase activity. Activation of the actin-activated ATPase activity also occurs with dephosphorylated myosin but at higher MgCl2 concentrations, between 10 and 40 mM added MgCl2. Viscosity and fluorescence measurements indicate that increasing Mg2+ levels over this concentration range favor the formation of the 6S conformation of dephosphorylated myosin, and it is proposed that the 10S to 6S transition is a prerequisite for the observed activation of ATPase activity. With glycerinated chicken gizzard fibers high MgCl2 concentrations (6-20 mM) promote tension in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Pegylation of Antimicrobial Peptides Maintains the Active Peptide Conformation, Model Membrane Interactions, and Antimicrobial Activity while Improving Lung Tissue Biocompatibility following Airway Delivery

    PubMed Central

    Morris, Christopher J.; Beck, Konrad; Fox, Marc A.; Ulaeto, David; Clark, Graeme C.

    2012-01-01

    Antimicrobial peptides (AMPs) have therapeutic potential, particularly for localized infections such as those of the lung. Here we show that airway administration of a pegylated AMP minimizes lung tissue toxicity while nevertheless maintaining antimicrobial activity. CaLL, a potent synthetic AMP (KWKLFKKIFKRIVQRIKDFLR) comprising fragments of LL-37 and cecropin A peptides, was N-terminally pegylated (PEG-CaLL). PEG-CaLL derivatives retained significant antimicrobial activity (50% inhibitory concentrations [IC50s] 2- to 3-fold higher than those of CaLL) against bacterial lung pathogens even in the presence of lung lining fluid. Circular dichroism and fluorescence spectroscopy confirmed that conformational changes associated with the binding of CaLL to model microbial membranes were not disrupted by pegylation. Pegylation of CaLL reduced AMP-elicited cell toxicity as measured using in vitro lung epithelial primary cell cultures. Further, in a fully intact ex vivo isolated perfused rat lung (IPRL) model, airway-administered PEG-CaLL did not result in disruption of the pulmonary epithelial barrier, whereas CaLL caused an immediate loss of membrane integrity leading to pulmonary edema. All AMPs (CaLL, PEG-CaLL, LL-37, cecropin A) delivered to the lung by airway administration showed limited (<3%) pulmonary absorption in the IPRL with extensive AMP accumulation in lung tissue itself, a characteristic anticipated to be beneficial for the treatment of pulmonary infections. We conclude that pegylation may present a means of improving the lung biocompatibility of AMPs designed for the treatment of pulmonary infections. PMID:22430978

  3. Conformational Landscape of Nicotinoids: Solving the "conformational - Rity" of Anabasine

    NASA Astrophysics Data System (ADS)

    Lesarri, Alberto; Cocinero, Emilio J.; Evangelisti, Luca; Suenram, Richard D.; Caminati, Walther; Grabow, Jens-Uwe

    2010-06-01

    The conformational landscape of the alkaloid anabasine (neonicotine) has been investigated using rotational spectroscopy and ab initio calculations. The results allow a detailed comparison of the structural properties of the prototype piperidinic and pyrrolidinic nicotinoids (anabasine vs. nicotine). Anabasine adopts two most stable conformations in isolation conditions, for which we determined accurate rotational and nuclear quadrupole coupling parameters. The preferred conformations are characterized by an equatorial pyridine moiety and additional N-H equatorial stereochemistry at the piperidine ring (Eq-Eq). The two rings of anabasine are close to a bisecting arrangement, with the observed conformations differing in a ca. 180° rotation of the pyridine subunit, denoted either Syn or Anti. The preference of anabasine for the Eq-Eq-Syn conformation has been established by relative intensity measurements (Syn/Anti˜5(2)). The conformational preferences of free anabasine are directed by a N\\cdot\\cdot\\cdotH-C weak hydrogen bond interaction between the nitrogen lone pair at piperidine and the closest hydrogen bond in pyridine, with N\\cdot\\cdot\\cdotN distances ranging from 4.750 Å (Syn) to 4.233 Å (Anti). R. J. Lavrich, R. D. Suenram, D. F. Plusquellic and S. Davis, 58^th OSU Int. Symp. on Mol. Spectrosc., Columbus, OH, 2003, Comm. RH13.

  4. 1,4-disubstituted-[1,2,3]triazolyl-containing analogues of MT-II: design, synthesis, conformational analysis, and biological activity.

    PubMed

    Testa, Chiara; Scrima, Mario; Grimaldi, Manuela; D'Ursi, Anna M; Dirain, Marvin L; Lubin-Germain, Nadège; Singh, Anamika; Haskell-Luevano, Carrie; Chorev, Michael; Rovero, Paolo; Papini, Anna M

    2014-11-26

    Side chain-to-side chain cyclizations represent a strategy to select a family of bioactive conformations by reducing the entropy and enhancing the stabilization of functional ligand-induced receptor conformations. This structural manipulation contributes to increased target specificity, enhanced biological potency, improved pharmacokinetic properties, increased functional potency, and lowered metabolic susceptibility. The Cu(I)-catalyzed azide-alkyne 1,3-dipolar Huisgen's cycloaddition, the prototypic click reaction, presents a promising opportunity to develop a new paradigm for an orthogonal bioorganic and intramolecular side chain-to-side chain cyclization. In fact, the proteolytic stable 1,4- or 4,1-disubstituted [1,2,3]triazolyl moiety is isosteric with the peptide bond and can function as a surrogate of the classical side chain-to-side chain lactam forming bridge. Herein we report the design, synthesis, conformational analysis, and functional biological activity of a series of i-to-i+5 1,4- and 4,1-disubstituted [1,2,3]triazole-bridged cyclopeptides derived from MT-II, the homodetic Asp(5) to Lys(10) side chain-to-side chain bridged heptapeptide, an extensively studied agonist of melanocortin receptors. PMID:25347033

  5. 1,4-Disubstituted-[1,2,3]triazolyl-Containing Analogues of MT-II: Design, Synthesis, Conformational Analysis, and Biological Activity

    PubMed Central

    2015-01-01

    Side chain-to-side chain cyclizations represent a strategy to select a family of bioactive conformations by reducing the entropy and enhancing the stabilization of functional ligand-induced receptor conformations. This structural manipulation contributes to increased target specificity, enhanced biological potency, improved pharmacokinetic properties, increased functional potency, and lowered metabolic susceptibility. The CuI-catalyzed azide–alkyne 1,3-dipolar Huisgen’s cycloaddition, the prototypic click reaction, presents a promising opportunity to develop a new paradigm for an orthogonal bioorganic and intramolecular side chain-to-side chain cyclization. In fact, the proteolytic stable 1,4- or 4,1-disubstituted [1,2,3]triazolyl moiety is isosteric with the peptide bond and can function as a surrogate of the classical side chain-to-side chain lactam forming bridge. Herein we report the design, synthesis, conformational analysis, and functional biological activity of a series of i-to-i+5 1,4- and 4,1-disubstituted [1,2,3]triazole-bridged cyclopeptides derived from MT-II, the homodetic Asp5 to Lys10 side chain-to-side chain bridged heptapeptide, an extensively studied agonist of melanocortin receptors. PMID:25347033

  6. Conformational dimorphism of isochroman-1-ones in the solid state

    NASA Astrophysics Data System (ADS)

    Babjaková, Eva; Hanulíková, Barbora; Dastychová, Lenka; Kuřitka, Ivo; Nečas, Marek; Vícha, Robert

    2014-12-01

    Isochroman-1-one derivatives, which are relatives of coumarins, display a broad spectrum of biological activity; therefore, these derivatives attract the attention of chemists. A series of new isochroman-1-ones were prepared by the reaction of benzyl-derived Grignard reagents with acyl chlorides. All of the prepared compounds were characterized using single-crystal X-ray diffraction as well as FT-IR, NMR and MS techniques. Single crystal X-ray diffraction analysis revealed that the isochromanones can adopt two distinct conformations in the solid state. For one of the compounds, two polymorphs with unique forms crystallized separately under different temperatures. The packing of all of the examined crystals is stabilized via weak intramolecular C-H⋯π and/or C-H⋯O interactions. Although the closed conformer was predominantly found in the actual crystals, the open conformer is thermochemically more stable for all of the examined compounds according to DFT calculations.

  7. Developmental regulation of chromatin conformation by Hox proteins in Drosophila

    PubMed Central

    Agelopoulos, Marios; McKay, Daniel J.; Mann, Richard S.

    2012-01-01

    Summary We present a strategy to examine the chromatin conformation of individual loci in specific cell types during Drosophila embryogenesis. Regulatory DNA is tagged with binding sites (lacO) for LacI, which is used to immunopreciptiate the tagged chromatin from specific cell types. We applied this approach to Distalless (Dll), a gene required for limb development in Drosophila. We show that the local chromatin conformation at Dll depends on the cell type: in cells that express Dll, the 5’ regulatory region is in close proximity to the Dll promoter. In Dll nonexpressing cells this DNA is in a more extended configuration. In addition, transcriptional activators and repressors are bound to Dll regulatory DNA in a cell type specific manner. The pattern of binding by GAGA factor and the variant histone H2Av suggest that they play a role in the regulation of Dll chromatin conformation in expressing and non-expressing cell types, respectively. PMID:22523743

  8. Spontaneous inactivation of human tryptase involves conformational changes consistent with conversion of the active site to a zymogen-like structure.

    PubMed

    Selwood, T; McCaslin, D R; Schechter, N M

    1998-09-22

    The conformational changes accompanying spontaneous inactivation and dextran sulfate (DS) mediated reactivation of the serine protease human tryptase were investigated by analysis of (i) intrinsic fluorescence, (ii) inhibitor binding, and (iii) catalytic efficiency. Spontaneous inactivation produced a marked decrease in fluorescence emission intensity that was reversed by the addition of DS. Fluorescence decreases at high (4.0 microM) and low (0.1 microM) tryptase concentrations were similar at early times and coincided with loss of enzymatic activity but deviated significantly from activity loss at later times by showing a difference in the extent of change. The fluorescence losses were best described by a two-step kinetic model in which the major decrease correlated to activity loss (t1/2 of 4.3 min in 0.2 M NaCl, pH 6.8, 30 degrees C) and was followed by a further decrease (t1/2 approximately 60 min) whose extent differed with tryptase concentration. The ability to bind the competitive inhibitor p-aminobenzamidine was reversibly lost upon spontaneous inactivation, providing evidence for conformational changes affecting the major substrate binding site (S1-pocket). Estimation of catalytic efficiency using an active site titrant showed that the specific activity of tryptase remained unchanged upon inactivation and reactivation. Return of enzymatic activity, intrinsic fluorescence, and the S1 pocket appeared to occur in the same time frame (t1/2 approximately 3 min). These studies indicate that spontaneous inactivation involves reversible changes which convert the active site to a nonfunctional state. The association of activity loss with an intrinsic fluorescence decrease and loss of the S1-pocket is consistent with the disruption of a critical ionic bond at the active site. Formation of this ionic bond is the basis of zymogen activation for the chymotrypsin family of serine proteases. PMID:9748324

  9. Small-Angle Neutron Scattering Reveals pH-Dependent Conformational Changes in Trichoderma reesei Cellobiohydrolase I: Implications for Enzymatic Activity

    SciTech Connect

    Pingali, Sai Venkatesh; O'Neill, Hugh Michael; McGaughey, Joseph; Urban, Volker S; Rempe, Caroline S; Petridis, Loukas; Smith, Jeremy C; Evans, Barbara R; Heller, William T

    2011-01-01

    Cellobiohydrolase I (Cel7A) of the fungus Trichoderma reesei (now classified as an anamorph of Hypocrea jecorina) hydrolyzes crystalline cellulose to soluble sugars, making it of key interest for producing fermentable sugars from biomass for biofuel production. The activity of the enzyme is pH-dependent, with its highest activity occurring at pH 4 5. To probe the response of the solution structure of Cel7A to changes in pH, we measured small angle neutron scattering of it in a series of solutions having pH values of 7.0, 6.0, 5.3, and 4.2. As the pH decreases from 7.0 to 5.3, the enzyme structure remains well defined, possessing a spatial differentiation between the cellulose binding domain and the catalytic core that only changes subtly. At pH 4.2, the solution conformation of the enzyme changes to a structure that is intermediate between a properly folded enzyme and a denatured, unfolded state, yet the secondary structure of the enzyme is essentially unaltered. The results indicate that at the pH of optimal activity, the catalytic core of the enzyme adopts a structure in which the compact packing typical of a fully folded polypeptide chain is disrupted and suggest that the increased range of structures afforded by this disordered state plays an important role in the increased activity of Cel7A through conformational selection.

  10. Helix 3 acts as a conformational hinge in Class A GPCR activation: An analysis of interhelical interaction energies in crystal structures.

    PubMed

    Lans, Isaias; Dalton, James A R; Giraldo, Jesús

    2015-12-01

    A collection of crystal structures of rhodopsin, β2-adrenergic and adenosine A2A receptors in active, intermediate and inactive states were selected for structural and energetic analyses to identify the changes involved in the activation/deactivation of Class A GPCRs. A set of helix interactions exclusive to either inactive or active/intermediate states were identified. The analysis of these interactions distinguished some local conformational changes involved in receptor activation, in particular, a packing between the intracellular domains of transmembrane helices H3 and H7 and a separation between those of H2 and H6. Also, differential movements of the extracellular and intracellular domains of these helices are apparent. Moreover, a segment of residues in helix H3, including residues L/I3.40 to L3.43, is identified as a key component of the activation mechanism, acting as a conformational hinge between extracellular and intracellular regions. Remarkably, the influence on the activation process of some glutamic and aspartic acidic residues and, as a consequence, the influence of variations on local pH is highlighted. Structural hypotheses that arose from the analysis of rhodopsin, β2-adrenergic and adenosine A2A receptors were tested on the active and inactive M2 muscarinic acetylcholine receptor structures and further discussed in the context of the new mechanistic insights provided by the recently determined active and inactive crystal structures of the μ-opioid receptor. Overall, the structural and energetic analyses of the interhelical interactions present in this collection of Class A GPCRs suggests the existence of a common general activation mechanism featuring a chemical space useful for drug discovery exploration.

  11. Suppressing epileptic activity in a neural mass model using a closed-loop proportional-integral controller

    NASA Astrophysics Data System (ADS)

    Wang, Junsong; Niebur, Ernst; Hu, Jinyu; Li, Xiaoli

    2016-06-01

    Closed-loop control is a promising deep brain stimulation (DBS) strategy that could be used to suppress high-amplitude epileptic activity. However, there are currently no analytical approaches to determine the stimulation parameters for effective and safe treatment protocols. Proportional-integral (PI) control is the most extensively used closed-loop control scheme in the field of control engineering because of its simple implementation and perfect performance. In this study, we took Jansen’s neural mass model (NMM) as a test bed to develop a PI-type closed-loop controller for suppressing epileptic activity. A graphical stability analysis method was employed to determine the stabilizing region of the PI controller in the control parameter space, which provided a theoretical guideline for the choice of the PI control parameters. Furthermore, we established the relationship between the parameters of the PI controller and the parameters of the NMM in the form of a stabilizing region, which provided insights into the mechanisms that may suppress epileptic activity in the NMM. The simulation results demonstrated the validity and effectiveness of the proposed closed-loop PI control scheme.

  12. Suppressing epileptic activity in a neural mass model using a closed-loop proportional-integral controller

    PubMed Central

    Wang, Junsong; Niebur, Ernst; Hu, Jinyu; Li, Xiaoli

    2016-01-01

    Closed-loop control is a promising deep brain stimulation (DBS) strategy that could be used to suppress high-amplitude epileptic activity. However, there are currently no analytical approaches to determine the stimulation parameters for effective and safe treatment protocols. Proportional-integral (PI) control is the most extensively used closed-loop control scheme in the field of control engineering because of its simple implementation and perfect performance. In this study, we took Jansen’s neural mass model (NMM) as a test bed to develop a PI-type closed-loop controller for suppressing epileptic activity. A graphical stability analysis method was employed to determine the stabilizing region of the PI controller in the control parameter space, which provided a theoretical guideline for the choice of the PI control parameters. Furthermore, we established the relationship between the parameters of the PI controller and the parameters of the NMM in the form of a stabilizing region, which provided insights into the mechanisms that may suppress epileptic activity in the NMM. The simulation results demonstrated the validity and effectiveness of the proposed closed-loop PI control scheme. PMID:27273563

  13. Structure-activity relationships of the antimicrobial peptide gramicidin S and its analogs: aqueous solubility, self-association, conformation, antimicrobial activity and interaction with model lipid membranes.

    PubMed

    Abraham, Thomas; Prenner, Elmar J; Lewis, Ruthven N A H; Mant, Colin T; Keller, Sandro; Hodges, Robert S; McElhaney, Ronald N

    2014-05-01

    GS10 [cyclo-(VKLdYPVKLdYP)] is a synthetic analog of the naturally occurring antimicrobial peptide gramicidin (GS) in which the two positively charged ornithine (Orn) residues are replaced by two positively charged lysine (Lys) residues and the two less polar aromatic phenylalanine (Phe) residues are replaced by the more polar tyrosine (Tyr) residues. In this study, we examine the effects of these seemingly conservative modifications to the parent GS molecule on the physical properties of the peptide, and on its interactions with lipid bilayer model and biological membranes, by a variety of biophysical techniques. We show that although GS10 retains the largely β-sheet conformation characteristic of GS, it is less structured in both water and membrane-mimetic solvents. GS10 is also more water soluble and less hydrophobic than GS, as predicted, and also exhibits a reduced tendency for self-association in aqueous solution. Surprisingly, GS10 associates more strongly with zwitterionic and anionic phospholipid bilayer model membranes than does GS, despite its greater water solubility, and the presence of anionic phospholipids and cholesterol (Chol) modestly reduces the association of both GS10 and GS to these model membranes. The strong partitioning of both peptides into lipid bilayers is driven by a large favorable entropy change opposed by a much smaller unfavorable enthalpy change. However, GS10 is also less potent than GS at inducing inverted cubic phases in phospholipid bilayer model membranes and at inhibiting the growth of the cell wall-less bacterium Acholeplasma laidlawii B. These results are discussed in terms of the comparative antibiotic and hemolytic activities of these peptides.

  14. Exploring conformational states of the bacterial voltage-gated sodium channel NavAb via molecular dynamics simulations.

    PubMed

    Amaral, Cristiano; Carnevale, Vincenzo; Klein, Michael L; Treptow, Werner

    2012-12-26

    The X-ray structure of the bacterial voltage-gated sodium channel NavAb has been reported in a conformation with a closed conduction pore. Comparison between this structure and the activated-open and resting-closed structures of the voltage-gated Kv1.2 potassium channel suggests that the voltage-sensor domains (VSDs) of the reported structure are not fully activated. Using the aforementioned structures of Kv1.2 as templates, molecular dynamics simulations are used to identify analogous functional conformations of NavAb. Specifically, starting from the NavAb crystal structure, conformations of the membrane-bound channel are sampled along likely pathways for activation of the VSD and opening of the pore domain. Gating charge computations suggest that a structural rearrangement comparable to that occurring between activated-open and resting-closed states is required to explain experimental values of the gating charge, thereby confirming that the reported VSD structure is likely an intermediate along the channel activation pathway. Our observation that the X-ray structure exhibits a low pore domain-opening propensity further supports this notion. The present molecular dynamics study also identifies conformations of NavAb that are seemingly related to the resting-closed and activated-open states. Our findings are consistent with recent structural and functional studies of the orthologous channels NavRh, NaChBac, and NavMs and offer possible structures for the functionally relevant conformations of NavAb.

  15. Conformational flexibility of aspartame.

    PubMed

    Toniolo, Claudio; Temussi, Pierandrea

    2016-05-01

    L-Aspartyl-L-phenylalanine methyl ester, better known as aspartame, is not only one of the most used artificial sweeteners, but also a very interesting molecule with respect to the correlation between molecular structure and taste. The extreme conformational flexibility of this dipeptide posed a huge difficulty when researchers tried to use it as a lead compound to design new sweeteners. In particular, it was difficult to take advantage of its molecular model as a mold to infer the shape of the, then unknown, active site of the sweet taste receptor. Here, we follow the story of the 3D structural aspects of aspartame from early conformational studies to recent docking into homology models of the receptor. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 376-384, 2016. PMID:27038223

  16. Conformational flexibility of aspartame.

    PubMed

    Toniolo, Claudio; Temussi, Pierandrea

    2016-05-01

    L-Aspartyl-L-phenylalanine methyl ester, better known as aspartame, is not only one of the most used artificial sweeteners, but also a very interesting molecule with respect to the correlation between molecular structure and taste. The extreme conformational flexibility of this dipeptide posed a huge difficulty when researchers tried to use it as a lead compound to design new sweeteners. In particular, it was difficult to take advantage of its molecular model as a mold to infer the shape of the, then unknown, active site of the sweet taste receptor. Here, we follow the story of the 3D structural aspects of aspartame from early conformational studies to recent docking into homology models of the receptor. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 376-384, 2016.

  17. Structural analysis of the active sites of dihydrofolate reductase from two species of Candida uncovers ligand-induced conformational changes shared among species

    PubMed Central

    Paulsen, Janet L.; Viswanathan, Kishore; Wright, Dennis L.; Anderson, Amy C.

    2013-01-01

    A novel strategy for targeting the pathogenic organisms Candida albicans and Candida glabrata focuses on the development of potent and selective antifolates effective against dihydrofolate reductase. Crystal structure analysis suggested that an essential loop at the active site (Thr 58-Phe 66) differs from the analogous residues in the human enzyme, potentially providing a mechanism for achieving selectivity. In order to probe the role of this loop, we employed chemical synthesis, crystal structure determination and molecular dynamics simulations. The results of these analyses show that the loop residues undergo ligand-induced conformational changes that are similar among the fungal and human species. PMID:23375226

  18. Comparison of body-powered voluntary opening and voluntary closing prehensor for activities of daily life.

    PubMed

    Berning, Kelsey; Cohick, Sarah; Johnson, Reva; Miller, Laura Ann; Sensinger, Jonathon W

    2014-01-01

    Persons with an upper-limb amputation who use a body-powered prosthesis typically control the prehensor through contralateral shoulder movement, which is transmitted through a Bowden cable. Increased cable tension either opens or closes the prehensor; when tension is released, some passive element, such as a spring, returns the prehensor to the default state (closed or open). In this study, we used the Southampton Hand Assessment Procedure to examine functional differences between these two types of prehensors in 29 nondisabled subjects (who used a body-powered bypass prosthesis) and 2 persons with unilateral transradial amputations (who used a conventional body-powered device). We also administered a survey to determine whether subjects preferred one prehensor or the other for specific tasks, with a long-term goal of assessing whether a prehensor that could switch between both modes would be advantageous. We found that using the voluntary closing prehensor was 1.3 s faster (p = 0.02) than using the voluntary opening prehensor, across tasks, and that there was consensus among subjects on which types of tasks they preferred to do with each prehensor type. Twenty-five subjects wanted a device that could switch between the two modes in order to perform particular tasks.

