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Sample records for active site protonation

  1. Key Role of Active-Site Water Molecules in Bacteriorhodopsin Proton-Transfer Reactions

    SciTech Connect

    Bondar, A.N.; Baudry, Jerome Y; Suhai, Sandor; Fischer, S.; Smith, Jeremy C

    2008-10-01

    The functional mechanism of the light-driven proton pump protein bacteriorhodopsin depends on the location of water molecules in the active site at various stages of the photocycle and on their roles in the proton-transfer steps. Here, free energy computations indicate that electrostatic interactions favor the presence of a cytoplasmic-side water molecule hydrogen bonding to the retinal Schiff base in the state preceding proton transfer from the retinal Schiff base to Asp85. However, the nonequilibrium nature of the pumping process means that the probability of occupancy of a water molecule in a given site depends both on the free energies of insertion of the water molecule in this and other sites during the preceding photocycle steps and on the kinetic accessibility of these sites on the time scale of the reaction steps. The presence of the cytoplasmic-side water molecule has a dramatic effect on the mechanism of proton transfer: the proton is channeled on the Thr89 side of the retinal, whereas the transfer on the Asp212 side is hindered. Reaction-path simulations and molecular dynamics simulations indicate that the presence of the cytoplasmic-side water molecule permits a low-energy bacteriorhodopsin conformer in which the water molecule bridges the twisted retinal Schiff base and the proton acceptor Asp85. From this low-energy conformer, proton transfer occurs via a concerted mechanism in which the water molecule participates as an intermediate proton carrier.

  2. The controlled relay of multiple protons required at the active site of nitrogenase.

    PubMed

    Dance, Ian

    2012-07-01

    The enzyme nitrogenase, when reducing natural and unnatural substrates, requires large numbers of protons per chemical catalytic cycle. The active face of the catalytic site (the FeMo-cofactor, FeMo-co) is situated in a protein domain which is largely hydrophobic and anhydrous, and incapable of serial provision of multiple protons. Through detailed analysis of the high quality protein crystal structures available the characteristics of a chain of water molecules leading from the protein surface to a key sulfur atom (S3B) of FeMo-co are described. The first half of the water chain from the surface inwards is branched, slightly variable, and able to accommodate exogenous small molecules: this is dubbed the proton bay. The second half, from the proton bay to S3B, is comprised of a single chain of eight hydrogen bonded water molecules. This section is strictly conserved, and is intimately involved in hydrogen bonds with homocitrate, an essential component that chelates Mo. This is the proton wire, and a detailed Grotthuss mechanism for serial translocation of protons through this proton wire to S3B is proposed. This controlled serial proton relay from the protein surface to S3B is an essential component of the intramolecular hydrogenation paradigm for the complete chemical mechanisms of nitrogenase. Each proton reaching S3B, instigated by electron transfer to FeMo-co, becomes a hydrogen atom that migrates to other components of the active face of FeMo-co and to bound substrates and intermediates, allowing subsequent multiple proton transfers along the proton wire. Experiments to test the proposed mechanism of proton supply are suggested. The water chain in nitrogenase is comparable with the purported proton pumping pathway of cytochrome c oxidase.

  3. Intramembrane Proton Binding Site Linked to Activation of Bacterial Pentameric Ion Channel*

    PubMed Central

    Wang, Hai-Long; Cheng, Xiaolin; Sine, Steven M.

    2012-01-01

    Prokaryotic orthologs of eukaryotic Cys-loop receptor channels recently emerged as structural and mechanistic surrogates to investigate this superfamily of intercellular signaling proteins. Here, we examine proton activation of the prokaryotic ortholog GLIC using patch clamp electrophysiology, mutagenesis, and molecular dynamics (MD) simulations. Whole-cell current recordings from human embryonic kidney (HEK) 293 cells expressing GLIC show half-maximal activation at pH 6, close to the pKa of histidine, implicating the three native His residues in proton sensing linked to activation. The mutation H235F abolishes proton activation, H277Y is without effect, and all nine mutations of His-127 prevent expression on the cell surface. In the GLIC crystal structure, His-235 on transmembrane (TM) α-helix 2, hydrogen bonds to the main chain carbonyl oxygen of Ile-259 on TM α-helix 3. MD simulations show that when His-235 is protonated, the hydrogen bond persists, and the channel remains in the open conformation, whereas when His-235 is deprotonated, the hydrogen bond dissociates, and the channel closes. Mutations of the proximal Tyr-263, which also links TM α-helices 2 and 3 via a hydrogen bond, alter proton sensitivity over a 1.5 pH unit range. MD simulations show that mutations of Tyr-263 alter the hydrogen bonding capacity of His-235. The overall findings show that His-235 in the TM region of GLIC is a novel proton binding site linked to channel activation. PMID:22084238

  4. Active site proton delivery and the lyase activity of human CYP17A1

    SciTech Connect

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G.