  19. The structure of truncated recombinant human bile salt-stimulated lipase reveals bile salt-independent conformational flexibility at the active-site loop and provides insights into heparin binding.

    PubMed

    Moore, S A; Kingston, R L; Loomes, K M; Hernell, O; Bläckberg, L; Baker, H M; Baker, E N

    2001-09-21

    a search model. This structure reveals the position of the C-terminal helix, missing in the truncated variant, and also shows the active-site loop to be in a closed conformation. PMID:11563913

  20. Experimental investigation of the vortical activity in the close wake of a simplified military transport aircraft

    NASA Astrophysics Data System (ADS)

    Bury, Yannick; Jardin, Thierry; Klöckner, Andreas

    2013-05-01

    This paper focuses on the experimental characterization of the vortex structures that develop in the aft fuselage region and in the wake of a simplified geometry of a military transport aircraft. It comes within the framework of the military applications of airflow influence on airdrop operations. This work relies on particle image velocimetry measurements combined with a vortex-tracking approach. Complex vortex dynamics is revealed, in terms of vortex positions, intensities, sizes, shapes and fluctuation levels, for both closed and opened cargo-door and ramp airdrop configurations.

  1. 29 CFR 1620.4 - “Closely related” and “directly essential” activities.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...; employees in the personnel, labor relations, employee benefits, safety and health, advertising, promotion, and public relations activities of the producing enterprise; work instructors for the...

  2. Conformal operators in QCD

    SciTech Connect

    Makeenko, Y.M.

    1981-03-01

    Utilizing the properties of the representations of the conformal group, we obtain new expressions for the conformal operators composed of spinor or scalar fields of arbitrary dimension in terms of Jacobi polynomials. These expressions generalize the known formulas in terms of Gegenbauer polynomials. Using the conformal Ward identities, we prove the multiplicative renormalizability of conformal operators in the leading logarithmic approximation.

  3. Sequence, structure, and active site analyses of p38 MAP kinase: exploiting DFG-out conformation as a strategy to design new type II leads.

    PubMed

    Badrinarayan, Preethi; Sastry, G Narahari

    2011-01-24

    A new knowledge, structure, and sequence based strategy involving the effective exploitation of the DFG-out conformation is delineated. A comprehensive analysis of the structure, sequence, cocrystals, and active sites of p38 MAP kinase crystal structures present in Protein Data Bank (PDB) and the FDA approved MAP kinase drugs has been done, and the information is used for the design of type II leads. The 98 crystal structures, 138 cocrystals, and 31 FDA drugs comprise of 7 different sequences of 2 organisms viz., Homo sapiens and Mus musculus differing in sequence length, constituting both homo- and heterochains. Multiple sequence alignment with ClustalW showed >95% sequence similarity with highly conserved domains and a high propensity for mutations in the activation loop. The bound ligands were extracted, and their interactions with DFG in and out conformations were studied. These cocrystals and FDA drugs were fragmented on the basis of their binding interactions and their affinity to ATP and allosteric sites. The fragment library thus generated contains 106 fragments with overlapping drug fragments. A blue print constituting three main parts viz., head (ATP region), linker (DFG region), and tail (allosteric region) has thus been formulated and used to design 64 type II p38 MAP kinase inhibitors. The above strategy has been employed to design potent type II p38 MAP kinase inhibitors, which are shown to be very promising. PMID:21141877

  4. Closed-loop control of epileptiform activities in a neural population model using a proportional-derivative controller

    NASA Astrophysics Data System (ADS)

    Wang, Jun-Song; Wang, Mei-Li; Li, Xiao-Li; Ernst, Niebur

    2015-03-01

    Epilepsy is believed to be caused by a lack of balance between excitation and inhibitation in the brain. A promising strategy for the control of the disease is closed-loop brain stimulation. How to determine the stimulation control parameters for effective and safe treatment protocols remains, however, an unsolved question. To constrain the complex dynamics of the biological brain, we use a neural population model (NPM). We propose that a proportional-derivative (PD) type closed-loop control can successfully suppress epileptiform activities. First, we determine the stability of root loci, which reveals that the dynamical mechanism underlying epilepsy in the NPM is the loss of homeostatic control caused by the lack of balance between excitation and inhibition. Then, we design a PD type closed-loop controller to stabilize the unstable NPM such that the homeostatic equilibriums are maintained; we show that epileptiform activities are successfully suppressed. A graphical approach is employed to determine the stabilizing region of the PD controller in the parameter space, providing a theoretical guideline for the selection of the PD control parameters. Furthermore, we establish the relationship between the control parameters and the model parameters in the form of stabilizing regions to help understand the mechanism of suppressing epileptiform activities in the NPM. Simulations show that the PD-type closed-loop control strategy can effectively suppress epileptiform activities in the NPM. Project supported by the National Natural Science Foundation of China (Grant Nos. 61473208, 61025019, and 91132722), ONR MURI N000141010278, and NIH grant R01EY016281.

  5. Conformational dynamics of Thermus aquaticus DNA polymerase I during catalysis.

    PubMed

    Xu, Cuiling; Maxwell, Brian A; Suo, Zucai

    2014-08-12

    Despite the fact that DNA polymerases have been investigated for many years and are commonly used as tools in a number of molecular biology assays, many details of the kinetic mechanism they use to catalyze DNA synthesis remain unclear. Structural and kinetic studies have characterized a rapid, pre-catalytic open-to-close conformational change of the Finger domain during nucleotide binding for many DNA polymerases including Thermus aquaticus DNA polymerase I (Taq Pol), a thermostable enzyme commonly used for DNA amplification in PCR. However, little has been performed to characterize the motions of other structural domains of Taq Pol or any other DNA polymerase during catalysis. Here, we used stopped-flow Förster resonance energy transfer to investigate the conformational dynamics of all five structural domains of the full-length Taq Pol relative to the DNA substrate during nucleotide binding and incorporation. Our study provides evidence for a rapid conformational change step induced by dNTP binding and a subsequent global conformational transition involving all domains of Taq Pol during catalysis. Additionally, our study shows that the rate of the global transition was greatly increased with the truncated form of Taq Pol lacking the N-terminal domain. Finally, we utilized a mutant of Taq Pol containing a de novo disulfide bond to demonstrate that limiting protein conformational flexibility greatly reduced the polymerization activity of Taq Pol. PMID:24931550

  6. Variability of atmospheric krypton-85 activity concentrations observed close to the ITCZ in the southern hemisphere.

    PubMed

    Bollhöfer, A; Schlosser, C; Ross, J O; Sartorius, H; Schmid, S

    2014-01-01

    Krypton-85 activity concentrations in surface air have been measured at Darwin, which is located in northern Australia and is influenced by seasonal monsoonal activity. Measurements between August 2007 and May 2010 covered three wet seasons. The mean activity concentration of krypton-85 measured during this period was 1.31±0.02Bqm(-3). A linear model fitted to the average monthly data, using month and monsoon as predictors, shows that krypton-85 activity concentration measured during the sampling period has declined by 0.01Bqm(-3) per year. Although there is no statistically significant difference in mean activity concentration of krypton-85 between wet and dry season, the model implies that activity concentration is higher by about 0.015Bqm(-3) during months influenced by the monsoon when a north westerly flow prevails. Backward dispersion runs using the Lagrangian particle dispersion model Hysplit4 highlight possible source regions during an active monsoon located deep in the northern hemisphere, and include reprocessing facilities in Japan and India. However, the contribution of these facilities to krypton-85 activity concentrations in Darwin would be less than 0.003Bqm(-3).

  7. Targeting Inactive Enzyme Conformation

    PubMed Central

    Liu, Sijiu; Zeng, Li-Fan; Wu, Li; Yu, Xiao; Xue, Ting; Gunawan, Andrea M.; Ya-Qiu, Long; Zhang, Zhong-Yin

    2009-01-01

    There has been considerable interest in protein tyrosine phosphatase 1B (PTP1B) as a therapeutic target for diabetes, obesity, as well as cancer. Identifying inhibitory compounds with good bioavailability is a major challenge of drug discovery programs targeted toward PTPs. Most current PTP active site-directed pharmacophores are negatively charged pTyr mimetics which cannot readily enter the cell. This lack of cell permeability limits the utility of such compounds in signaling studies and further therapeutic development. We identify aryl diketoacids as novel pTyr surrogates and show that neutral amide-linked aryl diketoacid dimers also exhibit excellent PTP inhibitory activity. Kinetic studies establish that these aryl diketoacid derivatives act as noncompetitive inhibitors of PTP1B. Crystal structures of ligand-bound PTP1B reveal that both the aryl diketoacid and its dimeric derivative bind PTP1B at the active site, albeit with distinct modes of interaction, in the catalytically inactive, WPD loop open conformation. Furthermore, dimeric aryl diketoacids are cell permeable and enhance insulin signaling in hepatoma cells, suggesting that targeting the inactive conformation may provide a unique opportunity for creating active site-directed PTP1B inhibitors with improved pharmacological properties. PMID:19012396

  8. Prediction of sea water intrusion for mining activity in close precincts of sea shore.

    PubMed

    Singh, Awanindra Pratap; Gupta, Prem Kumar; Khandelwal, Manoj

    2013-01-01

    The mining lease area of Surka [District Bhavnagar, Gujarat (India)] is located within 6-12 km horizontal distance of sea shore of Gulf of Cambay. Whenever, there will be onset of lignite extraction, there will be always a threat of sea water intrusion into the mining lease area due to its close proximity to seashore. This article describes the prediction of sea water intrusion into the lease area of whole mining block using Ghyben-Herzberg relation between fresh and saline water, Remote Sensing, Ground Truth verification, Electrical Resistivity Survey and groundwater table monitored during the year 2004. As per the Ghyben-Herzberg relation, results show that there will not be sea water intrusion. If there is excess pumping of water then also the basement rock below the lignite seam will put hindrance to any possible upconing of saline water interface. PMID:24083098

  9. Physical Activity Capture Technology With Potential for Incorporation Into Closed-Loop Control for Type 1 Diabetes.

    PubMed

    Dadlani, Vikash; Levine, James A; McCrady-Spitzer, Shelly K; Dassau, Eyal; Kudva, Yogish C

    2015-10-18

    Physical activity is an important determinant of glucose variability in type 1 diabetes (T1D). It has been incorporated as a nonglucose input into closed-loop control (CLC) protocols for T1D during the last 4 years mainly by 3 research groups in single center based controlled clinical trials involving a maximum of 18 subjects in any 1 study. Although physical activity data capture may have clinical benefit in patients with T1D by impacting cardiovascular fitness and optimal body weight achievement and maintenance, limited number of such studies have been conducted to date. Clinical trial registries provide information about a single small sample size 2 center prospective study incorporating physical activity data input to modulate closed-loop control in T1D that are seeking to build on prior studies. We expect an increase in such studies especially since the NIH has expanded support of this type of research with additional grants starting in the second half of 2015. Studies (1) involving patients with other disorders that have lasted 12 weeks or longer and tracked physical activity and (2) including both aerobic and resistance activity may offer insights about the user experience and device optimization even as single input CLC heads into real-world clinical trials over the next few years and nonglucose input is introduced as the next advance.

  10. Physical Activity Capture Technology With Potential for Incorporation Into Closed-Loop Control for Type 1 Diabetes.

    PubMed

    Dadlani, Vikash; Levine, James A; McCrady-Spitzer, Shelly K; Dassau, Eyal; Kudva, Yogish C

    2015-11-01

    Physical activity is an important determinant of glucose variability in type 1 diabetes (T1D). It has been incorporated as a nonglucose input into closed-loop control (CLC) protocols for T1D during the last 4 years mainly by 3 research groups in single center based controlled clinical trials involving a maximum of 18 subjects in any 1 study. Although physical activity data capture may have clinical benefit in patients with T1D by impacting cardiovascular fitness and optimal body weight achievement and maintenance, limited number of such studies have been conducted to date. Clinical trial registries provide information about a single small sample size 2 center prospective study incorporating physical activity data input to modulate closed-loop control in T1D that are seeking to build on prior studies. We expect an increase in such studies especially since the NIH has expanded support of this type of research with additional grants starting in the second half of 2015. Studies (1) involving patients with other disorders that have lasted 12 weeks or longer and tracked physical activity and (2) including both aerobic and resistance activity may offer insights about the user experience and device optimization even as single input CLC heads into real-world clinical trials over the next few years and nonglucose input is introduced as the next advance. PMID:26481641

  11. Physical Activity Capture Technology With Potential for Incorporation Into Closed-Loop Control for Type 1 Diabetes

    PubMed Central

    Dadlani, Vikash; Levine, James A.; McCrady-Spitzer, Shelly K.; Dassau, Eyal; Kudva, Yogish C.

    2015-01-01

    Physical activity is an important determinant of glucose variability in type 1 diabetes (T1D). It has been incorporated as a nonglucose input into closed-loop control (CLC) protocols for T1D during the last 4 years mainly by 3 research groups in single center based controlled clinical trials involving a maximum of 18 subjects in any 1 study. Although physical activity data capture may have clinical benefit in patients with T1D by impacting cardiovascular fitness and optimal body weight achievement and maintenance, limited number of such studies have been conducted to date. Clinical trial registries provide information about a single small sample size 2 center prospective study incorporating physical activity data input to modulate closed-loop control in T1D that are seeking to build on prior studies. We expect an increase in such studies especially since the NIH has expanded support of this type of research with additional grants starting in the second half of 2015. Studies (1) involving patients with other disorders that have lasted 12 weeks or longer and tracked physical activity and (2) including both aerobic and resistance activity may offer insights about the user experience and device optimization even as single input CLC heads into real-world clinical trials over the next few years and nonglucose input is introduced as the next advance. PMID:26481641

  12. Low-order design and high-order simulation of active closed-loop control for aerospace structures under construction

    NASA Technical Reports Server (NTRS)

    Balas, Mark J.

    1989-01-01

    Partially constructed/assembled structures in space are complicated enough but their dynamics will also be operating in closed-loop with feedback controllers. The dynamics of such structures are modeled by large-scale finite element models. The model dimension L is extremely large (approximately 10,000) while the numbers of actuators (M) and sensors (P) are small. The model parameters M(sub m) mass matrix, D(sub o) damping matrix, and K(sub o) stiffness matrix, are all symmetric and sparse (banded). Thus simulation of open-loop structure models of very large dimension can be accomplished by special integration techniques for sparse matrices. The problem of simulation of closed-loop control of such structures is complicated by the addition of controllers. Simulation of closed-loop controlled structures is an essential part of the controller design and evaluation process. Current research in the following areas is presented: high-order simulation of actively controlled aerospace structures; low-order controller design and SCI compensation for unmodeled dynamics; prediction of closed-loop stability using asymptotic eigenvalue series; and flexible robot manipulator control experiment.

  13. Muscle Activation and Onset Times of Hip Extensors during Various Loads of a Closed Kinetic Chain Exercise.

    PubMed

    Cochrane, Darryl J; Barnes, Matthew J

    2015-01-01

    The aim of this study was to determine the muscle activity and onset time of hip extensors during a closed kinetic chain exercise (deadlift) at sub-maximal loads of 30%, 40%, 50%, and 75% of one repetition maximum (1RM). Twelve healthy males with at least three years of resistance training experience volunteered for the study. Biceps femoris (BF) and gluteus maximus (GM) muscle activity, onset time, peak and mean power were measured during the concentric phase of the deadlift. There was no main effect (p > 0.05) or no interaction effect for the onset time in BF and GM and for each load BF and GM had similar muscle activity. Increasing the external load during deadlift had no adverse effect on the relative onset time and it did not promote BF onset to occur before GM onset, thus both muscles were simultaneously activated, which should not compromise a delay in GM. PMID:25656945

  14. Ionic liquid-induced all-α to α + β conformational transition in cytochrome c with improved peroxidase activity in aqueous medium.

    PubMed

    Bharmoria, Pankaj; Trivedi, Tushar J; Pabbathi, Ashok; Samanta, Anunay; Kumar, Arvind

    2015-04-21

    Choline dioctylsulfosuccinate [Cho][AOT] (a surface active ionic liquid) has been found to induce all-α to α + β conformational transition in the secondary structure of enzyme cytochrome c (Cyt c) with an enhanced peroxidase activity in its aqueous vesicular phase at pH 7.0. [Cho][AOT] interacted with Cyt c distinctly at three critical concentrations (aggregation C1, saturation C2 and vesicular C3) as detected from isothermal titration calorimetric analysis. Oxidation of heme iron was observed from the disappearance of the Q band in the UV-vis spectra of Cyt c upon [Cho][AOT] binding above C3. Circular dichroism analysis (CD) has shown the loss in both the secondary (190-240 nm) and tertiary (250-300 nm) structure of Cyt c in the monomeric regime until C1, followed by their stabilization until the pre-vesicular regime (C1 → C3). Loss in both the secondary and tertiary structure has been observed in the post-vesicular regime with the change in Cyt c conformation from all-α to α + β which is similar to the conformational changes of Cyt c upon binding with mitochondrial membrane (Biochemistry 1998, 37, 6402-6409), thus citing the potential utility of [Cho][AOT] membranes as an artificial analog for in vitro bio-mimicking. Fluorescence correlation spectroscopy (FCS) measurements confirm the unfolding of Cyt c in the vesicular phase. Dynamic light scattering experiments have shown the contraction of [Cho][AOT] vesicles upon Cyt c binding driven by electrostatic interactions observed by charge neutralization from zeta potential measurements. [Cho][AOT] has been found to enhance the peroxidase activity of Cyt c with maximum activity at C3, observed using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt as the substrate in the presence of hydrogen peroxide. This result shows the relevance of tuning ionic liquids to surfactants for bio-mimicking of specific membrane protein-lipid interactions. PMID:25798458

  15. Structure and cytotoxic activity of sesquiterpene glycoside esters from Calendula officinalis L.: Studies on the conformation of viridiflorol.

    PubMed

    D'Ambrosio, Michele; Ciocarlan, Alexandru; Colombo, Elisa; Guerriero, Antonio; Pizza, Cosimo; Sangiovanni, Enrico; Dell'Agli, Mario

    2015-09-01

    Topic applications of Calendula officinalis L. lipophilic extracts are used in phytotherapy to relieve skin inflammatory conditions whereas infusions are used as a remedy for gastric complaints. Such a different usage might be explained by some cytotoxicity of lipophilic extracts at gastric level but little is known about this. Therefore, we screened the CH2Cl2 extract from the flowers of C. officinalis by MTT and LDH assays in human epithelial gastric cells AGS. This bioassay-oriented approach led to the isolation of several sesquiterpene glycosides which were structurally characterized by spectroscopic measurements, chemical reactions and MM calculations. The conformational preferences of viridiflorol fucoside were established and a previously assigned stereochemistry was revised. The compounds 1a, 2a and 3f showed comparably high cytotoxicity in the MTT assays, whereas the effect on LDH release was lower. Our study provides new insights on the composition of C. officinalis extracts of medium polarity and identifies the main compounds that could be responsible for cytotoxic effects at gastric level. PMID:26057223

  16. Structure and cytotoxic activity of sesquiterpene glycoside esters from Calendula officinalis L.: Studies on the conformation of viridiflorol.

    PubMed

    D'Ambrosio, Michele; Ciocarlan, Alexandru; Colombo, Elisa; Guerriero, Antonio; Pizza, Cosimo; Sangiovanni, Enrico; Dell'Agli, Mario

    2015-09-01

    Topic applications of Calendula officinalis L. lipophilic extracts are used in phytotherapy to relieve skin inflammatory conditions whereas infusions are used as a remedy for gastric complaints. Such a different usage might be explained by some cytotoxicity of lipophilic extracts at gastric level but little is known about this. Therefore, we screened the CH2Cl2 extract from the flowers of C. officinalis by MTT and LDH assays in human epithelial gastric cells AGS. This bioassay-oriented approach led to the isolation of several sesquiterpene glycosides which were structurally characterized by spectroscopic measurements, chemical reactions and MM calculations. The conformational preferences of viridiflorol fucoside were established and a previously assigned stereochemistry was revised. The compounds 1a, 2a and 3f showed comparably high cytotoxicity in the MTT assays, whereas the effect on LDH release was lower. Our study provides new insights on the composition of C. officinalis extracts of medium polarity and identifies the main compounds that could be responsible for cytotoxic effects at gastric level.

  17. Applications of hydrogen deuterium exchange (HDX) for the characterization of conformational dynamics in light-activated photoreceptors

    PubMed Central

    Lindner, Robert; Heintz, Udo; Winkler, Andreas

    2015-01-01

    Rational design of optogenetic tools is inherently linked to the understanding of photoreceptor function. Structural analysis of elements involved in signal integration in individual sensor domains provides an initial idea of their mode of operation, but understanding how local structural rearrangements eventually affect signal transmission to output domains requires inclusion of the effector regions in the characterization. However, the dynamic nature of these assemblies renders their structural analysis challenging and therefore a combination of high- and low-resolution techniques is required to appreciate functional aspects of photoreceptors. This review focuses on the potential of hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) for complementing the structural characterization of photoreceptors. In this respect, the ability of HDX-MS to provide information on conformational dynamics and the possibility to address multiple functionally relevant states in solution render this methodology ideally suitable. We highlight recent examples demonstrating the potential of HDX-MS and discuss how these results can help to improve existing optogenetic systems or guide the design of novel optogenetic tools. PMID:26157802

  18. P450cam visits an open conformation in the absence of substrate.

    PubMed

    Lee, Young-Tae; Wilson, Richard F; Rupniewski, Igor; Goodin, David B

    2010-04-27

    P450cam from Pseudomonas putida is the best characterized member of the vast family of cytochrome P450s, and it has long been believed to have a more rigid and closed active site relative to other P450s. Here we report X-ray structures of P450cam crystallized in the absence of substrate and at high and low [K(+)]. The camphor-free structures are observed in a distinct open conformation characterized by a water-filled channel created by the retraction of the F and G helices, disorder of the B' helix, and loss of the K(+) binding site. Crystallization in the presence of K(+) alone does not alter the open conformation, while crystallization with camphor alone is sufficient for closure of the channel. Soaking crystals of the open conformation in excess camphor does not promote camphor binding or closure, suggesting resistance to conformational change by the crystal lattice. This open conformation is remarkably similar to that seen upon binding large tethered substrates, showing that it is not the result of a perturbation by the ligand. Redissolved crystals of the open conformation are observed as a mixture of P420 and P450 forms, which is converted to the P450 form upon addition of camphor and K(+). These data reveal that P450cam can dynamically visit an open conformation that allows access to the deeply buried active site without being induced by substrate or ligand. PMID:20297780

  19. Catalase activity as a potential indicator of the reducer component of small closed ecosystems.

    PubMed

    Sarangova, A B; Somova, L A; Pisman, T I

    1997-01-01

    Dynamics of catalase activity has been shown to reflect the growth curve of microorganisms in batch cultivation (celluloselythic bacteria Bacillus acidocaldarius and bacteria of the associated microflora Chlorella vulgaris). Gas and substrate closure of the three component ecosystems with spatially separated components "producer-consumer-reducer" (Chl. vulgaris-Paramecium caudatum-B. acidocaldarius, two bacterial strains isolated from the associated microflora Chl. vulgaris) demonstrated that the functioning of the reducer component can be estimated by the catalase activity of mciroorganisms of this component.

  20. Reversible competitive α-ketoheterocycle inhibitors of fatty acid amide hydrolase containing additional conformational constraints in the acyl side chain: orally active, long-acting analgesics.

    PubMed

    Ezzili, Cyrine; Mileni, Mauro; McGlinchey, Nicholas; Long, Jonathan Z; Kinsey, Steven G; Hochstatter, Dustin G; Stevens, Raymond C; Lichtman, Aron H; Cravatt, Benjamin F; Bilsky, Edward J; Boger, Dale L

    2011-04-28

    A series of α-ketooxazoles containing conformational constraints in the C2 acyl side chain of 2 (OL-135) were examined as inhibitors of fatty acid amide hydrolase (FAAH). Only one of the two possible enantiomers displayed potent FAAH inhibition (S vs R enantiomer), and their potency is comparable or improved relative to 2, indicating that the conformational restriction in the C2 acyl side chain is achievable. A cocrystal X-ray structure of the α-ketoheterocycle 12 bound to a humanized variant of rat FAAH revealed its binding details, confirmed that the (S)-enantiomer is the bound active inhibitor, shed light on the origin of the enantiomeric selectivity, and confirmed that the catalytic Ser241 is covalently bound to the electrophilic carbonyl as a deprotonated hemiketal. Preliminary in vivo characterization of the inhibitors 12 and 14 is reported demonstrating that they raise brain anandamide levels following either intraperitoneal (ip) or oral (po) administration indicative of effective in vivo FAAH inhibition. Significantly, the oral administration of 12 caused dramatic accumulation of anandamide in the brain, with peak levels achieved between 1.5 and 3 h, and these elevations were maintained over 9 h. Additional studies of these two representative members of the series (12 and 14) in models of thermal hyperalgesia and neuropathic pain are reported, including the demonstration that 12 administered orally significantly attenuated mechanical (>6 h) and cold (>9 h) allodynia for sustained periods consistent with its long-acting effects in raising the endogenous concentration of anandamide.

  1. The impact of β-azido(or 1-piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues.

    PubMed

    Maciejczyk, Maciej; Lasota, Anika; Frączak, Oliwia; Kosson, Piotr; Misicka, Aleksandra; Nowakowski, Michał; Ejchart, Andrzej; Olma, Aleksandra

    2016-08-01

    The synthesis of new dermorphin analogues is described. The (R)-alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α-methyl-β-azidoalanine or α-benzyl-β-azido(1-piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [(3) H]DAMGO (a μ ligand) and [(3) H]DELT (a δ ligand). The most active analogue in this series, Tyr-(R)-Ala-(R)-α-benzyl-β-azidoAla-Gly-Tyr-Pro-Ser-NH2 and its epimer were analysed by (1) H and (13) C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C(α) in the α-benzyl-β-azidoAla residue in position 3. The (R) configuration led to the formation of a type I β-turn, whilst switching to the (S) configuration gave rise to an inverse β-turn of type I', followed by the formation of a very short β-sheet. The selectivity of Tyr-(R)-Ala-(R) and (S)-α-benzyl-β-azidoAla-Gly-Tyr-Pro-Ser-NH2 was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27443980

  2. The impact of β-azido(or 1-piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues.

    PubMed

    Maciejczyk, Maciej; Lasota, Anika; Frączak, Oliwia; Kosson, Piotr; Misicka, Aleksandra; Nowakowski, Michał; Ejchart, Andrzej; Olma, Aleksandra

    2016-08-01

    The synthesis of new dermorphin analogues is described. The (R)-alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α-methyl-β-azidoalanine or α-benzyl-β-azido(1-piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [(3) H]DAMGO (a μ ligand) and [(3) H]DELT (a δ ligand). The most active analogue in this series, Tyr-(R)-Ala-(R)-α-benzyl-β-azidoAla-Gly-Tyr-Pro-Ser-NH2 and its epimer were analysed by (1) H and (13) C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C(α) in the α-benzyl-β-azidoAla residue in position 3. The (R) configuration led to the formation of a type I β-turn, whilst switching to the (S) configuration gave rise to an inverse β-turn of type I', followed by the formation of a very short β-sheet. The selectivity of Tyr-(R)-Ala-(R) and (S)-α-benzyl-β-azidoAla-Gly-Tyr-Pro-Ser-NH2 was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  3. Highly Active Chiral Ruthenium Catalysts for Asymmetric Ring-Closing Olefin Metathesis

    PubMed Central

    Funk, Timothy W.; Berlin, Jacob M.

    2008-01-01

    The synthesis of olefin metathesis catalysts containing chiral, monodentate N-heterocyclic carbenes and their application to asymmetric ring-closing metathesis (ARCM) is reported. These catalysts retain the high levels of reactivity found in the related achiral variants (1a and 1b). Using the parent chiral catalysts 2a and 2b and derivatives that contain steric bulk in the meta positions of the N-bound aryl rings (catalysts 3-5), five- through seven-membered rings were formed in up to 92% ee. The addition of sodium iodide to catalysts 2a-4a (to form 2b-4bin situ) caused a dramatic increase in enantioselectivity for many substrates. Catalyst 5a, which gave high enantiomeric excesses for certain substrates without the addition of NaI, could be used in loadings of ≤1 mol %. Mechanistic explanations for the large sodium iodide effect as well as possible mechanistic pathways leading to the observed products are discussed. PMID:16464082

  4. Analysis of cytochrome P450 CYP119 ligand-dependent conformational dynamics by two-dimensional NMR and X-ray crystallography.

    PubMed

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N; Nishida, Clinton R; de Montellano, Paul R Ortiz

    2015-04-17

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional (1)H,(15)N HSQC chemical shift perturbation mapping of (15)N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.

  5. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography*

    PubMed Central

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N.; Nishida, Clinton R.; de Montellano, Paul R. Ortiz

    2015-01-01

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. PMID:25670859

  6. Effects of High Pressure Homogenization on the Activity, Stability, Kinetics and Three-Dimensional Conformation of a Glucose Oxidase Produced by Aspergillus niger

    PubMed Central

    Tribst, Alline Artigiani Lima; Cota, Júnio; Murakami, Mario Tyago; Cristianini, Marcelo

    2014-01-01

    High pressure homogenization (HPH) is a non-thermal method, which has been employed to change the activity and stability of biotechnologically relevant enzymes. This work investigated how HPH affects the structural and functional characteristics of a glucose oxidase (GO) from Aspergillus niger. The enzyme was homogenized at 75 and 150 MPa and the effects were evaluated with respect to the enzyme activity, stability, kinetic parameters and molecular structure. The enzyme showed a pH-dependent response to the HPH treatment, with reduction or maintenance of activity at pH 4.5–6.0 and a remarkable activity increase (30–300%) at pH 6.5 in all tested temperatures (15, 50 and 75°C). The enzyme thermal tolerance was reduced due to HPH treatment and the storage for 24 h at high temperatures (50 and 75°C) also caused a reduction of activity. Interestingly, at lower temperatures (15°C) the activity levels were slightly higher than that observed for native enzyme or at least maintained. These effects of HPH treatment on function and stability of GO were further investigated by spectroscopic methods. Both fluorescence and circular dichroism revealed conformational changes in the molecular structure of the enzyme that might be associated with the distinct functional and stability behavior of GO. PMID:25061935

  7. Role of cytochrome B5 in modulating peroxide-supported cyp3a4 activity: evidence for a conformational transition and cytochrome P450 heterogeneity.

    PubMed

    Kumar, Santosh; Davydov, Dmitri R; Halpert, James R

    2005-08-01

    The role of cytochrome b(5) (b(5)) in the alpha-naphthoflavone (alpha-NF)-mediated inhibition of H(2)O(2)-supported 7-benzyloxyquinoline (7-BQ) debenzylation by heterologously expressed and purified cytochrome P450 3A4 (CYP3A4) was studied. Although alpha-NF showed negligible effect in an NADPH-dependent reconstituted system, inhibition of 7-BQ oxidation was observed in the H(2)O(2) system. Analysis of the effect of various constituents of a standard reconstituted system on H(2)O(2)-supported activity showed that b(5) alone resulted in a 2.5-fold increase in the k(cat) value and reversed the inhibitory effect of alpha-NF. In addition, titration with b(5) suggested that only 65% of the CYP3A4 participated in the interaction with b(5), consistent with cytochrome P450 (P450) heterogeneity. Study of the influence of b(5) on the kinetics of H(2)O(2)-dependent destruction of the P450 heme moiety suggested two distinct conformers of CYP3A4 with different sensitivity to heme loss. In the absence of b(5), 66% of the wild-type enzyme was bleached in the fast phase, whereas the addition of b(5) decreased the fraction of the fast phase to 16%. Finally, to locate amino acid residues that might influence b(5) action, several active site mutants were tested. Substitution of Ser-119, Ile-301, Ala-305, Ile-369, or Ala-370 with the larger Phe or Trp decreased or even abolished the activation by b(5). Ser-119 is in the B'-C loop, a predicted b(5)-P450 interaction site, and Ile-301 and Ala-305 are closest to the heme. In conclusion, the interaction of b(5) with P450 apparently leads to a conformational transition, which results in redistribution of the CYP3A4 pool. PMID:15870379

  8. Caspase Allostery and Conformational Selection.

    PubMed

    Clark, A Clay

    2016-06-01

    The role of caspase proteases in regulated processes such as apoptosis and inflammation has been studied for more than two decades, and the activation cascades are known in detail. Apoptotic caspases also are utilized in critical developmental processes, although it is not known how cells maintain the exquisite control over caspase activity in order to retain subthreshold levels required for a particular adaptive response while preventing entry into apoptosis. In addition to active site-directed inhibitors, caspase activity is modulated by post-translational modifications or metal binding to allosteric sites on the enzyme, which stabilize inactive states in the conformational ensemble. This review provides a comprehensive global view of the complex conformational landscape of caspases and mechanisms used to select states in the ensemble. The caspase structural database provides considerable detail on the active and inactive conformations in the ensemble, which provide the cell multiple opportunities to fine tune caspase activity. In contrast, the current database on caspase modifications is largely incomplete and thus provides only a low-resolution picture of global allosteric communications and their effects on the conformational landscape. In recent years, allosteric control has been utilized in the design of small drug compounds or other allosteric effectors to modulate caspase activity.

  9. Close-spaced thermionic converters with active spacing control and heat-pipe isothermal emitters

    SciTech Connect

    Fitzpatrick, G.O.; Koester, J.K.; Chang, J.; Britt, E.J.; McVey, J.B.

    1996-12-31

    Thermionic converters with interelectrode gaps smaller than 10 microns are capable of substantial performance improvements over conventional ignited mode diodes. Previous devices which have demonstrated operation at such small gaps have done so at low power densities and emitter temperatures. Higher power operation requires overcoming two primary design issues: thermal distortion of the emitter due to temperature gradients and degradation of the in-gap spacers at higher emitter temperatures. This work describes two innovations for solution of these issues. The issue of thermal distortion was addressed by an isothermal emitter incorporating a heat-pipe into its structure. Such a heat-pipe emitter, with a single-crystal emitting surface, was fabricated and characterized. Finite-element computational modeling was used to analyze its distortion with an applied heat flux. The calculations suggested that thermal distortion would be significantly reduced as compared with a solid emitter. Ongoing work and preliminary experimental results are described for a system of active interelectrode gap control. In the present design an integral transducer determines the interelectrode gap of the converter. Initial designs for spacing actuators and their required cesium vapor seals are discussed. A novel hot-shell converter design incorporating active spacing control and low-temperature seals is presented. A converter incorporating the above features would be capable of near ideal-converter performance at high power densities. In addition, active spacing control can potentially completely eliminate short-circuit failures in thermionic converter systems.

  10. Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes

    PubMed Central

    Pena, Darlene Aparecida; Andrade, Victor Piana de; Silva, Gabriela Ávila Fernandes; Neves, José Ivanildo; Oliveira, Paulo Sergio Lopes de; Alves, Maria Julia Manso; Devi, Lakshmi A.; Schechtman, Deborah

    2016-01-01

    Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCβ showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules. PMID:26911897

  11. Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes.

    PubMed

    Pena, Darlene Aparecida; Andrade, Victor Piana de; Silva, Gabriela Ávila Fernandes; Neves, José Ivanildo; Oliveira, Paulo Sergio Lopes de; Alves, Maria Julia Manso; Devi, Lakshmi A; Schechtman, Deborah

    2016-02-25

    Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCβ showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules.

  12. Active community profiling via capillary electrophoresis single-strand conformation polymorphism analysis of amplified 16S rRNA and 16S rRNA genes.

    PubMed

    Hiibel, Sage R; Pruden, Amy; Crimi, Barbara; Reardon, Kenneth F

    2010-12-01

    Here, we report the validation and advancement of a high-throughput method for fingerprinting the active members of a microbial community. This method, termed active community profiling (ACP), provides information about both the composition and the activity of mixed microbial cultures via comparative measurements of amplified 16S rRNA (RNA) and 16S rRNA genes (DNA). Capillary electrophoresis is used to resolve single-strand conformation polymorphisms of polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) products, producing electropherograms representative of the community structure. Active members of the community are distinguished by elevated RNA:DNA peak area ratios. Chemostat experiments with defined populations were conducted to validate the ACP approach. Using a pure culture of Escherichia coli, a direct correlation was found between the growth rate and the RNA:DNA peak ratio. In a second validation experiment, a binary culture of E. coli and Pseudomonas putida was subjected to a controlled environmental change consisting of a shift to anaerobic conditions. ACP revealed the expected cessation of growth of P. putida, an obligate aerobe, while the corresponding DNA-only analysis indicated no change in the culture. Finally, ACP was applied to a complex microbial community, and a novel binning approach was demonstrated for integrating the RNA and DNA electropherograms. ACP thus represents a significant advance from traditional DNA-based profiling techniques, which do not distinguish active from inactive or dead cells, and is well suited for high-throughput community analysis.

  13. Close, but no garlic: Perceptuomotor and event knowledge activation during language comprehension

    PubMed Central

    Amsel, Ben D.; DeLong, Katherine A.; Kutas, Marta

    2015-01-01

    Recent research has shown that language comprehension is guided by knowledge about the organization of objects and events in long-term memory. We use event-related brain potentials (ERPs) to determine the extent to which perceptuomotor object knowledge and event knowledge are immediately activated during incremental language processing. Event-related but anomalous sentence continuations preceded by single-sentence event descriptions elicited reduced N400s, despite their poor fit within local sentence contexts. Anomalous words sharing particular sensory or motor attributes with contextually expected words also elicited reduced N400s, despite being inconsistent with global context (i.e., event information). We rule out plausibility as an explanation for both relatedness effects. We show that perceptuomotor-related facilitation is not due to lexical priming between words in the local context and the target or to associative or categorical relationships between expected and unexpected targets. Overall our results are consistent with the immediate and incremental activation of perceptual and motor object knowledge and generalized event knowledge during sentence processing. PMID:25897182

  14. Conformational analysis of PKI(5-22)amide, the active inhibitory fragment of the inhibitor protein of the cyclic AMP-dependent protein kinase.

    PubMed

    Reed, J; De Ropp, J S; Trewhella, J; Glass, D B; Liddle, W K; Bradbury, E M; Kinzel, V; Walsh, D A

    1989-12-01

    Fourier-transform i.r. spectroscopy, 1H-n.m.r. spectroscopy and X-ray scattering were used to study the conformation and shape of the peptide PKI(5-22)amide, which contains the active site of the inhibitor protein of the cyclic AMP-dependent protein kinase [Cheng, Van Pattern, Smith & Walsh (1985) Biochem. J. 231, 655-661]. The X-ray-scattering solution studies show that the peptide has a compact structure with Rg 0.9 nm (9.0 A) and a linear maximum dimension of 2.5 nm (25A). Compatible with this, Fourier-transform i.r. and n.m.r. determinations indicate that the peptide contains approx. 26% alpha-helix located in the N-terminal one-third of the molecule. This region contains the phenylalanine residue that is one essential recognition determinant for high-affinity binding to the protein kinase catalytic site.

  15. Comparative study of two box H/ACA ribonucleoprotein pseudouridine-synthases: relation between conformational dynamics of the guide RNA, enzyme assembly and activity.

    PubMed

    Fourmann, Jean-Baptiste; Tillault, Anne-Sophie; Blaud, Magali; Leclerc, Fabrice; Branlant, Christiane; Charpentier, Bruno

    2013-01-01

    Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10-L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21.

  16. Comparative Study of Two Box H/ACA Ribonucleoprotein Pseudouridine-Synthases: Relation between Conformational Dynamics of the Guide RNA, Enzyme Assembly and Activity

    PubMed Central

    Leclerc, Fabrice; Branlant, Christiane; Charpentier, Bruno

    2013-01-01

    Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10–L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21. PMID:23922977

  17. Conformational Equilibrium of CDK/Cyclin Complexes by Molecular Dynamics with Excited Normal Modes

    PubMed Central

    Floquet, Nicolas; Costa, Mauricio G.S.; Batista, Paulo R.; Renault, Pedro; Bisch, Paulo M.; Raussin, Florent; Martinez, Jean; Morris, May C.; Perahia, David

    2015-01-01

    Cyclin-dependent kinases (CDKs) and their associated regulatory cyclins are central for timely regulation of cell-cycle progression. They constitute attractive pharmacological targets for development of anticancer therapeutics, since they are frequently deregulated in human cancers and contribute to sustained, uncontrolled tumor proliferation. Characterization of their structural/dynamic features is essential to gain in-depth insight into structure-activity relationships. In addition, the identification of druggable pockets or key intermediate conformations yields potential targets for the development of novel classes of inhibitors. Structural studies of CDK2/cyclin A have provided a wealth of information concerning monomeric/heterodimeric forms of this kinase. There is, however, much less structural information for other CDK/cyclin complexes, including CDK4/cyclin D1, which displays an alternative (open) position of the cyclin partner relative to CDK, contrasting with the closed CDK2/cyclin A conformation. In this study, we carried out normal-mode analysis and enhanced sampling simulations with our recently developed method, molecular dynamics with excited normal modes, to understand the conformational equilibrium on these complexes. Interestingly, the lowest-frequency normal mode computed for each complex described the transition between the open and closed conformations. Exploration of these motions with an explicit-solvent representation using molecular dynamics with excited normal modes confirmed that the closed conformation is the most stable for the CDK2/cyclin A complex, in agreement with their experimentally available structures. On the other hand, we clearly show that an open↔closed equilibrium may exist in CDK4/cyclin D1, with closed conformations resembling that captured for CDK2/cyclin A. Such conformational preferences may result from the distinct distributions of frustrated contacts in each complex. Using the same approach, the putative roles of

  18. The Evolutionary Conformation from Traditional Lecture to Active Learning in an Undergraduate Biology Course and Its Effects on Student Achievement

    ERIC Educational Resources Information Center

    Diederich, Kirsten Bakke

    2010-01-01

    In response to the declining number of students in the United States entering into the STEM (science, technology, engineering, and math) disciplines, there has been an attempt to retain student interest in the sciences through the implementation of more active learning in the classroom. Active learning is defined as any instructional method that…

  19. Adsorption of enzymes to stimuli-responsive polymer brushes: Influence of brush conformation on adsorbed amount and biocatalytic activity.

    PubMed

    Koenig, Meike; Bittrich, Eva; König, Ulla; Rajeev, Bhadra Lakshmi; Müller, Martin; Eichhorn, Klaus-Jochen; Thomas, Sabu; Stamm, Manfred; Uhlmann, Petra

    2016-10-01

    Polyelectrolyte brushes can be utilized to immobilize enzymes on macroscopic surfaces. This report investigates the influence of the pH value of the surrounding medium on the amount and the activity of enzymes adsorbed to poly(2-vinylpyridine) and poly(acrylic acid) brushes, as well as the creation of thermoresponsive biocatalytically active coatings via the adsorption of enzymes onto a mixed brush consisting of a polyelectrolyte and temperature-sensitive poly(N-isopropylacryl amide). Spectroscopic ellipsometry and attenuated total reflection-Fourier transform infrared spectroscopy are used to monitor the adsorption process. Additionally, infrared spectra are evaluated in terms of the secondary structure of the enzymes. Glucose oxidase is used as a model enzyme, where the enzymatic activity is measured after different adsorption conditions. Poly(acrylic acid) brushes generally adsorb larger amounts of enzyme, while less glucose oxidase is found on poly(2-vinylpyridine), which however exhibits higher specific activity. This difference in activity could be attributed to a difference in secondary structure of the adsorbed enzyme. For glucose oxidase adsorbed to mixed brushes, switching of enzymatic activity between an active state at 20°C and a less active state at 40°C as compared to the free enzyme in solution is observed. However, this switching is strongly depending on pH in mixed brushes of poly(acrylic acid) and poly(N-isopropylacryl amide) due to interactions between the polymers.