    2014-01-03

    equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon–carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.

  5. Limitations of the potentiometric titration technique in determining the proton active site density of goethite surfaces

    NASA Astrophysics Data System (ADS)

    Lützenkirchen, Johannes; Boily, Jean-François; Lövgren, Lars; Sjöberg, Staffan

    2002-10-01

    Density of proton active surface sites at mineral surfaces is a property of fundamental importance in equilibrium modeling of surface complexation reactions. In this article, methods for an experimental determination of these sites at the surface of α-FeOOH (goethite) are explored. It is shown that previously obtained saturation data of goethite with respect to protons do not yield a site density that can be considered as an intrinsic sorbent property: the results are below crystallographically expected values and values for different ionic media in terms of composition and concentration yield different numbers - for example, chloride would yield higher values than nitrate at the same concentration, and higher electrolyte concentration would favor higher apparent maxima. Although site saturation might be explained by electrostatic repulsion, which is more efficient at high electrolyte concentration or for certain ions, further independent experimental results show that no saturation occurs on goethite down to ph ≡ -log[H +] = 2.2 and possibly to ph = 1.0 in 0.6 M NaCl. For those very low pH values, the experimental charging curve was obtained by coulometric back titration (using the Gran plot) or titrations with tris (hydroxymethyl)-aminomethane of the supernatant of acidified goethite suspension. These experimental data are to our knowledge the first high quality data at such low pHs. However, small errors in the determination of proton concentrations (1%) are shown to strongly affect the shape of the charging curve for ph < 2. Furthermore, goethite dissolution (proton consumption and iron reduction in coulometric titrations) and liquid junction effects interfere at low ph, hampering the straightforward application of coulometric Gran titrations over the whole pH range. From these experiments, it can nonetheless be ascertained that a minimum of 2.5 protons/nm 2 can be adsorbed at the goethite surface from the point of zero charge (ph 9.4) to pH 0.9. Although

  6. Degenerin sites mediate proton activation of deltabetagamma-epithelial sodium channel.

    PubMed

    Ji, Hong-Long; Benos, Dale J

    2004-06-25

    The delta-subunit of epithelial Na(+) channels (ENaC) is predominately expressed in brain, heart, and pancreas. The amiloride sensitivity, Na(+) conductance, and critical domains for gating are characterized as a cross between proton-activated Na(+) channels and alpha-ENaC. The hypothesis that external protons may activate human delta-ENaC was addressed by expressing deltabetagamma-hENaC in Xenopus oocytes and evaluating proton-activated current with the two-electrode voltage clamp technique. Our results showed that protons transiently evoked a Na(+) current with an EC(50) of pH 6 overlapped on the basal current of deltabetagamma-hENaC. Proton-activated current was not observed in uninjected oocytes. Studies on gating kinetics revealed that activation, desensitization, and recovery times of proton-activated Na(+) current were 3.8 +/- 0.5 s, 253 +/- 9.5 s, and 10 +/- 3.6 s, respectively (n = 4-12). Alkali metal cation selectivity of the proton-activated current was identical to that of the basal current of deltabetagamma-hENaC. The metabolic acids, lactate, pyruvate, and formate, modified the proton-activated current, as did hypo-osmotic stress. EDTA, hypo-osmolarity, and lactate enhanced proton activation synergistically. Our results suggest that delta-hENaC subunit is essential for proton-activated current and gamma-subunit may potentially regulate the response of delta-hENaC to protons. We have concluded that deltabetagamma-hENaC is a proton-activated cation channel whose closing gate can be regulated by a proton-induced conformational change. Proton-sensitivity of deltabetagamma-hENaC may be an important mechanism for integrating external ischemic signals in inflamed and hypoxic tissues. PMID:15084585