  20. Adsorption of enzymes to stimuli-responsive polymer brushes: Influence of brush conformation on adsorbed amount and biocatalytic activity.

    PubMed

    Koenig, Meike; Bittrich, Eva; König, Ulla; Rajeev, Bhadra Lakshmi; Müller, Martin; Eichhorn, Klaus-Jochen; Thomas, Sabu; Stamm, Manfred; Uhlmann, Petra

    2016-10-01

    Polyelectrolyte brushes can be utilized to immobilize enzymes on macroscopic surfaces. This report investigates the influence of the pH value of the surrounding medium on the amount and the activity of enzymes adsorbed to poly(2-vinylpyridine) and poly(acrylic acid) brushes, as well as the creation of thermoresponsive biocatalytically active coatings via the adsorption of enzymes onto a mixed brush consisting of a polyelectrolyte and temperature-sensitive poly(N-isopropylacryl amide). Spectroscopic ellipsometry and attenuated total reflection-Fourier transform infrared spectroscopy are used to monitor the adsorption process. Additionally, infrared spectra are evaluated in terms of the secondary structure of the enzymes. Glucose oxidase is used as a model enzyme, where the enzymatic activity is measured after different adsorption conditions. Poly(acrylic acid) brushes generally adsorb larger amounts of enzyme, while less glucose oxidase is found on poly(2-vinylpyridine), which however exhibits higher specific activity. This difference in activity could be attributed to a difference in secondary structure of the adsorbed enzyme. For glucose oxidase adsorbed to mixed brushes, switching of enzymatic activity between an active state at 20°C and a less active state at 40°C as compared to the free enzyme in solution is observed. However, this switching is strongly depending on pH in mixed brushes of poly(acrylic acid) and poly(N-isopropylacryl amide) due to interactions between the polymers. PMID:27447452

  1. α-Lytic protease can exist in two separately stable conformations with different His57 mobilities and catalytic activities

    PubMed Central

    Haddad, Kristin Coffman; Sudmeier, James L.; Bachovchin, Daniel A.; Bachovchin, William W.

    2005-01-01

    α-Lytic protease is a bacterial serine protease widely studied as a model system of enzyme catalysis. Here we report that lyophilization induces a structural change in the enzyme that is not reversed by redissolution in water. The structural change reduces the mobility of the active-site histidine residue and the catalytic activity of the enzyme. The application of mild pressure to solutions of the altered enzyme reverses the lyophilization-induced structural change and restores the mobility of the histidine residue and the enzyme's catalytic activity. This effect of lyophilization permits a unique opportunity for investigating the relationship between histidine ring dynamics and catalytic activity. The results demonstrate that His57 in resting enzymes is more mobile than previously thought, especially when protonated. The histidine motion and its correlation to enzyme activity lend support to the reaction-driven ring flip hypothesis. PMID:15657134

  2. Measurement of multisite oxidation kinetics reveals an active site conformational change in Spo0F as a result of protein oxidation.

    PubMed

    Sharp, Joshua S; Sullivan, Daniel M; Cavanagh, John; Tomer, Kenneth B

    2006-05-23

    When most proteins undergo oxidative damage, they yield a variety of products containing oxidative damage at a large number of sites, most of which are modified substoichiometrically. The resulting complex mixture of products is not amenable to high-resolution structural analyses. The previous methods of structural analysis have relied upon either very generalized structural analyses such as circular dichroism or the creation of a battery of mutants to try to isolate single-residue damage effects. We present a methodology using mass spectrometry to measure the kinetics of oxidation at many sites simultaneously. Previous studies have shown that these kinetics are determined by the chemical nature of the damage site and by the accessibility of that site to the radical. By measuring deviations in the rate of oxidation from the expected pseudo-zero-order kinetics, we can detect and characterize local structural changes due to the oxidative damage. We demonstrate the application of this new technique to the Spo0F protein, a regulator of sporulation in Bacillus subtilis. Circular dichroism studies suggest a partial loss of helical structure of Spo0F as a result of oxidative damage. We report that oxidation causes a three-stage conformational change in Spo0F. Furthermore, we find the dramatic structural changes affect only the region surrounding the active site, while the remainder of the structure remains relatively unperturbed. Finally, we are able to determine that the specific oxidation event that triggers the conformational change at the active site of Spo0F occurs at Met81, a partially conserved methionine in the CheY superfamily.

  3. Sls1p Stimulates Sec63p-Mediated Activation of Kar2p in a Conformation-Dependent Manner in the Yeast Endoplasmic Reticulum

    PubMed Central

    Kabani, Mehdi; Beckerich, Jean-Marie; Gaillardin, Claude

    2000-01-01

    We previously characterized the SLS1 gene in the yeast Yarrowia lipolytica and showed that it interacts physically with YlKar2p to promote translocation across the endoplasmic-reticulum membrane (A. Boisramé, M. Kabani, J. M. Beckerich, E. Hartmann, and C. Gaillardin, J. Biol. Chem. 273:30903–30908, 1998). A Y. lipolytica Kar2p mutant was isolated that restored interaction with an Sls1p mutant, suggesting that the interaction with Sls1p could be nucleotide and/or conformation dependent. This result was used as a working hypothesis for more accurate investigations in Saccharomyces cerevisiae. We show by two-hybrid an in vitro assays that the S. cerevisiae homologue of Sls1p interacts with ScKar2p. Using dominant lethal mutants of ScKar2p, we were able to show that ScSls1p preferentially interacts with the ADP-bound conformation of the molecular chaperone. Synthetic lethality was observed between ΔScsls1 and translocation-deficient kar2 or sec63-1 mutants, providing in vivo evidence for a role of ScSls1p in protein translocation. Synthetic lethality was also observed with ER-associated degradation and folding-deficient kar2 mutants, strongly suggesting that Sls1p functions are not restricted to the translocation process. We show that Sls1p stimulates in a dose-dependent manner the binding of ScKar2p on the lumenal J domain of Sec63p fused to glutathione S-transferase. Moreover, Sls1p is shown to promote the Sec63p-mediated activation of Kar2p's ATPase activity. Our data strongly suggest that Sls1p could be the first GrpE-like protein described in the endoplasmic reticulum. PMID:10958688

  4. Near Infrared Activity Close to the Crab Pulsar Correlated with Giant Gamma-ray Flares

    NASA Technical Reports Server (NTRS)

    Rudy, Alexander R.; Max, Claire E.; Weisskopf, Martin C.

    2014-01-01

    We describe activity observed in the near-infrared correlated with a giant gamma-ray flare in the Crab Pulsar. The Crab Pulsar has been observed by the Fermi and AGILE satellites to flare for a period of 3 to 7 days, once every 1-1.5 years, increasing in brightness by a factor of 3-10 between 100MeV and 1GeV. We used Keck NIRC2 laser guide star adaptive optics imaging to observe the Crab Pulsar and environs before and during the March 2013 flare. We discuss the evidence for the knot as the location of the flares, and the theoretical implications of these observations. Ongoing target-of-opportunity programs hope to confirm this correlation for future flares.

  5. ALLOSTERY AND SUBSTRATE CHANNELING IN THE TRYPTOPHAN SYNTHASE BIENZYME COMPLEX: EVIDENCE FOR TWO SUBUNIT CONFORMATIONS AND FOUR QUATERNARY STATES

    PubMed Central

    Niks, Dimitri; Hilario, Eduardo; Dierkers, Adam; Ngo, Huu; Borchardt, Dan; Neubauer, Thomas J.; Fan, Li; Mueller, Leonard J.; Dunn, Michael F.

    2014-01-01

    The allosteric regulation of substrate channeling in tryptophan synthase involves ligand-mediated allosteric signaling that switches the α- and β-subunits between open (low activity) and closed (high activity) conformations. This switching prevents the escape of the common intermediate, indole, and synchronizes the α- and β-catalytic cycles. 19F NMR studies of bound α-site substrate analogues, N-(4’-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4’-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), were found to be sensitive NMR probes of β-subunit conformation. Both the internal and external aldimine F6 complexes gave a single bound peak at the same chemical shift, while α-aminoacrylate and quinonoid F6 complexes all gave a different bound peak shifted by +1.07 ppm. The F9 complexes exhibited similar behavior, but with a corresponding shift of -0.12 ppm. X-ray crystal structures show the F6 and F9 CF3 groups located at the α-β subunit interface and report changes in both the ligand conformation and the surrounding protein microenvironment. Ab initio computational modeling suggests that the change in 19F chemical shift results primarily from changes in the α-site ligand conformation. Structures of α-aminoacrylate F6 and F9 complexes and quinonoid F6 and F9 complexes show the α- and β-subunits have closed conformations wherein access of ligands into the α- and β-sites from solution is blocked. Internal and external aldimine structures show the α- and β-subunits with closed and open global conformations, respectively. These results establish that β-subunits exist in two global conformation states, designated open, where the β-sites are freely accessible to substrates, and closed, where the β-site portal into solution is blocked. Switching between these conformations is critically important for the αβ-catalytic cycle. PMID:23952479

  6. Side chain conformational averaging in human dihydrofolate reductase.

    PubMed

    Tuttle, Lisa M; Dyson, H Jane; Wright, Peter E

    2014-02-25

    The three-dimensional structures of the dihydrofolate reductase enzymes from Escherichia coli (ecDHFR or ecE) and Homo sapiens (hDHFR or hE) are very similar, despite a rather low level of sequence identity. Whereas the active site loops of ecDHFR undergo major conformational rearrangements during progression through the reaction cycle, hDHFR remains fixed in a closed loop conformation in all of its catalytic intermediates. To elucidate the structural and dynamic differences between the human and E. coli enzymes, we conducted a comprehensive analysis of side chain flexibility and dynamics in complexes of hDHFR that represent intermediates in the major catalytic cycle. Nuclear magnetic resonance relaxation dispersion experiments show that, in marked contrast to the functionally important motions that feature prominently in the catalytic intermediates of ecDHFR, millisecond time scale fluctuations cannot be detected for hDHFR side chains. Ligand flux in hDHFR is thought to be mediated by conformational changes between a hinge-open state when the substrate/product-binding pocket is vacant and a hinge-closed state when this pocket is occupied. Comparison of X-ray structures of hinge-open and hinge-closed states shows that helix αF changes position by sliding between the two states. Analysis of χ1 rotamer populations derived from measurements of (3)JCγCO and (3)JCγN couplings indicates that many of the side chains that contact helix αF exhibit rotamer averaging that may facilitate the conformational change. The χ1 rotamer adopted by the Phe31 side chain depends upon whether the active site contains the substrate or product. In the holoenzyme (the binary complex of hDHFR with reduced nicotinamide adenine dinucleotide phosphate), a combination of hinge opening and a change in the Phe31 χ1 rotamer opens the active site to facilitate entry of the substrate. Overall, the data suggest that, unlike ecDHFR, hDHFR requires minimal backbone conformational rearrangement as

  7. Probing the interaction induced conformation transitions in acid phosphatase with cobalt ferrite nanoparticles: Relation to inhibition and bio-activity of Chlorella vulgaris acid phosphatase.

    PubMed

    Ahmad, Farooq; Zhou, Xing; Yao, Hongzhou; Zhou, Ying; Xu, Chao

    2016-09-01

    The present study explored the interaction and kinetics of cobalt ferrite nanoparticles (NPs) with acid phosphatase (ACP) by utilizing diverse range of spectroscopic techniques. The results corroborate, the CoFe2O4 NPs cause fluorescence quenching in ACP by static quenching mechanism. The negative values of van't Hoff thermodynamic expressions (ΔH=-0.3293Jmol(-1)K(-1) and ΔG=-3.960kJmol(-1)K(-1)) corroborate the spontaneity and exothermic nature of static quenching. The positive value of ΔS (13.2893Jmol(-1)K(-1)) corroborate that major contributors of higher and stronger binding affinity among CoFe2O4 NPs with ACP were electrostatic. In addition, FTIR, UV-CD, UV-vis spectroscopy and three dimensional fluorescence (3D) techniques confirmed that CoFe2O4 NPs binding induces microenvironment perturbations leading to secondary and tertiary conformation changes in ACP to a great extent. Furthermore, synchronous fluorescence spectroscopy (SFS) affirmed the comparatively significant changes in microenvironment around tryptophan (Trp) residue by CoFe2O4 NPs. The effect of CoFe2O4 NPs on the activation kinetics of ACP was further examined in Chlorella vulgaris. Apparent Michaelis constant (Km) values of 0.57 and 26.5mM with activation energy values of 0.538 and 3.428kJmol(-1) were determined without and with 200μM CoFe2O4 NPs. Apparent Vmax value of -7Umml(-1) corroborate that enzyme active sites were completely captured by the NPs leaving no space for the substrate. The results confirmed that CoFe2O4 NPs ceased the activity by unfolding of ACP enzyme. This suggests CoFe2O4 NPs perturbed the enzyme activity by transitions in conformation and hence the metabolic activity of ACP. This study provides the pavement for novel and simple approach of using sensitive biomarkers for sensing NPs in environment. PMID:27209386

  8. Probing protein multidimensional conformational fluctuations by single-molecule multiparameter photon stamping spectroscopy.

    PubMed

    Lu, Maolin; Lu, H Peter

    2014-10-16

    Conformational motions of proteins are highly dynamic and intrinsically complex. To capture the temporal and spatial complexity of conformational motions and further to understand their roles in protein functions, an attempt is made to probe multidimensional conformational dynamics of proteins besides the typical one-dimensional FRET coordinate or the projected conformational motions on the one-dimensional FRET coordinate. T4 lysozyme hinge-bending motions between two domains along α-helix have been probed by single-molecule FRET. Nevertheless, the domain motions of T4 lysozyme are rather complex involving multiple coupled nuclear coordinates and most likely contain motions besides hinge-bending. It is highly likely that the multiple dimensional protein conformational motions beyond the typical enzymatic hinged-bending motions have profound impact on overall enzymatic functions. In this report, we have developed a single-molecule multiparameter photon stamping spectroscopy integrating fluorescence anisotropy, FRET, and fluorescence lifetime. This spectroscopic approach enables simultaneous observations of both FRET-related site-to-site conformational dynamics and molecular rotational (or orientational) motions of individual Cy3-Cy5 labeled T4 lysozyme molecules. We have further observed wide-distributed rotational flexibility along orientation coordinates by recording fluorescence anisotropy and simultaneously identified multiple intermediate conformational states along FRET coordinate by monitoring time-dependent donor lifetime, presenting a whole picture of multidimensional conformational dynamics in the process of T4 lysozyme open-close hinge-bending enzymatic turnover motions under enzymatic reaction conditions. By analyzing the autocorrelation functions of both lifetime and anisotropy trajectories, we have also observed the dynamic and static inhomogeneity of T4 lysozyme multidimensional conformational fluctuation dynamics, providing a fundamental

  9. How Can Active Region Plasma Escape into the Solar Wind from Below a Closed Helmet Streamer?

    NASA Astrophysics Data System (ADS)

    Mandrini, C. H.; Nuevo, F. A.; Vásquez, A. M.; Démoulin, P.; van Driel-Gesztelyi, L.; Baker, D.; Culhane, J. L.; Cristiani, G. D.; Pick, M.

    2014-11-01

    Recent studies show that active-region (AR) upflowing plasma, observed by the EUV-Imaging Spectrometer (EIS) onboard Hinode, can gain access to open-field lines and be released into the solar wind (SW) via magnetic-interchange reconnection at magnetic null-points in pseudo-streamer configurations. When only one bipolar AR is present on the Sun and is fully covered by the separatrix of a streamer, such as AR 10978 in December 2007, it seems unlikely that the upflowing AR plasma can find its way into the slow SW. However, signatures of plasma with AR composition have been found at 1 AU by Culhane et al. ( Solar Phys. 289, 3799, 2014) that apparently originated west of AR 10978. We present a detailed topology analysis of AR 10978 and the surrounding large-scale corona based on a potential-field source-surface (PFSS) model. Our study shows that it is possible for the AR plasma to move around the streamer separatrix and be released into the SW via magnetic reconnection, which occurs in at least two main steps. We analyse data from the Nançay Radioheliograph (NRH) in a search for evidence of the chain of magnetic reconnections that we propose. We find a noise storm above the AR and several varying sources at 150.9 MHz. Their locations suggest that they might be associated with particles accelerated during the first-step reconnection process at a null point well outside of the AR. We find no evidence of the second reconnection step in the radio data, however. Our results demonstrate that even when it appears highly improbable for the AR plasma to reach the SW, indirect channels involving a sequence of reconnections can make it possible.

  10. Association between food mixing ability and electromyographic activity of jaw-closing muscles during chewing of a wax cube.

    PubMed

    Fueki, K; Sugiura, T; Yoshida, E; Igarashi, Y

    2008-05-01

    The purpose of this study was to clarify association between food mixing ability and activity of jaw-closing muscles during chewing of a wax cube. Twenty subjects with complete dentitions (mean age 24.1 years) were directed to chew a two-coloured paraffin wax cube for 10 strokes on preferred chewing side. Surface electromyograms (EMG) were recorded from the right and left masseter and anterior temporalis muscles during chewing of the wax cube. Maximum voltage, duration and muscle work for burst of each chewing cycle were measured on integrated EMG in each muscle. Food mixing ability was estimated as mixing ability index determined from the colour mixture and shape of the chewed wax cube. Some EMG parameters of all muscles except for masseter muscle on non-chewing side showed significant positive correlations with the mixing ability index (r = 0.45-0.56, P < 0.05). However, most of the EMG parameters correlated with one another. As a result, only muscle work of masseter muscle on the chewing side was identified as a significant predictor accounting for 28% interindividual variation in the mixing ability index (P < 0.01). These results suggest that activity of jaw-closing muscles during chewing the wax cube seems to be weakly related to food mixing ability.

  11. Non-dimensionalised closed-form parametric analysis of semi-active vehicle suspensions using a quarter-car model

    NASA Astrophysics Data System (ADS)

    Ahmadian, Mehdi; Blanchard, Emmanuel

    2011-02-01

    This article provides a non-dimensionalised closed-form analysis of semi-active vehicle suspensions, using a quarter-car model. The derivation of the closed-form solutions for three indices that can be used for ride comfort, vehicle handling, and stability are presented based on non-dimensionalised suspension parameters. The behaviour of semi-active vehicle suspensions is evaluated using skyhook, groundhook, and hybrid control policies, and compared with passive suspensions. The relationship between vibration isolation, suspension deflection, and road holding is studied, using three performance indices based on the mean square of the sprung mass acceleration, rattle space, and tyre deflection, respectively. The results of the study indicate that the hybrid control policy yields significantly better comfort than a passive suspension, without reducing the road-holding quality or increasing the suspension displacement for typical passenger cars. The results also indicate that for typical passenger cars, the hybrid control policy results in a better compromise between comfort, road holding and suspension travel requirements than both the skyhook and groundhook control methods.

  12. A spectroscopic investigation on the interaction of a magnetic ferrofluid with a model plasma protein: effect on the conformation and activity of the protein.

    PubMed

    Paul, Bijan Kumar; Bhattacharjee, Kaustav; Bose, Subhrangsu; Guchhait, Nikhil

    2012-11-28

    The understanding of the interaction of nanomaterials with relevant biological targets e.g., proteins is of paramount importance in biological and pharmaceutical fields of research. In a biological fluid, proteins can associate with nanomaterials which can subsequently exert a significant impact on the conformation and functionality of the protein. Here we report the binding interaction of a model plasma protein Bovine Serum Albumin (BSA) with a magnetic nanoparticle of mixed spinel origin (Ni(0.5)Zn(0.5)Fe(2)O(4), abbreviated as NZFO from now and onwards). The thermodynamic parameters (ΔH, ΔS and ΔG) for the protein-nanoparticle binding interaction have been evaluated from the van't Hoff equation to unveil that the binding interaction is enthalpically as well as entropically driven (ΔH < 0 and ΔS > 0), with an overall favorable Gibbs free energy change (ΔG < 0). Also the thermodynamic parameters delineate the predominant role of electrostatic interaction in the BSA-NZFO binding process. The results of temperature dependent fluorescence quenching and time-resolved fluorescence decay measurements indicate a static quenching mechanism in the present case. Steady-state absorption, synchronous fluorescence, three-dimensional (3D) fluorescence and circular dichroism (CD) spectroscopic techniques have been employed to unveil the conformational changes in BSA induced by the binding of NZFO. Disruption of the native conformation of the protein upon binding with NZFO is reflected through a reduced functionality (in terms of esterase activity) of the protein-NZFO conjugate system in comparison to the native protein. Based on the experimental findings the probable binding location of NZFO is argued to be the hydrophilic domain IB. This seems physically realizable since domain I of BSA is characterized by a net negative charge and hence can serve as a favorable binding site for NZFO carrying a positive surface charge. The key role of electrostatic forces in the BSA

  13. A spectroscopic investigation on the interaction of a magnetic ferrofluid with a model plasma protein: effect on the conformation and activity of the protein.

    PubMed

    Paul, Bijan Kumar; Bhattacharjee, Kaustav; Bose, Subhrangsu; Guchhait, Nikhil

    2012-11-28

    The understanding of the interaction of nanomaterials with relevant biological targets e.g., proteins is of paramount importance in biological and pharmaceutical fields of research. In a biological fluid, proteins can associate with nanomaterials which can subsequently exert a significant impact on the conformation and functionality of the protein. Here we report the binding interaction of a model plasma protein Bovine Serum Albumin (BSA) with a magnetic nanoparticle of mixed spinel origin (Ni(0.5)Zn(0.5)Fe(2)O(4), abbreviated as NZFO from now and onwards). The thermodynamic parameters (ΔH, ΔS and ΔG) for the protein-nanoparticle binding interaction have been evaluated from the van't Hoff equation to unveil that the binding interaction is enthalpically as well as entropically driven (ΔH < 0 and ΔS > 0), with an overall favorable Gibbs free energy change (ΔG < 0). Also the thermodynamic parameters delineate the predominant role of electrostatic interaction in the BSA-NZFO binding process. The results of temperature dependent fluorescence quenching and time-resolved fluorescence decay measurements indicate a static quenching mechanism in the present case. Steady-state absorption, synchronous fluorescence, three-dimensional (3D) fluorescence and circular dichroism (CD) spectroscopic techniques have been employed to unveil the conformational changes in BSA induced by the binding of NZFO. Disruption of the native conformation of the protein upon binding with NZFO is reflected through a reduced functionality (in terms of esterase activity) of the protein-NZFO conjugate system in comparison to the native protein. Based on the experimental findings the probable binding location of NZFO is argued to be the hydrophilic domain IB. This seems physically realizable since domain I of BSA is characterized by a net negative charge and hence can serve as a favorable binding site for NZFO carrying a positive surface charge. The key role of electrostatic forces in the BSA

  14. Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation

    SciTech Connect

    Boudreaux, David A.; Maiti, Tushar K.; Davies, Christopher W.; Das, Chittaranjan

    2010-07-06

    Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 {angstrom} from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and I93M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarily at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal {beta}-hairpin at the distal site - a surface-exposed hydrophobic crevice 17 {angstrom} away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 {angstrom} of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate.

  15. The evolutionary conformation from traditional lecture to active learning in an undergraduate biology course and its effects on student achievement

    NASA Astrophysics Data System (ADS)

    Diederich, Kirsten Bakke

    In response to the declining number of students in the United States entering into the STEM (science, technology, engineering, and math) disciplines, there has been an attempt to retain student interest in the sciences through the implementation of more active learning in the classroom. Active learning is defined as any instructional method that requires students do something in the classroom rather than simply listen to a lecture (Herreid, 2006). These student centered approaches provide the students with the opportunity to work cooperatively while developing the skills required for critical inquiry. They also help the students make the connections between what is being taught and how it can be applied in a real world setting. Science education researchers have attempted to analyze the efficacy of active learning. Although they find it difficult to compare the data, they state unequivocally that "Active learning is a better strategy for learning than the traditional didactic lecture format" (Prince, 2004). However, even though research supports the efficacy of active learning, instructors find it difficult to adopt this pedagogy into their classrooms due to concerns such as loss of content knowledge and student resistance. This three year qualitative and quantitative study addressed the level of student learning and satisfaction in an introductory vertebrate biology class at a small liberal arts college. The courses were taught by the same instructor using three pedagogical methods; traditional lecture (TL), problem-based learning (PBL), and case-based learning (CBL). Student grades and levels of assessment were compared between the TL and PBL, while student attrition rates, course satisfaction and views of active and group learning were analyzed across all three sections. The evolutionary confirmations from TL to PBL and ultimately the adoption of CBL as the method of choice are discussed from the view of both the faculty member and the students.

  16. Interaction of Rio1 Kinase with Toyocamycin Reveals a Conformational Switch That Controls Oligomeric State and Catalytic Activity

    PubMed Central

    Kiburu, Irene N.; LaRonde-LeBlanc, Nicole

    2012-01-01

    Rio1 kinase is an essential ribosome-processing factor required for proper maturation of 40 S ribosomal subunit. Although its structure is known, several questions regarding its functional remain to be addressed. We report that both Archaeoglobus fulgidus and human Rio1 bind more tightly to an adenosine analog, toyocamycin, than to ATP. Toyocamycin has antibiotic, antiviral and cytotoxic properties, and is known to inhibit ribosome biogenesis, specifically the maturation of 40 S. We determined the X-ray crystal structure of toyocamycin bound to Rio1 at 2.0 Å and demonstrated that toyocamycin binds in the ATP binding pocket of the protein. Despite this, measured steady state kinetics were inconsistent with strict competitive inhibition by toyocamycin. In analyzing this interaction, we discovered that Rio1 is capable of accessing multiple distinct oligomeric states and that toyocamycin may inhibit Rio1 by stabilizing a less catalytically active oligomer. We also present evidence of substrate inhibition by high concentrations of ATP for both archaeal and human Rio1. Oligomeric state studies show both proteins access a higher order oligomeric state in the presence of ATP. The study revealed that autophosphorylation by Rio1 reduces oligomer formation and promotes monomerization, resulting in the most active species. Taken together, these results suggest the activity of Rio1 may be modulated by regulating its oligomerization properties in a conserved mechanism, identifies the first ribosome processing target of toyocamycin and presents the first small molecule inhibitor of Rio1 kinase activity. PMID:22629386

  17. Interaction of Rio1 Kinase with Toyocamycin Reveals a Conformational Switch That Controls Oligomeric State and Catalytic Activity

    SciTech Connect

    Kiburu, Irene N.; LaRonde-LeBlanc, Nicole

    2012-10-10

    Rio1 kinase is an essential ribosome-processing factor required for proper maturation of 40 S ribosomal subunit. Although its structure is known, several questions regarding its functional remain to be addressed. We report that both Archaeoglobus fulgidus and human Rio1 bind more tightly to an adenosine analog, toyocamycin, than to ATP. Toyocamycin has antibiotic, antiviral and cytotoxic properties, and is known to inhibit ribosome biogenesis, specifically the maturation of 40 S. We determined the X-ray crystal structure of toyocamycin bound to Rio1 at 2.0 {angstrom} and demonstrated that toyocamycin binds in the ATP binding pocket of the protein. Despite this, measured steady state kinetics were inconsistent with strict competitive inhibition by toyocamycin. In analyzing this interaction, we discovered that Rio1 is capable of accessing multiple distinct oligomeric states and that toyocamycin may inhibit Rio1 by stabilizing a less catalytically active oligomer. We also present evidence of substrate inhibition by high concentrations of ATP for both archaeal and human Rio1. Oligomeric state studies show both proteins access a higher order oligomeric state in the presence of ATP. The study revealed that autophosphorylation by Rio1 reduces oligomer formation and promotes monomerization, resulting in the most active species. Taken together, these results suggest the activity of Rio1 may be modulated by regulating its oligomerization properties in a conserved mechanism, identifies the first ribosome processing target of toyocamycin and presents the first small molecule inhibitor of Rio1 kinase activity.

  18. Conformational transitions linked to active site ligation in human thrombin: effect on the interaction with fibrinogen and the cleavable platelet receptor.

    PubMed

    De Cristofaro, R; De Candia, E; Picozzi, M; Landolfi, R

    1995-01-27

    An experimental strategy based on solution viscosity perturbation allowed us to study the energetics of amide-substrates, p-aminobenzamidine (p-ABZ) and proflavin binding to the catalytic site of two proteolyzed forms of alpha-thrombin, i.e. zeta- and gamma T-thrombin. These thrombin derivatives are cleaved at the Leu144-Gly150 loop and at the fibrinogen recognition exosite (FRS), respectively. A phenomenological analysis of thermodynamic data showed that the amide substrates and p-ABZ interactions with zeta-thrombin were respectively, associated with a chemical compensation (i.e. the linear relationship between entropy and enthalpy of binding) and a hydrophobic phenomenon (i.e. a change in the standard heat capacity). The latter was slightly lower than that previously observed for a alpha-thrombin (0.78 +/- 0.25 versus 1.01 +/- 0.17 kcal/mol K). Both phenomenon were absent in gamma T-thrombin. The interaction of a alpha-, zeta- and gamma T-thrombin with macromolecular substrates that "bridge-bind" to both the catalytic site (CS) and fibrinogen recognition exosite (FRS), such as fibrinogen and the cleavable platelet receptor (CPR), was also evaluated. These interactions were studied by following fibrinopeptide A (FpA) release and by measuring intraplatelet Ca2+ changes induced by thrombin-CPR interaction. It was found that the free energy of activation (RT ln Kcat/Km) for both fibrinogen and CPR hydrolysis followed the same hierarchy, i.e. alpha > zeta > gamma. Moreover, the values of delta Cp for alpha-, zeta- and gamma T-thrombin interaction with p-ABZ were found to be linearly correlated to the free energy of activation for both fibrinogen and CPR cleavage. In conclusion, these data demonstrate that: (1) the Leu144-Gly150 loop and the FRS are both involved in the conformational transition linked to the binding of p-aminobenzamidine to the thrombin active site; (2) the extent of thrombin's capacity to undergo conformational transitions in alpha-, zeta- and gamma

  19. Effects of high hydrostatic pressure treatments on haemagglutination activity and structural conformations of phytohemagglutinin from red kidney bean (Phaseolus vulgaris).

    PubMed

    Liu, Cencen; Zhao, Mouming; Sun, Weizheng; Ren, Jiaoyan

    2013-02-15

    Red kidney beans were subjected to high hydrostatic pressure (HHP) treatment (50, 150, 250, 350, 450 MPa) and phytohaemagglutinin (PHA) was then extracted by affinity chromatography. It appeared that HHP treatment could increase crude extract yield and decrease its haemagglutination activity. For purified samples, PHA yield was not affected at pressures <450 MPa while the haemagglutination activity was noticeably reduced at 450 MPa. The structural changes were investigated using electrophoresis, size exclusion chromatography (SEC), Fourier transform infrared (FTIR) and differencial scanning calorimetry (DSC). Electrophoresis and SEC profiles revealed a new high molecular weight polymer after 450 MPa treatment. At pressures <450 MPa, FTIR showed an increase in β-sheet structure and a decrease in α-helix. At 450 MPa, the bands at 1688 cm(-1), representing aggregate strands and random coils, increased. The conclusions are that pressures <450 MPa can cause PHA unfolding and induce PHA aggregation at 450 MPa.

  20. Effects of high hydrostatic pressure treatments on haemagglutination activity and structural conformations of phytohemagglutinin from red kidney bean (Phaseolus vulgaris).

    PubMed

    Liu, Cencen; Zhao, Mouming; Sun, Weizheng; Ren, Jiaoyan

    2013-02-15

    Red kidney beans were subjected to high hydrostatic pressure (HHP) treatment (50, 150, 250, 350, 450 MPa) and phytohaemagglutinin (PHA) was then extracted by affinity chromatography. It appeared that HHP treatment could increase crude extract yield and decrease its haemagglutination activity. For purified samples, PHA yield was not affected at pressures <450 MPa while the haemagglutination activity was noticeably reduced at 450 MPa. The structural changes were investigated using electrophoresis, size exclusion chromatography (SEC), Fourier transform infrared (FTIR) and differencial scanning calorimetry (DSC). Electrophoresis and SEC profiles revealed a new high molecular weight polymer after 450 MPa treatment. At pressures <450 MPa, FTIR showed an increase in β-sheet structure and a decrease in α-helix. At 450 MPa, the bands at 1688 cm(-1), representing aggregate strands and random coils, increased. The conclusions are that pressures <450 MPa can cause PHA unfolding and induce PHA aggregation at 450 MPa. PMID:23194535

  1. Structure of substrate-free human insulin-degrading enzyme (IDE) and biophysical analysis of ATP-induced conformational switch of IDE.

    PubMed

    Im, Hookang; Manolopoulou, Marika; Malito, Enrico; Shen, Yuequan; Zhao, Ji; Neant-Fery, Marie; Sun, Ching-Yu; Meredith, Stephen C; Sisodia, Sangram S; Leissring, Malcolm A; Tang, Wei-Jen

    2007-08-31

    Insulin-degrading enzyme (IDE) is a zinc metalloprotease that hydrolyzes amyloid-beta (Abeta) and insulin, which are peptides associated with Alzheimer disease (AD) and diabetes, respectively. Our previous structural analysis of substrate-bound human 113-kDa IDE reveals that the N- and C-terminal domains of IDE, IDE-N and IDE-C, make substantial contact to form an enclosed catalytic chamber to entrap its substrates. Furthermore, IDE undergoes a switch between the closed and open conformations for catalysis. Here we report a substrate-free IDE structure in its closed conformation, revealing the molecular details of the active conformation of the catalytic site of IDE and new insights as to how the closed conformation of IDE may be kept in its resting, inactive conformation. We also show that Abeta is degraded more efficiently by IDE carrying destabilizing mutations at the interface of IDE-N and IDE-C (D426C and K899C), resulting in an increase in Vmax with only minimal changes to Km. Because ATP is known to activate the ability of IDE to degrade short peptides, we investigated the interaction between ATP and activating mutations. We found that these mutations rendered IDE less sensitive to ATP activation, suggesting that ATP might facilitate the transition from the closed state to the open conformation. Consistent with this notion, we found that ATP induced an increase in hydrodynamic radius, a shift in electrophoretic mobility, and changes in secondary structure. Together, our results highlight the importance of the closed conformation for regulating the activity of IDE and provide new molecular details that will facilitate the development of activators and inhibitors of IDE.

  2. Global attractors of complete conformal foliations

    SciTech Connect

    Zhukova, Nina I

    2012-03-31

    We prove that every complete conformal foliation (M,F) of codimension q{>=}3 is either Riemannian or a (Conf(S{sup q}), S{sup q})-foliation. We further prove that if (M,F) is not Riemannian, it has a global attractor which is either a nontrivial minimal set or a closed leaf or a union of two closed leaves. In this theorem we do not assume that the manifold M is compact. In particular, every proper conformal non-Riemannian foliation (M,F) has a global attractor which is either a closed leaf or a union of two closed leaves, and the space of all nonclosed leaves is a connected q-dimensional orbifold. We show that every countable group of conformal transformations of the sphere S{sup q} can be realized as the global holonomy group of a complete conformal foliation. Examples of complete conformal foliations with exceptional and exotic minimal sets as global attractors are constructed. Bibliography: 20 titles.

  3. Oil removal and effects of spilled oil on active microbial communities in close to salt-saturation brines.

    PubMed

    Corsellis, Yannick Y; Krasovec, Marc M; Sylvi, Léa L; Cuny, Philippe P; Militon, Cécile C

    2016-05-01

    Abiotic and biotic processes associated with the degradation of a light petroleum in brines close to the salt-saturation (~31 %) and the effect of labile organic matter (LOM) supply (casaminoacids/citrate; 0.2 and 0.1 % w/v, respectively) were followed during an incubation of 30 days. After 4-week incubation at 40 °C under light/dark cycles, a 24 % of abiotic degradation was observed in untreated brines. The stimulation of native brines community with LOM addition allowed an additional 12.8 % oil attenuation due to biodegradation processes. Successional changes in the active microbial community structure due to the oil contamination (16S rRNA DGGE approach) showed the selection of one phylotype affiliated to Salinibacter and the disappearance of Haloquadratum walsbyi in untreated brines. In LOM-amended microcosms, phylotypes related to Salinibacter, Haloarcula, Haloterrigena and Halorhabdus were selected. An effect of hydrocarbon contamination was only observed in the bacterial community with the inhibition of two dominant proteobacterial phylotypes. This study further confirms that short-term and moderate oil biodegradation is possible in LOM-stimulated brines. Biodegradation should be much more reduced under in situ conditions. Self-cleaning capacities of close to saturation hypersaline lakes appears, therefore very limited compared to non-extreme haline environments. PMID:26955981

  4. A comparative analysis between active and passive techniques for underwater 3D reconstruction of close-range objects.

    PubMed

    Bianco, Gianfranco; Gallo, Alessandro; Bruno, Fabio; Muzzupappa, Maurizio

    2013-01-01

    In some application fields, such as underwater archaeology or marine biology, there is the need to collect three-dimensional, close-range data from objects that cannot be removed from their site. In particular, 3D imaging techniques are widely employed for close-range acquisitions in underwater environment. In this work we have compared in water two 3D imaging techniques based on active and passive approaches, respectively, and whole-field acquisition. The comparison is performed under poor visibility conditions, produced in the laboratory by suspending different quantities of clay in a water tank. For a fair comparison, a stereo configuration has been adopted for both the techniques, using the same setup, working distance, calibration, and objects. At the moment, the proposed setup is not suitable for real world applications, but it allowed us to conduct a preliminary analysis on the performances of the two techniques and to understand their capability to acquire 3D points in presence of turbidity. The performances have been evaluated in terms of accuracy and density of the acquired 3D points. Our results can be used as a reference for further comparisons in the analysis of other 3D techniques and algorithms. PMID:23966193

  5. A comparative analysis between active and passive techniques for underwater 3D reconstruction of close-range objects.

    PubMed

    Bianco, Gianfranco; Gallo, Alessandro; Bruno, Fabio; Muzzupappa, Maurizio

    2013-08-20

    In some application fields, such as underwater archaeology or marine biology, there is the need to collect three-dimensional, close-range data from objects that cannot be removed from their site. In particular, 3D imaging techniques are widely employed for close-range acquisitions in underwater environment. In this work we have compared in water two 3D imaging techniques based on active and passive approaches, respectively, and whole-field acquisition. The comparison is performed under poor visibility conditions, produced in the laboratory by suspending different quantities of clay in a water tank. For a fair comparison, a stereo configuration has been adopted for both the techniques, using the same setup, working distance, calibration, and objects. At the moment, the proposed setup is not suitable for real world applications, but it allowed us to conduct a preliminary analysis on the performances of the two techniques and to understand their capability to acquire 3D points in presence of turbidity. The performances have been evaluated in terms of accuracy and density of the acquired 3D points. Our results can be used as a reference for further comparisons in the analysis of other 3D techniques and algorithms.

  6. A Comparative Analysis between Active and Passive Techniques for Underwater 3D Reconstruction of Close-Range Objects

    PubMed Central

    Bianco, Gianfranco; Gallo, Alessandro; Bruno, Fabio; Muzzupappa, Maurizio

    2013-01-01

    In some application fields, such as underwater archaeology or marine biology, there is the need to collect three-dimensional, close-range data from objects that cannot be removed from their site. In particular, 3D imaging techniques are widely employed for close-range acquisitions in underwater environment. In this work we have compared in water two 3D imaging techniques based on active and passive approaches, respectively, and whole-field acquisition. The comparison is performed under poor visibility conditions, produced in the laboratory by suspending different quantities of clay in a water tank. For a fair comparison, a stereo configuration has been adopted for both the techniques, using the same setup, working distance, calibration, and objects. At the moment, the proposed setup is not suitable for real world applications, but it allowed us to conduct a preliminary analysis on the performances of the two techniques and to understand their capability to acquire 3D points in presence of turbidity. The performances have been evaluated in terms of accuracy and density of the acquired 3D points. Our results can be used as a reference for further comparisons in the analysis of other 3D techniques and algorithms. PMID:23966193

  7. The Tail of KdsC: Conformational Changes Control the Activity of a Haloacid Dehalogenase Superfamily Phosphatase

    SciTech Connect

    Biswas, Tapan; Yi, Li; Aggarwal, Parag; Wu, Jing; Rubin, John R.; Stuckey, Jeanne A.; Woodard, Ronald W.; Tsodikov, Oleg V.

    2010-01-28

    The phosphatase KdsC cleaves 3-deoxy-d-manno-octulosonate 8-phosphate to generate a molecule of inorganic phosphate and Kdo. Kdo is an essential component of the lipopolysaccharide envelope in Gram-negative bacteria. Because lipopolysaccharide is an important determinant of bacterial resistance and toxicity, KdsC is a potential target for novel antibacterial agents. KdsC belongs to the broad haloacid dehalogenase superfamily. In haloacid dehalogenase superfamily enzymes, substrate specificity and catalytic efficiency are generally dictated by a fold feature called the cap domain. It is therefore not clear why KdsC, which lacks a cap domain, is catalytically efficient and highly specific to 3-deoxy-d-manno-octulosonate 8-phosphate. Here, we present a set of seven structures of tetrameric Escherichia coli KdsC (ranging from 1.4 to 3.06 {angstrom} in resolution) that model different intermediate states in its catalytic mechanism. A crystal structure of product-bound E. coli KdsC shows how the interface between adjacent monomers defines the active site pocket. Kdo is engaged in a network of polar and nonpolar interactions with residues at this interface, which explains substrate specificity. Furthermore, this structural and kinetic analysis strongly suggests that the binding of the flexible C-terminal region (tail) to the active site makes KdsC catalytically efficient by facilitating product release.

  8. Estrogen receptor alpha somatic mutations Y537S and D538G confer breast cancer endocrine resistance by stabilizing the activating function-2 binding conformation

    PubMed Central

    Fanning, Sean W; Mayne, Christopher G; Dharmarajan, Venkatasubramanian; Carlson, Kathryn E; Martin, Teresa A; Novick, Scott J; Toy, Weiyi; Green, Bradley; Panchamukhi, Srinivas; Katzenellenbogen, Benita S; Tajkhorshid, Emad; Griffin, Patrick R; Shen, Yang; Chandarlapaty, Sarat; Katzenellenbogen, John A; Greene, Geoffrey L

    2016-01-01

    Somatic mutations in the estrogen receptor alpha (ERα) gene (ESR1), especially Y537S and D538G, have been linked to acquired resistance to endocrine therapies. Cell-based studies demonstrated that these mutants confer ERα constitutive activity and antiestrogen resistance and suggest that ligand-binding domain dysfunction leads to endocrine therapy resistance. Here, we integrate biophysical and structural biology data to reveal how these mutations lead to a constitutively active and antiestrogen-resistant ERα. We show that these mutant ERs recruit coactivator in the absence of hormone while their affinities for estrogen agonist (estradiol) and antagonist (4-hydroxytamoxifen) are reduced. Further, they confer antiestrogen resistance by altering the conformational dynamics of the loop connecting Helix 11 and Helix 12 in the ligand-binding domain of ERα, which leads to a stabilized agonist state and an altered antagonist state that resists inhibition. DOI: http://dx.doi.org/10.7554/eLife.12792.001 PMID:26836308

  9. A plant scaffold attached region detected close to a T-DNA integration site is active in mammalian cells.

    PubMed Central

    Dietz, A; Kay, V; Schlake, T; Landsmann, J; Bode, J

    1994-01-01

    Integration of foreign genes into plant genomes by the Agrobacterium T-DNA transfer system has been considered to occur at random. It has been speculated that the chromosomal structure of the integration site might affect the expression pattern of the introduced genes. To gain insight into the molecular structure of T-DNA integration sites and its possible impact on gene expression, we have examined plant DNA sequences in the vicinity of T-DNA borders. Analysis of a transgenic petunia plant containing a chloramphenicol acetyltransferase (CAT) gene regulated by the hemoglobin promoter (PAR) from Parasponia andersonii revealed a scaffold attachment region (SAR) close to one T-DNA end. In addition to having strong binding affinities for both animal and plant nuclear scaffolds this petunia SAR element is as active in mammalian cells as the authentic elements from mammalian sources. Images PMID:8052530

  10. Conformational, spectroscopic and nonlinear optical properties of biologically active N,N-dimethyltryptamine molecule: A theoretical study

    NASA Astrophysics Data System (ADS)

    Öner, Nazmiye; Tamer, Ömer; Avcı, Davut; Atalay, Yusuf

    2014-12-01

    The effective psychoactive properties of N,N-dimethyltryptamine (DMT) known as the near-death molecule have encouraged the imagination of many research disciplines for several decades. Although there is no theoretical study, a number of paper composed by experimental techniques have been reported for DMT molecule. In this study, the molecular modeling of DMT was carried out using B3LYP and HSEh1PBE levels of density functional theory (DFT). Our calculations showed that the energy gap between HOMO and LUMO is low, demonstrating that DMT is a biologically active molecule. Large hyperconjugation interaction energies imply that molecular charge transfer occurs in DMT. Moreover, NLO analysis indicates that DMT can be used an effective NLO material.