  7. Proton nuclear Overhauser effect study of the heme active site structure of Coprinus macrorhizus peroxidase.

    PubMed

    Dugad, L B; Goff, H M

    1992-07-13

    Proton nuclear Overhauser effect and paramagnetic relaxation measurements have been used to define more extensively the heme active site structure of Coprinus macrorhizus peroxidase, CMP (previously known as Coprinus cinereus peroxidase), as the ferric low-spin cyanide ligated complex. The results are compared with other well-characterized peroxidase enzymes. The NMR spectrum of CMPCN shows changes in the paramagnetically shifted resonances as a function of time, suggesting a significant heme disorder for CMP. The presence of proximal and distal histidine amino acid residues are common to the heme environments of both CMPCN and HRPCN. However, the upfield distal arginine signals of HRPCN are not evident in the 1H-NMR spectra of CMPCN.

  8. 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

  9. How active site protonation state influences the reactivity and ligation of the heme in chlorite dismutase

    PubMed Central

    Streit, Bennett R.; Blanc, Béatrice; Lukat-Rodgers, Gudrun S.; Rodgers, Kenton R.; DuBois, Jennifer L.

    2010-01-01

    Chlorite dismutase catalyzes O2 release from chlorite with exquisite efficiency and specificity. The spectroscopic properties, ligand binding affinities, and steady state kinetics of chlorite dismutase from Dechloromonas aromatica were examined over pH 3–11.5 to gain insight into how the protonation state of the heme environment influences dioxygen formation. An acid/base transition was observed by UV/visible and resonance Raman spectroscopy with a pKa of 8.7, 2–3 pH units below analogous transitions observed in typical His-ligated peroxidases. This transition marks the conversion of a five coordinate high spin Fe(III) to a mixed high/low spin ferric-hydroxide, as confirmed by resonance Raman (rR) spectroscopy. The two Fe–OH stretching frequencies are quite low, consistent with a weak Fe–OH bond, despite the nearly neutral imidazole side chain of the proximal histidine ligand. The hydroxide is proposed to interact strongly with a distal H-bond donor, thereby weakening the Fe–OH bond. The rR spectra of Cld-CO as a function of pH reveal two forms of the complex, one in which there is minimal interaction of distal residues with the carbonyl oxygen and another, acidic form in which the oxygen is under the influence of positive charge. Recent crystallographic data reveal arginine 183 as the lone H-bond donating residue in the distal pocket. It is likely that this Arg is the strong, positively charged H-bond donor implicated by vibrational data to interact with exogenous axial heme ligands. The same Arg in its neutral (pKa ~ 6.5) form also appears to act as the active site base in binding reactions of protonated ligands, such as HCN, to ferric Cld. The steady state profile for the rate of chlorite decomposition is characterized by these same pKas. The 5 coordinate high spin acidic Cld is more active than the alkaline hydroxide-bound form. The acid form decomposes chlorite most efficiently when the distal Arg is protonated/cationic (maximum kcat = 2.0 (±0.6)

  10. Distinguishing two groups of flavin reductases by analyzing the protonation state of an active site carboxylic acid.

    PubMed

    Dumit, Verónica I; Cortez, Néstor; Matthias Ullmann, G

    2011-07-01

    Flavin-containing reductases are involved in a wide variety of physiological reactions such as photosynthesis, nitric oxide synthesis, and detoxification of foreign compounds, including therapeutic drugs. Ferredoxin-NADP(H)-reductase (FNR) is the prototypical enzyme of this family. The fold of this protein is highly conserved and occurs as one domain of several multidomain enzymes such as the members of the diflavin reductase family. The enzymes of this family have emerged as fusion of a FNR and a flavodoxin. Although the active sites of these enzymes are very similar, different enzymes function in opposite directions, that is, some reduce oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)) and some oxidize reduced nicotinamide adenine dinucleotide phosphate (NADPH). In this work, we analyze the protonation behavior of titratable residues of these enzymes through electrostatic calculations. We find that a highly conserved carboxylic acid in the active site shows a different titration behavior in different flavin reductases. This residue is deprotonated in flavin reductases present in plastids, but protonated in bacterial counterparts and in diflavin reductases. The protonation state of the carboxylic acid may also influence substrate binding. The physiological substrate for plastidic enzymes is NADP(+), but it is NADPH for the other mentioned reductases. In this article, we discuss the relevance of the environment of this residue for its protonation and its importance in catalysis. Our results allow to reinterpret and explain experimental data. PMID:21538544

  11. The pathway for serial proton supply to the active site of nitrogenase: enhanced density functional modeling of the Grotthuss mechanism.

    PubMed

    Dance, Ian

    2015-11-01

    Nitrogenase contains a well defined and conserved chain of water molecules leading to the FeMo cofactor (FeMo-co, an [Fe7MoCS9] cluster with bidentate chelation of Mo by homocitrate) that is the active site where N2 and other substrates are sequentially hydrogenated using multiple protons and electrons. The function of this chain is proposed to be a proton wire, serially translocating protons to triply-bridging S3B of FeMo-co, where, concomitant with electron transfer to FeMo-co, an H atom is generated on S3B. Density functional simulations of this proton translocation mechanism are reported here, using a large 269-atom model that includes all residues hydrogen bonded to and surrounding the water chain, and likely to influence proton transfer: three carboxylate O atoms of obligatory homocitrate are essential. The mechanism involves the standard two components of the Grotthuss mechanism, namely H atom slides that shift H3O(+) from one water site to the next, and HOH molecular rotations that convert backward (posterior) OH bonds in the water chain to forward (anterior) OH bonds. The topography of the potential energy surface for each of these steps has been mapped. H atom slides pass through very short (ca. 2.5 Å) O-H-O hydrogen bonds, while HOH rotations involve the breaking of O-HO hydrogen bonds, and the occurrence of long (up to 3.6 Å) separations between contiguous water molecules. Both steps involve low potential energy barriers, <7 kcal mol(-1). During operation of the Grotthuss mechanism in nitrogenase there are substantial displacements of water molecules along the chain, occurring as ripples. These characteristics of the 'Grotthuss two-step', coupled with a buffering ability of two carboxylate O atoms of homocitrate, and combined with density functional characterisation of the final proton slide from the ultimate water molecule to S3B (including electron addition), have been choreographed into a complete mechanism for serial hydrogenation of FeMo-co. The

  12. Multiple active site histidine protonation states in Acetobacter aceti N5-carboxyaminoimidazole ribonucleotide mutase detected by REDOR NMR.

    PubMed

    Schaefer, Jacob; Jiang, Hong; Ransome, Aaron E; Kappock, T Joseph

    2007-08-21

    Class I PurE (N5-carboxyaminoimidazole mutase) catalyzes a chemically unique mutase reaction. A working mechanistic hypothesis involves a histidine (His45 in Escherichia coli PurE) functioning as a general acid, but no evidence for multiple protonation states has been obtained. Solution NMR is a peerless tool for this task but has had limited application to enzymes, most of which are larger than its effective molecular size limit. Solid-state NMR is not subject to this limit. REDOR NMR studies of a 151 kDa complex of uniformly 15N-labeled Acetobacter aceti PurE (AaPurE) and the active site ligand [6-13C]citrate probed a single ionization equilibrium associated with the key histidine (AaPurE His59). In the AaPurE complex, the citrate central carboxylate C6 13C peak moves upfield, indicating diminution of negative charge, and broadens, indicating heterogeneity. Histidine 15N chemical shifts indicate His59 exists in approximately equimolar amounts of an Ndelta-unprotonated (pyridine-like) form and an Ndelta-protonated (pyrrole-like) form, each of which is approximately 4 A from citrate C6. The spectroscopic data are consistent with proton transfers involving His59 Ndelta that are invoked in the class I PurE mechanism.

  13. Proton NMR investigation of the heme active site structure of an engineered cytochrome c peroxidase that mimics manganese peroxidase.