  11. Conformational, spectroscopic and nonlinear optical properties of biologically active N,N-dimethyltryptamine molecule: a theoretical study.

    PubMed

    Öner, Nazmiye; Tamer, Ömer; Avcı, Davut; Atalay, Yusuf

    2014-12-10

    The effective psychoactive properties of N,N-dimethyltryptamine (DMT) known as the near-death molecule have encouraged the imagination of many research disciplines for several decades. Although there is no theoretical study, a number of paper composed by experimental techniques have been reported for DMT molecule. In this study, the molecular modeling of DMT was carried out using B3LYP and HSEh1PBE levels of density functional theory (DFT). Our calculations showed that the energy gap between HOMO and LUMO is low, demonstrating that DMT is a biologically active molecule. Large hyperconjugation interaction energies imply that molecular charge transfer occurs in DMT. Moreover, NLO analysis indicates that DMT can be used an effective NLO material.

  12. Closed bore XMR (CBXMR) systems for aortic valve replacement: active magnetic shielding of x-ray tubes.

    PubMed

    Bracken, John A; DeCrescenzo, Giovanni; Komljenovic, Philip; Lillaney, Prasheel V; Fahrig, Rebecca; Rowlands, J A

    2009-05-01

    Hybrid closed bore x-ray/MRI systems are being developed to improve the safety and efficacy of percutaneous aortic valve replacement procedures by harnessing the complementary strengths of the x-ray and MRI modalities in a single interventional suite without requiring patient transfer between two rooms. These systems are composed of an x-ray C-arm in close proximity (approximately 1 m) to an MRI scanner. The MRI magnetic fringe field can cause the electron beam in the x-ray tube to deflect. The deflection causes the x-ray field of view to shift position on the detector receptacle. This could result in unnecessary radiation exposure to the patient and the staff in the cardiac catheterization laboratory. Therefore, the electron beam deflection must be corrected. The authors developed an active magnetic shielding system that can correct for electron beam deflection to within an accuracy of 5% without truncating the field of view or increasing exposure to the patient. This system was able to automatically adjust to different field strengths as the external magnetic field acting on the x-ray tube was changed. Although a small torque was observed on the shielding coils of the active shielding system when they were placed in a magnetic field, this torque will not impact their performance if they are securely mounted on the x-ray tube and the C-arm. The heating of the coils of the shielding system for use in the clinic caused by electric current was found to be slow enough not to require a dedicated cooling system for one percutaneous aortic valve replacement procedure. However, a cooling system will be required if multiple procedures are performed in one session. PMID:19544789

  13. Closed bore XMR (CBXMR) systems for aortic valve replacement: Active magnetic shielding of x-ray tubes

    PubMed Central

    Bracken, John A.; DeCrescenzo, Giovanni; Komljenovic, Philip; Lillaney, Prasheel V.; Fahrig, Rebecca; Rowlands, J. A.

    2009-01-01

    Hybrid closed bore x-ray∕MRI systems are being developed to improve the safety and efficacy of percutaneous aortic valve replacement procedures by harnessing the complementary strengths of the x-ray and MRI modalities in a single interventional suite without requiring patient transfer between two rooms. These systems are composed of an x-ray C-arm in close proximity (≈1 m) to an MRI scanner. The MRI magnetic fringe field can cause the electron beam in the x-ray tube to deflect. The deflection causes the x-ray field of view to shift position on the detector receptacle. This could result in unnecessary radiation exposure to the patient and the staff in the cardiac catheterization laboratory. Therefore, the electron beam deflection must be corrected. The authors developed an active magnetic shielding system that can correct for electron beam deflection to within an accuracy of 5% without truncating the field of view or increasing exposure to the patient. This system was able to automatically adjust to different field strengths as the external magnetic field acting on the x-ray tube was changed. Although a small torque was observed on the shielding coils of the active shielding system when they were placed in a magnetic field, this torque will not impact their performance if they are securely mounted on the x-ray tube and the C-arm. The heating of the coils of the shielding system for use in the clinic caused by electric current was found to be slow enough not to require a dedicated cooling system for one percutaneous aortic valve replacement procedure. However, a cooling system will be required if multiple procedures are performed in one session. PMID:19544789

  14. Activation and Transformation of Ethane by Au2 VO3(+) Clusters with Closed-Shell Electronic Structures.

    PubMed

    Li, Ya-Ke; Li, Zi-Yu; Zhao, Yan-Xia; Liu, Qing-Yu; Meng, Jing-Heng; He, Sheng-Gui

    2016-01-26

    The study of chemical reactions between gold-containing heteronuclear oxide clusters and small molecules can provide molecular level mechanisms to understand the excellent activity of gold supported by metal oxides. While the promotion role of gold in alkane transformation was identified in the clusters with atomic oxygen radicals (O(-.)), the role of gold in the systems without O(-.) is not clear. By employing mass spectrometry and quantum chemistry calculations, the reactivity of Au2 VO3(+) clusters with closed-shell electronic structures toward ethane was explored. Both the dehydrogenation and ethene elimination channels were identified. It is gold rather than oxygen species initiating the C-H activation. The Au-Au dimer formed during the reactions plays important roles in ethane transformation. The reactivity comparison between Au2 VO3(+) and bare Au2(+) demonstrates that Au2 VO3(+) not only retains the property of bare Au2(+) that transforming ethane to dihydrogen, but also exhibits new functions in converting ethane to ethene, which reveals the importance of the composite system. This study provides a further understanding of the reactivity of metal oxide supported gold in alkane activation and transformation. PMID:26679978

  15. Effects of water miscible organic solvents on the activity and conformation of the Baeyer-Villiger monooxygenases from Thermobifida fusca and Acinetobacter calcoaceticus: a comparative study.

    PubMed

    Secundo, Francesco; Fialà, Stefano; Fraaije, Marco W; de Gonzalo, Gonzalo; Meli, Massimiliano; Zambianchi, Francesca; Ottolina, Gianluca

    2011-03-01

    A broader exploitation of enzymes in organic synthesis can be achieved by increasing their tolerance toward organic solvents. In this study, the stability and activity of Baeyer-Villiger monooxygenases from Thermobifida fusca (PAMO) and Acinetobacter sp. (CHMO) in the presence of water miscible organic solvents were compared. PAMO was more stable than CHMO. The concentration of solvent (v/v) at which it halved its activity (C(50) ) was 4- to 16-fold higher than that observed for CHMO. For PAMO, the C(50) varied from 16% to 55% of solvent and followed the destabilizing order methanol < ethanol < 1,4-dioxane < acetonitrile < trifluoroethanol. In the case of CHMO, the maximal C(50) was 7% with methanol and even lower with the other solvents. Therefore, methanol was the most tolerated solvent. In the case of PAMO, methanol induced a significant increase of enzyme activity (up to fivefold), which was optimal at 20% (v/v) solvent. Only minor spectral variations were observed with PAMO in 20% methanol, suggesting that the increase of activity observed in this condition is not due to marked conformational changes. Fluorescence and circular dichroism analyses showed that the lower stability of CHMO toward organic solvent correlates with a more pronounced destructive effect on its secondary and tertiary structure. A possible rationale for the higher stability of PAMO could be inferred from inspection of the PAMO and CHMO (two enzymes of similar size) structure, which revealed a higher (up to twofold) number of ionic bridges in PAMO with respect to CHMO.

  16. The Clusters-in-a-Liquid Approach for Solvation: New Insights from the Conformer Specific Gas Phase Spectroscopy and Vibrational Optical Activity Spectroscopy

    PubMed Central

    Perera, Angelo S.; Thomas, Javix; Poopari, Mohammad R.; Xu, Yunjie

    2016-01-01

    Vibrational optical activity spectroscopies, namely vibrational circular dichroism (VCD) and Raman optical activity (ROA), have been emerged in the past decade as powerful spectroscopic tools for stereochemical information of a wide range of chiral compounds in solution directly. More recently, their applications in unveiling solvent effects, especially those associated with water solvent, have been explored. In this review article, we first select a few examples to demonstrate the unique sensitivity of VCD spectral signatures to both bulk solvent effects and explicit hydrogen-bonding interactions in solution. Second, we discuss the induced solvent chirality, or chiral transfer, VCD spectral features observed in the water bending band region in detail. From these chirality transfer spectral data, the related conformer specific gas phase spectroscopic studies of small chiral hydration clusters, and the associated matrix isolation VCD experiments of hydrogen-bonded complexes in cold rare gas matrices, a general picture of solvation in aqueous solution emerges. In such an aqueous solution, some small chiral hydration clusters, rather than the chiral solutes themselves, are the dominant species and are the ones that contribute mainly to the experimentally observed VCD features. We then review a series of VCD studies of amino acids and their derivatives in aqueous solution under different pHs to emphasize the importance of the inclusion of the bulk solvent effects. These experimental data and the associated theoretical analyses are the foundation for the proposed “clusters-in-a-liquid” approach to account for solvent effects effectively. We present several approaches to identify and build such representative chiral hydration clusters. Recent studies which applied molecular dynamics simulations and the subsequent snapshot averaging approach to generate the ROA, VCD, electronic CD, and optical rotatory dispersion spectra are also reviewed. Challenges associated with

  17. The clusters-in-a-liquid approach for solvation: New insights from the conformer specific gas phase spectroscopy and vibrational optical activity spectroscopy

    NASA Astrophysics Data System (ADS)

    Xu, Yunjie; Perera, Angelo; Thomas, Javix; Poopari, Mohammad

    2016-02-01

    Vibrational optical activity spectroscopies, namely vibrational circular dichroism (VCD) and Raman optical activity (ROA), have been emerged in the past decade as a powerful spectroscopic tool for stereochemical information of a wide range of chiral compounds in solution directly. More recently, their applications in unveiling solvent effects, especially those associated with water solvent, have been explored. In this review article, we first select a few examples to demonstrate the unique sensitivity of VCD spectral signatures to both bulk solvent effects and explicit hydrogen-bonding interactions in solution. Second, we discuss the induced solvent chirality, or chiral transfer, VCD spectral features observed at the water bending band region in detail. From these chirality transfer spectral data, the related conformer specific gas phase spectroscopic studies of small chiral hydration clusters, and the associated matrix isolation VCD experiments of hydrogen-bonded complexes in cold rare gas matrices, a general picture of solvation in aqueous solution emerges. In such an aqueous solution, some small chiral hydration clusters, rather than the chiral solutes themselves, are the dominant species and are the ones who contribute mainly to the experimentally observed VCD features. We then review a series of VCD studies of amino acids and their derivatives in aqueous solution under different pHs to emphasize the importance of the inclusion of the bulk solvent effects. These experimental data and the associated theoretical analyses are the foundation for the proposed “clusters-in-a-liquid” approach to account for solvent effects effectively. We present several approaches to identify and build such representative chiral hydration clusters. Recent studies which applied molecular dynamics simulations and the subsequent snapshot averaging approach to generate the ROA, electronic CD, and optical rotatory dispersion spectra are also reviewed. Challenges associated with the

  18. Structural basis for the antipolymer activity of Hb ζ2βs2 trapped in a tense conformation

    NASA Astrophysics Data System (ADS)

    Safo, Martin K.; Ko, Tzu-Ping; Schreiter, Eric R.; Eric Russell, J.

    2015-11-01

    The phenotypical severity of sickle cell disease (SCD) can be mitigated by modifying mutant hemoglobin S (Hb s, Hb α2β 2s) to contain embryonic ζ globin in place of adult α-globin subunits (Hb ζ2β2s). Crystallographical analyses of liganded Hb ζζ2β2s, though, demonstrate a tense (T-state) quaternary structure that paradoxically predicts its participation in--rather than its exclusion from--pathological deoxyHb S polymers. We resolved this structure-function conundrum by examining the effects of α → ζ exchange on the characteristics of specific amino acids that mediate sickle polymer assembly. Superposition analyses of the βs subunits of T-state deoxyHb α2β2s and T-state CO-liganded Hb ζ2β2s reveal significant displacements of both mutant βsVal6 and conserved β-chain contact residues, predicting weakening of corresponding polymer-stabilizing interactions. Similar comparisons of the α- and ζ-globin subunits implicate four amino acids that are either repositioned or undergo non-conservative substitution, abrogating critical polymer contacts. CO-Hb ζ2βs2 additionally exhibits a unique trimer-of-heterotetramers crystal packing that is sustained by novel intermolecular interactions involving the pathological βsVal6, contrasting sharply with the classical double-stranded packing of deoxyHb S. Finally, the unusually large buried solvent-accessible surface area for CO-Hb ζ2β2s suggests that it does not co-assemble with deoxyHb S in vivo. In sum, the antipolymer activities of Hb ζ203b2;2s appear to arise from both repositioning and replacement of specific α- and βs-chain residues, favoring an alternate T-state solution structure that is excluded from pathological deoxyHb S polymers. These data account for the antipolymer activity of Hb ζ2β2s, and recommend the utility of SCD therapeutics that capitalize on α-globin exchange strategies.

  19. Assessment of conformational, spectral, antimicrobial activity, chemical reactivity and NLO application of Pyrrole-2,5-dicarboxaldehyde bis(oxaloyldihydrazone)

    NASA Astrophysics Data System (ADS)

    Rawat, Poonam; Singh, R. N.

    2015-04-01

    An orange colored pyrrole dihydrazone: Pyrrole-2,5-dicarboxaldehyde bis(oxaloyldihydrazone) (PDBO) has been synthesized by reaction of oxalic acid dihydrazide with 2,5 diformyl-1H-pyrrole and has been characterized by spectroscopic analysis (1H, 13C NMR, UV-visible, FT-IR and DART Mass). The properties of the compound has been evaluated using B3LYP functional and 6-31G(d,p)/6-311+G(d,p) basis set. The symmetric (3319, 3320 cm-1) and asymmetric (3389, 3382 cm-1) stretching wave number confirm free NH2 groups in PDBO. NBO analysis shows, inter/intra molecular interactions within the molecule. Topological parameters have been analyzed by QTAIM theory and provide the existence of intramolecular hydrogen bonding (N-H⋯O). The local reactivity descriptors analyses determine the reactive sites within molecule. The calculated first hyperpolarizability value (β0 = 23.83 × 10-30 esu) of pyrrole dihydrazone shows its suitability for non-linear optical (NLO) response. The preliminary bioassay suggested that the PDBO exhibits relatively good antibacterial and fungicidal activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Candida albicans, Aspergillus niger. The local reactivity descriptors - Fukui functions (fk+, fk-), local softnesses (sk+, sk-) and electrophilicity indices (ωk+, ωk-) analyses have been used to determine the reactive sites within molecule.

  20. Chi hotspots trigger a conformational change in the helicase-like domain of AddAB to activate homologous recombination

    PubMed Central

    Gilhooly, Neville S.; Carrasco, Carolina; Gollnick, Benjamin; Wilkinson, Martin; Wigley, Dale B.; Moreno-Herrero, Fernando; Dillingham, Mark S.

    2016-01-01

    In bacteria, the repair of double-stranded DNA breaks is modulated by Chi sequences. These are recognised by helicase-nuclease complexes that process DNA ends for homologous recombination. Chi activates recombination by changing the biochemical properties of the helicase-nuclease, transforming it from a destructive exonuclease into a recombination-promoting repair enzyme. This transition is thought to be controlled by the Chi-dependent opening of a molecular latch, which enables part of the DNA substrate to evade degradation beyond Chi. Here, we show that disruption of the latch improves Chi recognition efficiency and stabilizes the interaction of AddAB with Chi, even in mutants that are impaired for Chi binding. Chi recognition elicits a structural change in AddAB that maps to a region of AddB which resembles a helicase domain, and which harbours both the Chi recognition locus and the latch. Mutation of the latch potentiates the change and moderately reduces the duration of a translocation pause at Chi. However, this mutant displays properties of Chi-modified AddAB even in the complete absence of bona fide hotspot sequences. The results are used to develop a model for AddAB regulation in which allosteric communication between Chi binding and latch opening ensures quality control during recombination hotspot recognition. PMID:26762979

  1. Assessment of conformational, spectral, antimicrobial activity, chemical reactivity and NLO application of Pyrrole-2,5-dicarboxaldehyde bis(oxaloyldihydrazone).

    PubMed

    Rawat, Poonam; Singh, R N

    2015-04-01

    An orange colored pyrrole dihydrazone: Pyrrole-2,5-dicarboxaldehyde bis(oxaloyldihydrazone) (PDBO) has been synthesized by reaction of oxalic acid dihydrazide with 2,5 diformyl-1H-pyrrole and has been characterized by spectroscopic analysis (1H, 13C NMR, UV-visible, FT-IR and DART Mass). The properties of the compound has been evaluated using B3LYP functional and 6-31G(d,p)/6-311+G(d,p) basis set. The symmetric (3319, 3320 cm(-1)) and asymmetric (3389, 3382 cm(-1)) stretching wave number confirm free NH2 groups in PDBO. NBO analysis shows, inter/intra molecular interactions within the molecule. Topological parameters have been analyzed by QTAIM theory and provide the existence of intramolecular hydrogen bonding (N-H⋯O). The local reactivity descriptors analyses determine the reactive sites within molecule. The calculated first hyperpolarizability value (β0=23.83×10(-30) esu) of pyrrole dihydrazone shows its suitability for non-linear optical (NLO) response. The preliminary bioassay suggested that the PDBO exhibits relatively good antibacterial and fungicidal activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Candida albicans, Aspergillus niger. The local reactivity descriptors--Fukui functions (fk+, fk-), local softnesses (sk+, sk-) and electrophilicity indices (ωk+, ωk-) analyses have been used to determine the reactive sites within molecule.

  2. Dolastatin 11 conformations, analogues and pharmacophore.

    PubMed

    Ali, Md Ahad; Bates, Robert B; Crane, Zackary D; Dicus, Christopher W; Gramme, Michelle R; Hamel, Ernest; Marcischak, Jacob; Martinez, David S; McClure, Kelly J; Nakkiew, Pichaya; Pettit, George R; Stessman, Chad C; Sufi, Bilal A; Yarick, Gayle V

    2005-07-01

    Twenty analogues of the natural antitumor agent dolastatin 11, including majusculamide C, were synthesized and tested for cytotoxicity against human cancer cells and stimulation of actin polymerization. Only analogues containing the 30-membered ring were active. Molecular modeling and NMR evidence showed the low-energy conformations. The amide bonds are all trans except for the one between the Tyr and Val units, which is cis. Since an analogue restricted to negative 2-3-4-5 angles stimulated actin polymerization but was inactive in cells, the binding conformation (most likely the lowest-energy conformation in water) has a negative 2-3-4-5 angle, whereas a conformation with a positive 2-3-4-5 angle (most likely the lowest energy conformation in chloroform) goes through cell walls. The highly active R alcohol from borohydride reduction of dolastatin 11 is a candidate for conversion to prodrugs.

  3. A Case Study of After-School Activities in One School That Is Making Progress in Closing the Achievement Gap

    ERIC Educational Resources Information Center

    Shugerman, Susan Robin

    2013-01-01

    Closing the achievement gap has been a national conversation for several decades and a priority for educators and researchers. By looking closely at one school which is showing exceptional success with closing the achievement gap for low income students and English language learners, this study seeks to understand how school personnel and parents…

  4. Proteolytic degradation of the RGD-binding and non-RGD-binding conformers of human platelet integrin glycoprotein IIb/IIIa: clues for identification of regions involved in the receptor's activation.

    PubMed Central

    Calvete, J J; Mann, K; Schäfer, W; Fernandez-Lafuente, R; Guisán, J M

    1994-01-01

    The human integrin glycoprotein (GP)IIb/IIIa plays a central role in haemostasis as an inducible receptor for fibrinogen and other RGD-containing adhesive proteins at the platelet plasma membrane. Expression of the fibrinogen receptor on platelet activation involves conformational changes in the quaternary structure of GPIIb/IIIa. Little is known, however, about the nature of this conformational transition. Given that isolated GPIIb/IIIa contains a mixture of RGD-binding and non-RGD-binding heterodimers, we used limited proteolysis as a tool for investigating the structural differences between the two conformers. Comparison of their fragmentation patterns shows that, whereas in the non-RGD-binding form of GPIIb/IIIa the N-terminal half of the heavy chain of GPIIb (GPIIbH) and the central region of GPIIIa are cleaved by endoproteinase Arg-C, these domains associate tightly with one another in the RGD-binding GPIIb/IIIa and are thus protected from proteolysis. In addition, the C-terminal half of GPIIb becomes more susceptible to degradation in the non-RGD-binding GPIIb/IIIa conformer. Our interpretation, in the context of available structural and functional data, is that a major relative reorientation of the GPIIbH and GPIIIa extracellular domains takes place along the subunit interface during the conformational transition of the platelet integrin. Images Figure 1 PMID:8129707

  5. Active ocular vergence improves postural control in elderly as close viewing distance with or without a single cognitive task.

    PubMed

    Matheron, Eric; Yang, Qing; Delpit-Baraut, Vincent; Dailly, Olivier; Kapoula, Zoï

    2016-01-01

    Performance of the vestibular, visual, and somatosensory systems decreases with age, reducing the capacity of postural control, and increasing the risk of falling. The purpose of this study is to measure the effects of vision, active vergence eye movements, viewing distance/vergence angle and a simple cognitive task on postural control during an upright stance, in completely autonomous elderly individuals. Participated in the study, 23 elderly subjects (73.4 ± 6.8 years) who were enrolled in a center dedicated to the prevention of falling. Their body oscillations were measured with the DynaPort(®) device, with three accelerometers, placed at the lumbosacral level, near the center of mass. The conditions were the following: eyes open fixating on LED at 20 cm or 150 cm (vergence angle 17.0° and 2.3° respectively) with or without additional cognitive tasks (counting down from one hundred), performing active vergence by alternating the fixation between the far and the near LED (convergence and divergence), eyes closed after having fixated the far LED. The results showed that the postural stability significantly decreased when fixating on the LED at a far distance (weak convergence angle) with or without cognitive tasks; active convergence-divergence between the LEDs improved the postural stability while eye closure decreased it. The privilege of proximity (with increased convergence at near), previously established with foot posturography, is shown here to be valid for accelerometry with the center of mass in elderly. Another major result is the beneficial contribution of active vergence eye movements to better postural stability. The results bring new perspectives for the role of eye movement training to preserve postural control and autonomy in elderly.