    PubMed

    Wang, X; Lu, Y

    1999-07-13

    The heme active site structure of an engineered cytochrome c peroxidase [MnCcP; see Yeung, B. K., et al. (1997) Chem. Biol. 4, 215-221] that closely mimics manganese peroxidase (MnP) has been characterized by both one- and two-dimensional NMR spectroscopy. All hyperfine-shifted resonances from the heme pocket as well as resonances from catalytically relevant amino acid residues in the congested diamagnetic envelope have been assigned. From the NMR spectral assignment and the line broadening pattern of specific protons in NOESY spectra of MnCcP, the location of the engineered Mn(II) center is firmly identified. Furthermore, we found that the creation of the Mn(II)-binding site in CcP resulted in no detectable structural changes on the distal heme pocket of the protein. However, notable structural changes are observed at the proximal side of the heme cavity. Both CepsilonH shift of the proximal histidine and (15)N shift of the bound C(15)N(-) suggest a weaker heme Fe(III)-N(His) bond in MnCcP compared to WtCcP. Our results indicate that the engineered Mn(II)-binding site in CcP resulted in not only a similar Mn(II)-binding affinity and improved MnP activity, but also weakened the Fe(III)-N(His) bond strength of the template protein CcP so that its bond strength is similar to that of the target protein MnP. The results presented here help elucidate the impact of designing a metal-binding site on both the local and global structure of the enzyme, and provide a structural basis for engineering the next generation of MnCcP that mimics MnP more closely. PMID:10413489

  14. Fluorophenyl-substituted Fe-only hydrogenases active site ADT models: different electrocatalytic process for proton reduction in HOAc and HBF4/Et2O.

    PubMed

    Wang, Wen-Guang; Wang, Hong-Yan; Si, Gang; Tung, Chen-Ho; Wu, Li-Zhu

    2009-04-21

    A set of fluorophenyl-substituted adt-bridged Fe2S2 active site models of Fe-only hydrogenase, [(micro-SCH2)2NR]Fe2(CO)6 (, R=C6F4CF3-p; , R=C6H4CF3-p) and [(micro-SCH2)2NR]Fe2(CO)5(PPh3) (, R=C6F4CF3-p; , R=C6H4CF3-p), have been synthesized and well characterized. Spectroscopic and electrochemical studies demonstrate that the aryl-substituted complexes are stable toward a strong acid HBF4/Et2O, and electrocatalytic process for the hydrogen production is mostly dependent on the strength of the available proton source. When CH3COOH is used as the proton source, the electrocatalytic process begins with successively two one-electron reduction processes to produce H2 at Fe(0)Fe(0) (E2pc); whereas in the presence of strong acid, HBF4/Et2O, the process is initiated by protonation of a N-bridged atom followed by reduction of the protonated N-bridged atom around -1.29 V, and then release of H2 at Fe(0)Fe(I) (E1pc). Varying the strength of acid leads to the initial electron-transfer step from the reduction of a protonated N-bridged atom to the active site of [Fe(I)Fe(I)].

  15. Kinetic isotope effects for concerted multiple proton transfer: a direct dynamics study of an active-site model of carbonic anhydrase II.

    PubMed

    Smedarchina, Zorka; Siebrand, Willem; Fernández-Ramos, Antonio; Cui, Qiang

    2003-01-01

    The rate constant of the reaction catalyzed by the enzyme carbonic anhydrase II, which removes carbon dioxide from body fluids, is calculated for a model of the active site. The rate-determining step is proton transfer from a zinc-bound water molecule to a histidine residue via a bridge of two or more water molecules. The structure of the active site is known from X-ray studies except for the number and location of the water molecules. Model calculations are reported for a system of 58 atoms including a four-coordinated zinc ion connected to a methylimidazole molecule by a chain of two waters, constrained to reproduce the size of the active site. The structure and vibrational force field are calculated by an approximate density functional treatment of the proton-transfer step at the Self-Consistent-Charge Density Functional Tight Binding (SCC-DFTB) level. A single transition state is found indicating concerted triple proton transfer. Direct-dynamics calculations for proton and deuteron transfer and combinations thereof, based on the Approximate Instanton Method and on Variational Transition State Theory with Tunneling Corrections, are in fair agreement and yield rates that are considerably higher and kinetic isotope effects (KIEs) that are somewhat higher than experiment. Classical rate constants obtained from Transition State Theory are smaller than the quantum values but the corresponding KIEs are five times larger. For multiple proton transfer along water bridges classical KIEs are shown to be generally larger than quantum KIEs, which invalidates the standard method to distinguish tunneling and over-barrier transfer. In the present case, a three-way comparison of classical and quantum results with the observed data is necessary to conclude that proton transfer along the bridge proceeds by tunneling. The results suggest that the two-water bridge is present in low concentrations but makes a substantial contribution to proton transport because of its high

  16. Threonine-89 participates in the active site of bacteriorhodopsin: evidence for a role in color regulation and Schiff base proton transfer.