  6. NMR structure of the noncytotoxic α-sarcin mutant Δ(7-22): The importance of the native conformation of peripheral loops for activity

    PubMed Central

    García-Mayoral, Ma Flor; García-Ortega, Lucia; Lillo, Ma Pilar; Santoro, Jorge; Martínez Del Pozo, Álvaro; Gavilanes, José G.; Rico, Manuel; Bruix, Marta

    2004-01-01

    The deletion mutant Δ(7-22) of α-sarcin, unlike its wild-type protein counterpart, lacks the specific ability to degrade rRNA in intact ribosomes and exhibits an increased unspecific ribonuclease activity and decreased interaction with lipid vesicles. In trying to shed light on these differences, we report here on the three-dimensional structure of the Δ(7-22) α-sarcin mutant using NMR methods. We also evaluated its dynamic properties on the basis of theoretical models and measured its correlation time (6.2 nsec) by time-resolved fluorescence anisotropy. The global fold characteristic of ribotoxins is preserved in the mutant. The most significant differences with respect to the α-sarcin structure are concentrated in (1) loop 2, (2) loop 3, which adopts a new orientation, and (3) loop 5, which shows multiple conformations and an altered dynamics. The interactions between loop 5 and the N-terminal hairpin are lost in the mutant, producing increased solvent accessibility of the active-site residues. The degree of solvent exposure of the catalytic His 137 is similar to that shown by His 92 in RNase T1. Additionally, the calculated order parameters of residues belonging to loop 5 in the mutant correspond to an internal dynamic behavior more similar to RNase T1 than α-sarcin. On the other hand, changes in the relative orientation of loop 3 move the lysine-rich region 111–114, crucial for substrate recognition, away from the active site. All of the structural and dynamic data presented here reveal that the mutant is a hybrid of ribotoxins and noncytotoxic ribonucleases, consistent with its biological properties. PMID:15044731

  7. Effect of PEG surface conformation on anticancer activity and blood circulation of nanoemulsions loaded with tocotrienol-rich fraction of palm oil.

    PubMed

    Alayoubi, Alaadin; Alqahtani, Saeed; Kaddoumi, Amal; Nazzal, Sami

    2013-10-01

    Tocotrienol-rich fraction of palm oil, which contains the isomers of vitamin E, was shown to possess potent anticancer activity against mammary adenocarcinoma cell lines. Its clinical use, however, is limited by poor oral bioavailability and short half-life. Previously, we developed tocotrienol-rich lipid nanoemulsions for intravenous administration. The objective of this study was to investigate the effect of surface grafted polyethylene glycol (PEG) on the properties of the nanoemulsions. PEGylation was achieved by the addition of equimolar PEG groups using poloxamer or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)2000] (PEG2000-DSPE). The effect of PEG surface topography on the antiproliferative activity of nanoemulsions against mammary adenocarcinoma cells, their susceptibility to protein adsorption, and its effect on blood hemolysis and circulation time was investigated. Nanoemulsions PEGylated with poloxamer or PEG2000-DSPE were stable under physical stress. Poloxamer nanoemulsion, however, displayed higher uptake and potency against MCF-7 tumor cells in 2D and 3D culture and increased hemolytic effect and susceptibility to IgG adsorption, which was reflected in its rapid clearance and short circulation half-life (1.7 h). Conversely, PEGylation with PEG2000-DSPE led to a 7-fold increase in mean residence time (12.3 h) after IV injection in rats. Reduced activity in vitro and improved circulation time suggested strong shielding of plasma proteins from the droplets. Differences between the nanoemulsions were attributed to polymer imbibitions and the differences in PEG conformation and density on the surface of the droplets.

  8. Chromosome Conformation Capture in Drosophila.

    PubMed

    Li, Hua-Bing

    2016-01-01

    Linear chromatin fiber is packed inside the nuclei as a complex three-dimensional structure, and the organization of the chromatin has important roles in the appropriate spatial and temporal regulation of gene expression. To understand how chromatin organizes inside nuclei, and how regulatory proteins physically interact with genes, chromosome conformation capture (3C) technique provides a powerful and sensitive tool to detect both short- and long-range DNA-DNA interaction. Here I describe the 3C technique to detect the DNA-DNA interactions mediated by insulator proteins that are closely related to PcG in Drosophila, which is also broadly applicable to other systems. PMID:27659987

  9. SLITHER: a web server for generating contiguous conformations of substrate molecules entering into deep active sites of proteins or migrating through channels in membrane transporters.

    PubMed

    Lee, Po-Hsien; Kuo, Kuei-Ling; Chu, Pei-Ying; Liu, Eric M; Lin, Jung-Hsin

    2009-07-01

    Many proteins use a long channel to guide the substrate or ligand molecules into the well-defined active sites for catalytic reactions or for switching molecular states. In addition, substrates of membrane transporters can migrate to another side of cellular compartment by means of certain selective mechanisms. SLITHER (http://bioinfo.mc.ntu.edu.tw/slither/or http://slither.rcas.sinica.edu.tw/) is a web server that can generate contiguous conformations of a molecule along a curved tunnel inside a protein, and the binding free energy profile along the predicted channel pathway. SLITHER adopts an iterative docking scheme, which combines with a puddle-skimming procedure, i.e. repeatedly elevating the potential energies of the identified global minima, thereby determines the contiguous binding modes of substrates inside the protein. In contrast to some programs that are widely used to determine the geometric dimensions in the ion channels, SLITHER can be applied to predict whether a substrate molecule can crawl through an inner channel or a half-channel of proteins across surmountable energy barriers. Besides, SLITHER also provides the list of the pore-facing residues, which can be directly compared with many genetic diseases. Finally, the adjacent binding poses determined by SLITHER can also be used for fragment-based drug design.

  10. Structural studies of conformational changes of proteins upon phosphorylation: Structures of activated CheY, CheY-N16-FliM complex, and AAA {sup +} ATPase domain of NtrC1 in both inactive and active states

    SciTech Connect

    Lee, Seok-Yong

    2003-04-10

    Protein phosphorylation is a general mechanism for signal transduction as well as regulation of cellular function. Unlike phosphorylation in eukaryotic systems that uses Ser/Thr for the sites of modification, two-component signal transduction systems, which are prevalent in bacteria, archea, and lower eukaryotes, use an aspartate as the site of phosphorylation. Two-component systems comprise a histidine kinase and a receiver domain. The conformational change of the receiver domain upon phosphorylation leads to signal transfer to the downstream target, a process that had not been understood well at the molecular level. The transient nature of the phospho-Asp bond had made structural studies difficult. The discovery of an excellent analogue for acylphosphate, BeF{sub 3}{sup -}, enabled structural study of activated receiver domains. The structure of activated Chemotaxis protein Y (CheY) was determined both by NMR spectroscopy and X-ray crystallography. These structures revealed the molecular basis of the conformational change that is coupled to phosphorylation. Phosphorylation of the conserved Asp residue in the active site allows hydrogen bonding of the T87 O{gamma} to phospho-aspartate, which in turn leads to the rotation of Y106 into the ''in'' position (termed Y-T coupling). The structure of activated CheY complexed with the 16 N-terminal residues of FliM (N16-FliM), its target, was also determined by X-ray crystallography and confirmed the proposed mechanism of activation (Y-T coupling). First, N16-FliM binds to the region on CheY that undergoes a significant conformational change. Second, the ''in'' position of Y106 presents a better binding surface for FliM because the sidechain of Y106 in the inactive form of CheY (''out'' position) sterically interferes with binding of N16-FliM. In addition to confirmation of Y-T coupling, the structure of the activated CheY-N16-FliM complex suggested that the N16-FliM might be sandwiched between CheY and the remainder of

  11. Conformal Window and Correlation Functions in Lattice Conformal QCD

    NASA Astrophysics Data System (ADS)

    Iwasaki, Y.

    We discuss various aspects of Conformal Field Theories on the Lattice. We mainly investigate the SU(3) gauge theory with Nf degenerate fermions in the fundamental representation, employing the one-plaquette gauge action and the Wilson fermion action. First we make a brief review of our previous works on the phase structure of lattice gauge theories in terms of the gauge coupling constant and the quark mass. We thereby clarify the reason why we conjecture that the conformal window is 7 ≤ Nf ≤ 16. Secondly, we introduce a new concept, "conformal theories with IR cutof" and point out that any numerical simulation on a lattice is bounded by an IR cutoff ∧IR. Then we make predictions that when Nf is within the conformal window, the propagator of a meson G(t) behaves at large t, as G(t) = c exp (-mHt)/tα, that is, a modified Yukawa-type decay form, instead of the usual exponential decay form exp (-mHt), in the small quark mass region. This holds on an any lattice for any coupling constant g, as far as g is between 0 and g*, where g* is the IR fixed point. We verify that numerical results really satisfy the predictions for the Nf = 7 case and the Nf = 16 case. Thirdly, we discuss small number of flavors (Nf = 2 ˜ 6) QCD at finite temperatures. We point out theoretically and verify numerically that the correlation functions at T/Tc > 1 exhibit the characteristics of the conformal function with IR cutoff, an exponential decay with power correction. Investigating our numerical data by a new method which we call the "local-analysis" of propagators, we observe that the Nf = 7 case and the Nf = 2 at T ˜ 2Tc case are similar to each other, while the Nf = 16 case and the Nf = 2 at T = 102 ˜ 105Tc cases are similar to each other. Further, we observe our data are consistent with the picture that the Nf = 7 case and the Nf = 2 at T ˜ 2Tc case are close to the meson unparticle model. On the other hand, the Nf = 16 case and the Nf = 2 at T = 102 ˜ 105Tc cases are close to

  12. Nonequilibrium Relaxation of Conformational Dynamics Facilitates Catalytic Reaction in an Elastic Network Model of T7 DNA Polymerase.

    PubMed

    Zhao, Ziqing W; Xie, X Sunney; Ge, Hao

    2016-03-24

    Nucleotide-induced conformational closing of the finger domain of DNA polymerase is crucial for its catalytic action during DNA replication. Such large-amplitude molecular motion is often not fully accessible to either direct experimental monitoring or molecular dynamics simulations. However, a coarse-grained model can offer an informative alternative, especially for probing the relationship between conformational dynamics and catalysis. Here we investigate the dynamics of T7 DNA polymerase catalysis using a Langevin-type elastic network model incorporating detailed structural information on the open conformation without the substrate bound. Such a single-parameter model remarkably captures the induced conformational dynamics of DNA polymerase upon dNTP binding, and reveals its close coupling to the advancement toward transition state along the coordinate of the target reaction, which contributes to significant lowering of the activation energy barrier. Furthermore, analysis of stochastic catalytic rates suggests that when the activation energy barrier has already been significantly lowered and nonequilibrium relaxation toward the closed form dominates the catalytic rate, one must appeal to a picture of two-dimensional free energy surface in order to account for the full spectrum of catalytic modes. Our semiquantitative study illustrates the general role of conformational dynamics in achieving transition-state stabilization, and suggests that such an elastic network model, albeit simplified, possesses the potential to furnish significant mechanistic insights into the functioning of a variety of enzymatic systems.

  13. Representation of target-bound drugs by computed conformers: implications for conformational libraries

    PubMed Central

    Günther, Stefan; Senger, Christian; Michalsky, Elke; Goede, Andrean; Preissner, Robert

    2006-01-01

    Background The increasing number of known protein structures provides valuable information about pharmaceutical targets. Drug binding sites are identifiable and suitable lead compounds can be proposed. The flexibility of ligands is a critical point for the selection of potential drugs. Since computed 3D structures of millions of compounds are available, the knowledge of their binding conformations would be a great benefit for the development of efficient screening methods. Results Integration of two public databases allowed superposition of conformers for 193 approved drugs with 5507 crystallised target-bound counterparts. The generation of 9600 drug conformers using an atomic force field was carried out to obtain an optimal coverage of the conformational space. Bioactive conformations are best described by a conformational ensemble: half of all drugs exhibit multiple active states, distributed over the entire range of the reachable energy and conformational space. A number of up to 100 conformers per drug enabled us to reproduce the bound states within a similarity threshold of 1.0 Å in 70% of all cases. This fraction rises to about 90% for smaller or average sized drugs. Conclusion Single drugs adopt multiple bioactive conformations if they interact with different target proteins. Due to the structural diversity of binding sites they adopt conformations that are distributed over a broad conformational space and wide energy range. Since the majority of drugs is well represented by a predefined low number of conformers (up to 100) this procedure is a valuable method to compare compounds by three-dimensional features or for fast similarity searches starting with pharmacophores. The underlying 9600 generated drug conformers are downloadable from the Super Drug Web site [1]. All superpositions are visualised at the same source. Additional conformers (110,000) of 2400 classified WHO-drugs are also available. PMID:16764718

  14. Estimation of ligand efficacies of metabotropic glutamate receptors from conformational forces obtained from molecular dynamics simulations.

    PubMed

    Lakkaraju, Sirish Kaushik; Xue, Fengtian; Faden, Alan I; MacKerell, Alexander D

    2013-06-24

    Group 1 metabotropic glutamate receptors (mGluR) are G-protein coupled receptors with a large bilobate extracellular ligand binding region (LBR) that resembles a Venus fly trap. Closing of this LBR in the presence of a ligand is associated with the activation of the receptor. From conformational sampling of the LBR-ligand complexes using all-atom molecular dynamics (MD) simulations, we characterized the conformational minima related to the hinge like motion associated with the LBR closing/opening in the presence of known agonists and antagonists. By applying a harmonic restraint on the LBR, we also determined the conformational forces generated by the different ligands. The change in the location of the minima and the conformational forces were used to quantify the efficacies of the ligands. This analysis shows that efficacies can be estimated from the forces of a single conformation of the receptor, indicating the potential of MD simulations as an efficient and useful technique to quantify efficacies, thereby facilitating the rational design of mGluR agonists and antagonists.

  15. Conformational Electroresistance and Hysteresis in Nanoclusters

    SciTech Connect

    Li, Xiangguo; Zhang, Xiaoguang; Cheng, Hai-Ping

    2014-07-02

    Among many mechanisms proposed for electroresistance, ones involving structural changes are the least understood because of challenges of controllability and repeatability. Yet structural changes can cause dramatic changes in electronic properties, leading to multiple ways in which conduction paths can be opened and closed, not limited to filament movement or variation in molecular conductance. Here we show at least another way: conformational dependence of the Coulomb charging energy of a nanocluster, where charging induced conformational distortion changes the blockade voltage, which in turn leads to a giant electroresistance. This intricate interplay between charging and conformation change is demonstrated in a nanocluster Zn3O4 by combining a first-principles calculation with a temperature dependent transport model. The predicted hysteretic Coulomb blockade staircase in the current-voltage curve adds another dimension to the rich phenomenon of tunneling electroresistance. The new mechanism also provides a better controlled and repeatable platform to study conformational electroresistance.

  16. Conformational Electroresistance and Hysteresis in Nanoclusters

    DOE PAGES

    Li, Xiangguo; Zhang, Xiaoguang; Cheng, Hai-Ping

    2014-07-02

    Among many mechanisms proposed for electroresistance, ones involving structural changes are the least understood because of challenges of controllability and repeatability. Yet structural changes can cause dramatic changes in electronic properties, leading to multiple ways in which conduction paths can be opened and closed, not limited to filament movement or variation in molecular conductance. Here we show at least another way: conformational dependence of the Coulomb charging energy of a nanocluster, where charging induced conformational distortion changes the blockade voltage, which in turn leads to a giant electroresistance. This intricate interplay between charging and conformation change is demonstrated in amore » nanocluster Zn3O4 by combining a first-principles calculation with a temperature dependent transport model. The predicted hysteretic Coulomb blockade staircase in the current-voltage curve adds another dimension to the rich phenomenon of tunneling electroresistance. The new mechanism also provides a better controlled and repeatable platform to study conformational electroresistance.« less

  17. Kinetics and thermodyamics of the rate limiting conformational change in the actomyosin V mechanochemical cycle

    PubMed Central

    Jacobs, Donald J.; Trivedi, Darshan; David, Charles; Yengo, Christopher M.

    2011-01-01

    We used FRET to examine the kinetics and thermodynamics of structural changes associated with ADP release in myosin V, which is thought to be a strain sensitive step in many muscle and non-muscle myosins. We also explore essential dynamics using FIRST/FRODA starting with three different myosin V X-ray crystal structures to examine intrinsic flexibility and correlated motions. Our steady-state and time resolved FRET analysis demonstrates a temperature dependent reversible conformational change in the nucleotide binding pocket. Our kinetic results demonstrate that the nucleotide binding pocket goes from a closed to an open conformation prior to the release of ADP while the actin binding cleft remains closed. Interestingly, we find that the temperature dependence of the maximum actin-activated myosin V ATPase rate is similar to the pocket opening step, demonstrating this is the rate limiting structural transition in the ATPase cycle. Thermodynamic analysis demonstrates the transition from the open to closed nucleotide binding pocket conformation is unfavorable because of a decrease in entropy. The intrinsic flexibility analysis is consistent with conformational entropy playing a role in this transition as the MV.ADP structure is highly flexible compared to the MV.APO structure. Our experimental and modeling studies support the conclusion of a novel post-power-stroke actomyosin.ADP state in which the nucleotide binding pocket and actin binding cleft are closed. The novel state may be important for strain sensitivity as the transition from the closed to open nucleotide binding pocket conformation may be altered by lever arm position. PMID:21315083

  18. Structural Studies of E. coli Topoisomerase III-DNA Complexes Reveal a Novel Type IA Topoisomerase-DNA Conformational Intermediate

    SciTech Connect

    Changela, Anita; DiGate, Russell J.; Mondragon, Alfonso

    2010-03-05

    Escherichia coli DNA topoisomerase III belongs to the type IA family of DNA topoisomerases, which transiently cleave single-stranded DNA (ssDNA) via a 5{prime} phosphotyrosine intermediate. We have solved crystal structures of wild-type E. coli topoisomerase III bound to an eight-base ssDNA molecule in three different pH environments. The structures reveal the enzyme in three distinct conformational states while bound to DNA. One conformation resembles the one observed previously with a DNA-bound, catalytically inactive mutant of topoisomerase III where DNA binding realigns catalytic residues to form a functional active site. Another conformation represents a novel intermediate in which DNA is bound along the ssDNA-binding groove but does not enter the active site, which remains in a catalytically inactive, closed state. A third conformation shows an intermediate state where the enzyme is still in a closed state, but the ssDNA is starting to invade the active site. For the first time, the active site region in the presence of both the catalytic tyrosine and ssDNA substrate is revealed for a type IA DNA topoisomerase, although there is no evidence of ssDNA cleavage. Comparative analysis of the various conformational states suggests a sequence of domain movements undertaken by the enzyme upon substrate binding.

  19. CONSENSUS AND CONFORMITY.

    ERIC Educational Resources Information Center

    ALLEN, VERNON L.; LEVINE, JOHN M.

    IN THIS STUDY, PROFESSOR ALLEN EMPLOYS TWO METHODS OF BREAKING GROUP CONSENSUS, AND HE MEASURES THE EFFECTS ON THE RESPONSES OF COLLEGE SUBJECTS TO BOTH OBJECTIVE AND SUBJECTIVE STIMULI. THE RESULTS SUGGEST THE NEED FOR MODIFICATION OF EXISTING THEORIES OF CONFORMITY BEHAVIOR. IN ADDITION, THESE RESULTS EMPHASIZE THE DIFFERENCES IN CONFORMITY OF…

  20. Genetic dissection of independent and cooperative transcriptional activation by the LysR-type activator ThnR at close divergent promoters.

    PubMed

    Rivas-Marín, Elena; Floriano, Belén; Santero, Eduardo

    2016-04-18

    Regulation of tetralin biodegradation operons is one of the examples of unconventional LysR-type mediated transcriptional regulation. ThnR activates transcription from two divergent and closely located promoters PB and PC. Although ThnR activates each promoter independently, transcription from each one increases when both promoters are together. Mutational analysis of the intergenic region shows that cooperative transcription is achieved through formation of a ThnR complex when bound to its respective sites at each promoter, via formation of a DNA loop. Mutations also defined ThnR contact sites that are important for independent transcriptional activation at each promoter. A mutation at the PB promoter region, which abolishes its independent transcription, does not affect at all PB transcription in the presence of the divergent promoter PC, thus indicating that the complex formed via DNA loop can compensate for the deficiencies in the correct protein-DNA interaction at one of the promoters. Combination of mutations in both promoters identifies a region at PC that is not important for its independent transcription but it is essential for cooperative transcription from both promoters. This work provides new insights into the diversity and complexity of activation mechanisms used by the most abundant type of bacterial transcriptional regulators.