    PubMed

    Russell, T S; Coleman, M; Rath, P; Nilsson, A; Rothschild, K J

    1997-06-17

    Bacteriorhodopsin (bR) functions as a light-driven proton pump in the purple membrane of Halobacterium salinarium. A major feature of bR is the existence of an active site which includes a retinylidene Schiff base and amino acid residues Asp-85, Asp-212, and Arg-82. This active site participates in proton transfers and regulates the visible absorption of bacteriorhodopsin and its photointermediates. In this work we find evidence that Thr-89 also participates in this active site. The substitution Thr-89 --> Asn (T89N) results in changes in the properties of the all-trans retinylidene chromophore of light-adapted bR including a redshift of the visible lambda(max) and a downshift in C=N and C=C stretch frequencies. Changes are also found in the M and N intermediates of the T89N photocycle including shifts in lambda(max), a downshift of the Asp-85 carboxylic acid C=O stretch frequency by 10 cm(-1), and a 3-5-fold decrease in the rate of formation of the M intermediate. In contrast, the properties of the 13-cis retinylidene chromophore of dark-adapted T89N as well as the K and L intermediates of the T89N photocycle are similar to the wild-type bacteriorhodopsin. These results are consistent with an interaction of the hydroxyl group of Thr-89 with the protonated Schiff base of light-adapted bR and possibly the N intermediate but not the 13-cis chromophore of dark-adapted bR or the K and L intermediates. Thr-89 also appears to influence the rate of Schiff base proton transfer to Asp-85 during formation of the M intermediate, possibly through an interaction with Asp-85. In contrast, the hydroxyl group of Thr-89 is not obligatory for proton transfer from Asp-96 to the Schiff base during formation of the N intermediate.

  17. Site and mechanism of activation of proton-induced sodium current in chick dorsal root ganglion neurones.

    PubMed Central

    Davies, N W; Lux, H D; Morad, M

    1988-01-01

    1. In dissociated and cultured 1- to 2-day-old chick dorsal root ganglion cells, and in isolated outside-out membrane patches, a large transient current lasting 1-2 s could be activated upon step increases in [H+]o. The proton-induced current reversed direction at the Na+ equilibrium potential, was abolished completely in the absence of Na+, and was therefore labelled INa(H). 2. To investigate the activation and deactivation kinetics of INa(H) at the single-channel level, we employed isolated membrane patches and a method whereby we could change the external solution in less than 1 ms. 3. In outside-out membrane patches, INa(H) was fully activated within 2 ms between pH 6.7 and 5.7. Half-times of activation decreased with increasing [H+]o. The calculated association rate constant was 9.5 x 10(9) M-1 s-1. 4. Deactivation of INa(H), following a step reduction in [H+]o, occurred with half-times of within 1.3-2 ms. 5. In the continued presence of an activating solution (pH 6.7 and 1 mM-Ca2+), INa(H) inactivated slowly, with a time constant of about 300 ms. 6. Inactivation showed a limited dependence on [Ca2+]o. The time constant of inactivation increased from about 300-500 ms as [Ca2+]o was decreased from 5 to 0.1 mM. Further decrease in [Ca2+]o did not significantly increase the time course of inactivation. Increases in [Ca2+]i from 10(-9) to 10(-3) M had no effect on the activation or inactivation kinetics of INa(H). 7. Conditioning proton concentrations which by themselves failed to activate any channel openings, partially inactivated INa(H). 8. Recovery from inactivation appeared to follow a time course similar to that of inactivation itself. 9. INa(H) could not be activated in inside-out patches. A step increase in proton concentration outside a cell-attached patch was also ineffective at producing INa(H) in the patch. Intracellular pH between 7.9 and 6.7 had no effect on the activation or inactivation of INa(H). 10. The activation and inactivation kinetics were

  18. Active interrogation using energetic protons

    SciTech Connect

    Morris, Christopher L; Chung, Kiwhan; Greene, Steven J; Hogan, Gary E; Makela, Mark; Mariam, Fesseha; Milner, Edward C; Murray, Matthew; Saunders, Alexander; Spaulding, Randy; Wang, Zhehui; Waters, Laurie; Wysocki, Frederick

    2010-01-01

    Energetic proton beams provide an attractive alternative when compared to electromagnetic and neutron beams for active interrogation of nuclear threats because they have large fission cross sections, long mean free paths and high penetration, and they can be manipulated with magnetic optics. We have measured time-dependent cross sections and neutron yields for delayed neutrons and gamma rays using 800 MeV and 4 GeV proton beams with a set of bare and shielded targets. The results show significant signals from both unshielded and shielded nuclear materials. Measurements of neutron energies yield suggest a signature unique to fissile material. Results are presented in this paper.