  1. Genetic dissection of independent and cooperative transcriptional activation by the LysR-type activator ThnR at close divergent promoters

    PubMed Central

    Rivas-Marín, Elena; Floriano, Belén; Santero, Eduardo

    2016-01-01

    Regulation of tetralin biodegradation operons is one of the examples of unconventional LysR-type mediated transcriptional regulation. ThnR activates transcription from two divergent and closely located promoters PB and PC. Although ThnR activates each promoter independently, transcription from each one increases when both promoters are together. Mutational analysis of the intergenic region shows that cooperative transcription is achieved through formation of a ThnR complex when bound to its respective sites at each promoter, via formation of a DNA loop. Mutations also defined ThnR contact sites that are important for independent transcriptional activation at each promoter. A mutation at the PB promoter region, which abolishes its independent transcription, does not affect at all PB transcription in the presence of the divergent promoter PC, thus indicating that the complex formed via DNA loop can compensate for the deficiencies in the correct protein-DNA interaction at one of the promoters. Combination of mutations in both promoters identifies a region at PC that is not important for its independent transcription but it is essential for cooperative transcription from both promoters. This work provides new insights into the diversity and complexity of activation mechanisms used by the most abundant type of bacterial transcriptional regulators. PMID:27087658

  2. The Closing Mechanism of DNA Polymerase I at Atomic Resolution.

    PubMed

    Miller, Bill R; Beese, Lorena S; Parish, Carol A; Wu, Eugene Y

    2015-09-01

    DNA polymerases must quickly and accurately distinguish between similar nucleic acids to form Watson-Crick base pairs and avoid DNA replication errors. Deoxynucleoside triphosphate (dNTP) binding to the DNA polymerase active site induces a large conformational change that is difficult to characterize experimentally on an atomic level. Here, we report an X-ray crystal structure of DNA polymerase I bound to DNA in the open conformation with a dNTP present in the active site. We use this structure to computationally simulate the open to closed transition of DNA polymerase in the presence of a Watson-Crick base pair. Our microsecond simulations allowed us to characterize the key steps involved in active site assembly, and propose the sequence of events involved in the prechemistry steps of DNA polymerase catalysis. They also reveal new features of the polymerase mechanism, such as a conserved histidine as a potential proton acceptor from the primer 3'-hydroxyl. PMID:26211612

  3. Structure of Human Pancreatic Lipase-Related Protein 2 with the Lid in an Open Conformation

    SciTech Connect

    Eydoux, Cecilia; Spinelli, Silvia; Davis, Tara L.; Walker, John R.; Seitova, Alma; Dhe-Paganon, Sirano; De Caro, Alain; Cambillau, Christian; Carriere, Frederic

    2008-10-02

    Access to the active site of pancreatic lipase (PL) is controlled by a surface loop, the lid, which normally undergoes conformational changes only upon addition of lipids or amphiphiles. Structures of PL with their lids in the open and functional conformation have required cocrystallization with amphiphiles. Here we report two crystal structures of wild-type and unglycosylated human pancreatic lipase-related protein 2 (HPLRP2) with the lid in an open conformation in the absence of amphiphiles. These structures solved independently are strikingly similar, with some residues of the lid being poorly defined in the electron-density map. The open conformation of the lid is however different from that previously observed in classical liganded PL, suggesting different kinetic properties for HPLRP2. Here we show that the HPLRP2 is directly inhibited by E600, does not present interfacial activation, and acts preferentially on substrates forming monomers or small aggregates (micelles) dispersed in solution like monoglycerides, phospholipids and galactolipids, whereas classical PL displays reverse properties and a high specificity for unsoluble substrates like triglycerides and diglycerides forming oil-in-water interfaces. These biochemical properties imply that the lid of HPLRP2 is likely to spontaneously adopt in solution the open conformation observed in the crystal structure. This open conformation generates a large cavity capable of accommodating the digalactose polar head of galactolipids, similar to that previously observed in the active site of the guinea pig PLRP2, but absent from the classical PL. Most of the structural and kinetic properties of HPLRP2 were found to be different from those of rat PLRP2, the structure of which was previously obtained with the lid in a closed conformation. Our findings illustrate the essential role of the lid in determining the substrate specificity and the mechanism of action of lipases.

  4. Energetic and structural details of the trigger-loop closing transition in RNA polymerase II.

    PubMed

    Wang, Beibei; Predeus, Alexander V; Burton, Zachary F; Feig, Michael

    2013-08-01

    An evolutionarily conserved element in RNA polymerase II, the trigger loop (TL), has been suggested to play an important role in the elongation rate, fidelity of selection of the matched nucleoside triphosphate (NTP), catalysis of transcription elongation, and translocation in both eukaryotes and prokaryotes. In response to NTP binding, the TL undergoes large conformational changes to switch between distinct open and closed states to tighten the active site and avail catalysis. A computational strategy for characterizing the conformational transition pathway is presented to bridge the open and closed states of the TL. Information from a large number of independent all-atom molecular dynamics trajectories from Hamiltonian replica exchange and targeted molecular dynamics simulations is gathered together to assemble a connectivity map of the conformational transition. The results show that with a cognate NTP, TL closing should be a spontaneous process. One major intermediate state is identified along the conformational transition pathway, and the key structural features are characterized. The complete pathway from the open TL to the closed TL provides a clear picture of the TL closing.

  5. Novel conformationally constrained tropane analogues by 6-endo-trig radical cyclization and stille coupling - switch of activity toward the serotonin and/or norepinephrine transporter.

    PubMed

    Hoepping, A; Johnson, K M; George, C; Flippen-Anderson, J; Kozikowski, A P

    2000-05-18

    A novel class of tricyclic tropane analogues has been synthesized by making use of radical cyclization technology in combination with the Stille coupling reaction. As hybrids between tropanes and quinuclidines, these tropaquinuclidines represent a significant structural departure from many of the other classes of tropane ligands synthesized to date. This structure class is characterized by the boat conformation of the tropane ring and the orientation of the additional bridge (and therefore of the nitrogen lone pair) together with the unusual placement of the aromatic moiety. All compounds were tested for their ability to inhibit monoamine reuptake under identical conditions. The ability to inhibit reuptake of dopamine in comparison to cocaine is generally decreased in this series but for one compound. (1S,3R, 6S)-(Z)-9-(thienylmethylene)-7-azatricyclo[4.3.1.0(3, 7)]decane-2beta-carboxylic acid methyl ester (5h) exhibits reasonable activity at the dopamine transporter (DAT) (K(i) = 268 nM) and good activity at the norepinephrine transporter (NET) (K(i) = 26 nM). The potency and selectivity shown by some of these ligands for the NET, serotonine transporter (SERT), or NET/SERT is striking, particularly in view of the displacement of the aromatic ring in this series from its usual position at C-3 in the WIN analogues. Thus, (1S,3R,6S)-(Z)-9-(4-biphenylylmethylene)-7-azatricyclo[4.3.1 . 0(3,7)]decane-2beta-carboxylic acid methyl ester (5a) is a selective inhibitor of norepinephrine reuptake (K(i) = 12 nM). Its p-methoxy analogue 5c is a mixed inhibitor of norepinephrine and serotonin reuptake (K(i) = 187 nM at the NET and 56 nM at the SERT). The most active and selective compound we found in the present series is compound 8b [(1S,3R,6S)-2-(acetoxymethyl)-(Z)-9-(3, 4-dichlorophenylmethylene)-7-azatricyclo[4.3.1.0(3,7)]decane ]. This compound is a potent (K(i) = 1.6 nM) and selective inhibitor of serotonin reuptake into rat midbrain synaptosomes. Its selectivity is about

  6. THE CHROMOSPHERIC ACTIVITY OF [HH97] FS Aur-79: A CLOSE BINARY WITH LATE-TYPE ACTIVE (dK7e+dM3e) COMPONENTS

    SciTech Connect

    Austin, S. J.; Robertson, J. W.; De Souza, T. R.; Tycner, C.; Honeycutt, R. K. E-mail: jrobertson@atu.edu E-mail: c.tycner@cmich.edu

    2011-04-15

    Using Doppler tomography we show that FS Aur-79, a near-contact close binary system with late-type active dK7e+dM3e components, has chromospheric prominences in two distinct emission regions associated with the primary star and a larger amount of chromospheric activity associated with the cooler secondary star. The line profiles, equivalent widths, and equivalent width ratios of the H{alpha} and H{beta} emission lines as a function of orbital phase further support that the majority of the chromospheric emission originates above the secondary star and near the neck region. Analysis of high-resolution spectra using the technique of broadening functions has enabled us to determine the radial velocity of the secondary star near quadratures to be approximately 224 km s{sup -1}. A Wilson-Devinney model of the system fitting the UBV light curves and radial velocities shows that there are star spots near the chromospherically active regions. Finally, the absence of Li I {lambda}6708 in the spectra lets us put a lower limit on the age of this system to at least 500 Myr.

  7. Calnexin and calreticulin bind to enzymically active tissue-type plasminogen activator during biosynthesis and are not required for folding to the native conformation.

    PubMed Central

    Allen, S; Bulleid, N J

    1997-01-01

    The roles of the endoplasmic-reticulum lectins calnexin and calreticulin in the folding of tissue-type plasminogen activator (tPA) have been investigated using an in vitro translation system that reconstitutes these processes as they would occur in the intact cell. Using co-immunoprecipitation of newly synthesized tPA with antibodies to calnexin and calreticulin, it was demonstrated that the interaction of tPA with both lectins was dependent upon tPA glycosylation and glucosidase trimming. When tPA was synthesized in the presence of semi-permeabilized cells under conditions preventing complex formation with calnexin and calreticulin, the translation product had a specific plasminogenolytic activity identical with that when synthesized under conditions permitting interactions with both lectins. Furthermore, complexes of tPA bound to calnexin and calreticulin were shown to be enzymically active. These results demonstrate that calnexin and calreticulin can form a stable interaction with correctly folded tPA; however, such interactions are not required for the synthesis of enzymically active tPA. PMID:9359841

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

    PubMed Central

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

    2015-01-01

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

  9. Anticholinergic substances: A single consistent conformation

    PubMed Central

    Pauling, Peter; Datta, Narayandas

    1980-01-01

    An interactive computer-graphics analysis of 24 antagonists of acetylcholine at peripheral autonomic post-ganglionic (muscarinic) nervous junctions and at similar junctions in the central nervous system, the crystal structures of which are known, has led to the determination of a single, consistent, energetically favorable conformation for all 24 substances, although their observed crystal structure conformations vary widely. The absolute configuration and the single, consistent (ideal) conformation of the chemical groups required for maximum anticholinergic activity are described quantitatively. Images PMID:16592775

  10. Assemblies of Conformal Tanks

    NASA Technical Reports Server (NTRS)

    DeLay, Tom

    2009-01-01

    Assemblies of tanks having shapes that conform to each other and/or conform to other proximate objects have been investigated for use in storing fuels and oxidizers in small available spaces in upper stages of spacecraft. Such assemblies might also prove useful in aircraft, automobiles, boats, and other terrestrial vehicles in which space available for tanks is limited. The basic concept of using conformal tanks to maximize the utilization of limited space is not new in itself: for example, conformal tanks are used in some automobiles to store windshield -washer liquid and coolant that overflows from radiators. The novelty of the present development lies in the concept of an assembly of smaller conformal tanks, as distinguished from a single larger conformal tank. In an assembly of smaller tanks, it would be possible to store different liquids in different tanks. Even if the same liquid were stored in all the tanks, the assembly would offer an advantage by reducing the mechanical disturbance caused by sloshing of fuel in a single larger tank: indeed, the requirement to reduce sloshing is critical in some applications. The figure shows a prototype assembly of conformal tanks. Each tank was fabricated by (1) copper plating a wax tank mandrel to form a liner and (2) wrapping and curing layers of graphite/epoxy composite to form a shell supporting the liner. In this case, the conformal tank surfaces are flat ones where they come in contact with the adjacent tanks. A band of fibers around the outside binds the tanks together tightly in the assembly, which has a quasi-toroidal shape. For proper functioning, it would be necessary to maintain equal pressure in all the tanks.

  11. Pilot Non-Conformance to Alerting System Commands

    NASA Technical Reports Server (NTRS)

    Pritchett, Amy R.; Hansman, R. John

    1997-01-01

    Instances of pilot non-conformance to alerting system commands have been identified in previous studies. Pilot non-conformance changes the final behavior of the system, and therefore may reduce actual performance from that anticipated. A simulator study has examined pilot non-conformance, using the task of collision avoidance during closely spaced parallel approaches as a case study. Consonance between the display and the alerting system was found to significantly improve subject agreement with automatic alerts. Based on these results, a more general discussion of the factors involved in pilot conformance is given, and design guidelines for alerting systems are given.

  12. Mycobacterium tuberculosis acyl carrier protein synthase adopts two different pH-dependent structural conformations

    SciTech Connect

    Gokulan, Kuppan; Aggarwal, Anup; Shipman, Lance; Besra, Gurdyal S.; Sacchettini, James C.

    2011-07-01

    Bacterial acyl carrier protein synthase plays an essential role in the synthesis of fatty acids, nonribosomal peptides and polyketides. In Mycobacterium tuberculosis, AcpS or group I phosphopentatheine transferase exhibits two different structural conformations depending upon the pH. The crystal structures of acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) and Corynebacterium ammoniagenes determined at pH 5.3 and pH 6.5, respectively, are reported. Comparison of the Mtb apo-AcpS structure with the recently reported structure of the Mtb AcpS–ADP complex revealed that AcpS adopts two different conformations: the orthorhombic and trigonal space-group structures show structural differences in the α2 helix and in the conformation of the α3–α4 connecting loop, which is in a closed conformation. The apo-AcpS structure shows electron density for the entire model and was obtained at lower pH values (4.4–6.0). In contrast, at a higher pH value (6.5) AcpS undergoes significant conformational changes, resulting in disordered regions that show no electron density in the AcpS model. The solved structures also reveal that C. ammoniagenes AcpS undergoes structural rearrangement in two regions, similar to the recently reported Mtb AcpS–ADP complex structure. In vitro reconstitution experiments show that AcpS has a higher post-translational modification activity between pH 4.4 and 6.0 than at pH values above 6.5, where the activity drops owing to the change in conformation. The results show that apo-AcpS and AcpS–ADP adopt different conformations depending upon the pH conditions of the crystallization solution.

  13. [Closing diastemas].

    PubMed

    Vieira, L C; Pereira, J C; Coradazzi, J L; Francischone, C E

    1990-01-01

    The authors describe a clinical case of closing upper central incisives diastema, reconstructiva of a conoid upper lateral and the rechaping of an upper canine to a lateral incisive. The material used was composite resin.

  14. Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr215 in Aerococcus viridans lactate oxidase

    PubMed Central

    Stoisser, Thomas; Brunsteiner, Michael; Wilson, David K.; Nidetzky, Bernd

    2016-01-01

    L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr215 in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr215, effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr215 can thus lead to a kinetic bottleneck in product release. PMID:27302031

  15. Crystal structure analysis of the exocytosis-sensitive phosphoprotein, pp63/parafusin (phosphoglucomutase), from Paramecium reveals significant conformational variability.

    PubMed

    Müller, Simone; Diederichs, Kay; Breed, Jason; Kissmehl, Roland; Hauser, Karin; Plattner, Helmut; Welte, Wolfram

    2002-01-11

    During exocytosis of dense-core secretory vesicles (trichocysts) in Paramecium, the protein pp63/parafusin (pp63/pf) is transiently dephosphorylated. We report here the structures of two crystal forms of one isoform of this protein which has a high degree of homology with rabbit phosphoglucomutase, whose structure has been reported. As expected, both proteins possess highly similar structures, showing the same four domains forming two lobes with an active-site crevice in between. The two X-ray structures that we report here were determined after crystallization in the presence of sulfate and tartrate, and show the lobes arranged as a closed and an open conformation, respectively. While both conformations possess a bound divalent cation, only the closed (sulfate-bound) conformation shows bound sulfate ions in the "phosphate-transfer site" near the catalytic serine residue and in the "phosphate-binding site". Comparison with the open form shows that the latter dianion is placed in the centre of three arginine residues, one contributed by subunit II and two by subunit IV, suggesting that it causes a contraction of the arginine triangle, which establishes the observed conformational closure of the lobes. It is therefore likely that the closed conformation forms only when a phosphoryl group is bound to the phosphate-binding site. The previously published structure of rabbit phosphoglucomutase is intermediate between these two conformers. Several of the known reversible phosphorylation sites of pp63/pf-1 are at positions critical for transition between the conformations and for binding of the ligands and thus give hints as to possible roles of pp63/pf-1 in the course of exocytosis.

  16. The Structure of a High Fidelity DNA Polymerase Bound to a Mismatched Nucleotide Reveals an ;Ajar; Intermediate Conformation in the Nucleotide Selection Mechanism

    SciTech Connect

    Wu, Eugene Y.; Beese, Lorena S.

    2011-10-10

    To achieve accurate DNA synthesis, DNA polymerases must rapidly sample and discriminate against incorrect nucleotides. Here we report the crystal structure of a high fidelity DNA polymerase I bound to DNA primer-template caught in the act of binding a mismatched (dG:dTTP) nucleoside triphosphate. The polymerase adopts a conformation in between the previously established 'open' and 'closed' states. In this 'ajar' conformation, the template base has moved into the insertion site but misaligns an incorrect nucleotide relative to the primer terminus. The displacement of a conserved active site tyrosine in the insertion site by the template base is accommodated by a distinctive kink in the polymerase O helix, resulting in a partially open ternary complex. We suggest that the ajar conformation allows the template to probe incoming nucleotides for complementarity before closure of the enzyme around the substrate. Based on solution fluorescence, kinetics, and crystallographic analyses of wild-type and mutant polymerases reported here, we present a three-state reaction pathway in which nucleotides either pass through this intermediate conformation to the closed conformation and catalysis or are misaligned within the intermediate, leading to destabilization of the closed conformation.

  17. Notes on conformal invariance of gauge fields

    NASA Astrophysics Data System (ADS)

    Barnich, Glenn; Bekaert, Xavier; Grigoriev, Maxim

    2015-12-01

    In Lagrangian gauge systems, the vector space of global reducibility parameters forms a module under the Lie algebra of symmetries of the action. Since the classification of global reducibility parameters is generically easier than the classification of symmetries of the action, this fact can be used to constrain the latter when knowing the former. We apply this strategy and its generalization for the non-Lagrangian setting to the problem of conformal symmetry of various free higher spin gauge fields. This scheme allows one to show that, in terms of potentials, massless higher spin gauge fields in Minkowski space and partially massless (PM) fields in (A)dS space are not conformal for spin strictly greater than one, while in terms of curvatures, maximal-depth PM fields in four dimensions are also not conformal, unlike the closely related, but less constrained, maximal-depth Fradkin-Tseytlin fields.

  18. Models of Voltage-Dependent Conformational Changes in NaChBac Channels

    PubMed Central

    Shafrir, Yinon; Durell, Stewart R.; Guy, H. Robert

    2008-01-01

    Models of the transmembrane region of the NaChBac channel were developed in two open/inactivated and several closed conformations. Homology models of NaChBac were developed using crystal structures of Kv1.2 and a Kv1.2/2.1 chimera as templates for open conformations, and MlotiK and KcsA channels as templates for closed conformations. Multiple molecular-dynamic simulations were performed to refine and evaluate these models. A striking difference between the S4 structures of the Kv1.2-like open models and MlotiK-like closed models is the secondary structure. In the open model, the first part of S4 forms an α-helix, and the last part forms a 310 helix, whereas in the closed model, the first part of S4 forms a 310 helix, and the last part forms an α-helix. A conformational change that involves this type of transition in secondary structure should be voltage-dependent. However, this transition alone is not sufficient to account for the large gating charge movement reported for NaChBac channels and for experimental results in other voltage-gated channels. To increase the magnitude of the motion of S4, we developed another model of an open/inactivated conformation, in which S4 is displaced farther outward, and a number of closed models in which S4 is displaced farther inward. A helical screw motion for the α-helical part of S4 and a simple axial translation for the 310 portion were used to develop models of these additional conformations. In our models, four positively charged residues of S4 moved outwardly during activation, across a transition barrier formed by highly conserved hydrophobic residues on S1, S2, and S3. The S4 movement was coupled to an opening of the activation gate formed by S6 through interactions with the segment linking S4 to S5. Consistencies of our models with experimental studies of NaChBac and Kv channels are discussed. PMID:18641074

  19. Quantifying macromolecular conformational transition pathways

    NASA Astrophysics Data System (ADS)

    Seyler, Sean; Kumar, Avishek; Thorpe, Michael; Beckstein, Oliver

    2015-03-01

    Diverse classes of proteins function through large-scale conformational changes that are challenging for computer simulations. A range of fast path-sampling techniques have been used to generate transitions, but it has been difficult to compare paths from (and assess the relative strengths of) different methods. We introduce a comprehensive method (pathway similarity analysis, PSA) for quantitatively characterizing and comparing macromolecular pathways. The Hausdorff and Fréchet metrics (known from computational geometry) are used to quantify the degree of similarity between polygonal curves in configuration space. A strength of PSA is its use of the full information available from the 3 N-dimensional configuration space trajectory without requiring additional specific knowledge about the system. We compare a sample of eleven different methods for the closed-to-open transitions of the apo enzyme adenylate kinase (AdK) and also apply PSA to an ensemble of 400 AdK trajectories produced by dynamic importance sampling MD and the Geometrical Pathways algorithm. We discuss the method's potential to enhance our understanding of transition path sampling methods, validate them, and help guide future research toward deeper physical insights into conformational transitions.

  20. Conformal inflation coupled to matter

    SciTech Connect

    Brax, Philippe

    2014-05-01

    We formulate new conformal models of inflation and dark energy which generalise the Higgs-Dilaton scenario. We embed these models in unimodular gravity whose effect is to break scale invariance in the late time Universe. In the early Universe, inflation occurs close to a maximum of both the scalar potential and the scalar coupling to the Ricci scalar in the Jordan frame. At late times, the dilaton, which decouples from the dynamics during inflation, receives a potential term from unimodular gravity and leads to the acceleration of the Universe. We address two central issues in this scenario. First we show that the Damour-Polyalov mechanism, when non-relativistic matter is present prior to the start of inflation, sets the initial conditions for inflation at the maximum of the scalar potential. We then show that conformal invariance implies that matter particles are not coupled to the dilaton in the late Universe at the classical level. When fermions acquire masses at low energy, scale invariance is broken and quantum corrections induce a coupling between the dilaton and matter which is still small enough to evade the gravitational constraints in the solar system.

  1. The structure of apo tryptophanase from Escherichia coli reveals a wide-open conformation.

    PubMed

    Tsesin, Natalia; Kogan, Anna; Gdalevsky, Garik Y; Himanen, Juha Pekka; Cohen-Luria, Rivka; Parola, Abraham H; Goldgur, Yehuda; Almog, Orna

    2007-09-01

    The crystal structure of apo tryptophanase from Escherichia coli (space group F222, unit-cell parameters a = 118.4, b = 120.1, c = 171.2 A) was determined at 1.9 A resolution using the molecular-replacement method and refined to an R factor of 20.3% (R(free) = 23.2%). The structure revealed a significant shift in the relative orientation of the domains compared with both the holo form of Proteus vulgaris tryptophanase and with another crystal structure of apo E. coli tryptophanase, reflecting the internal flexibility of the molecule. Domain shifts were previously observed in tryptophanase and in the closely related enzyme tyrosine phenol-lyase, with the holo form found in an open conformation and the apo form in either an open or a closed conformation. Here, a wide-open conformation of the apo form of tryptophanase is reported. A conformational change is also observed in loop 297-303. The structure contains a hydrated Mg(2+) at the cation-binding site and a Cl(-) ion at the subunit interface. The enzyme activity depends