  19. Protonation States of the Tryptophan Synthase Internal Aldimine Active Site from Solid-State NMR Spectroscopy: Direct Observation of the Protonated Schiff Base Linkage to Pyridoxal-5′-Phosphate

    PubMed Central

    2015-01-01

    The acid–base chemistry that drives catalysis in pyridoxal-5′-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP’s role as a coenzyme in this extensive class of enzymes. It was first proposed over 50 years ago that the initial step in the catalytic cycle is facilitated by a protonated Schiff base form of the holoenzyme in which the linking lysine ε-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first 15N NMR chemical shift measurements of such a Schiff base linkage in the resting holoenzyme form, the internal aldimine state of tryptophan synthase. Double-resonance experiments confirm the assignment of the Schiff base nitrogen, and additional 13C, 15N, and 31P chemical shift measurements of sites on the PLP coenzyme allow a detailed model of coenzyme protonation states to be established. PMID:25148001

  20. ⁵¹V NMR Crystallography of Vanadium Chloroperoxidase and Its Directed Evolution P395D/L241V/T343A Mutant: Protonation Environments of the Active Site.

    PubMed

    Gupta, Rupal; Hou, Guangjin; Renirie, Rokus; Wever, Ron; Polenova, Tatyana

    2015-04-29

    Vanadium-dependent haloperoxidases (VHPOs) perform two-electron oxidation of halides using hydrogen peroxide. Their mechanism, including the factors determining the substrate specificity and the pH-dependence of the catalytic rates, is poorly understood. The vanadate cofactor in the active site of VHPOs contains "spectroscopically silent" V(V), which does not change oxidation state during the reaction. We employed an NMR crystallography approach based on (51)V magic angle spinning NMR spectroscopy and Density Functional Theory, to gain insights into the structure and coordination environment of the cofactor in the resting state of vanadium-dependent chloroperoxidases (VCPO). The cofactor environments in the wild-type VCPO and its P395D/L241V/T343A mutant exhibiting 5-100-fold improved catalytic activity are examined at various pH values. Optimal sensitivity attained due to the fast MAS probe technologies enabled the assignment of the location and number of protons on the vanadate as a function of pH. The vanadate cofactor changes its protonation from quadruply protonated at pH 6.3 to triply protonated at pH 7.3 to doubly protonated at pH 8.3. In contrast, in the mutant, the vanadate protonation is the same at pH 5.0 and 8.3, and the cofactor is doubly protonated. This methodology to identify the distinct protonation environments of the cofactor, which are also pH-dependent, could help explain the different reactivities of the wild-type and mutant VCPO and their pH-dependence. This study demonstrates that (51)V-based NMR crystallography can be used to derive the detailed coordination environments of vanadium centers in large biological molecules.

  1. Characterizing the proton loading site in cytochrome c oxidase.

    PubMed

    Lu, Jianxun; Gunner, M R

    2014-08-26

    Cytochrome c oxidase (CcO) uses the energy released by reduction of O2 to H2O to drive eight charges from the high pH to low pH side of the membrane, increasing the electrochemical gradient. Four electrons and protons are used for chemistry, while four more protons are pumped. Proton pumping requires that residues on a pathway change proton affinity through the reaction cycle to load and then release protons. The protonation states of all residues in CcO are determined in MultiConformational Continuum Electrostatics simulations with the protonation and redox states of heme a, a3, Cu(B), Y288, and E286 used to define the catalytic cycle. One proton is found to be loaded and released from residues identified as the proton loading site (PLS) on the P-side of the protein in each of the four CcO redox states. Thus, the same proton pumping mechanism can be used each time CcO is reduced. Calculations with structures of Rhodobacter sphaeroides, Paracoccus denitrificans, and bovine CcO derived by crystallography and molecular dynamics show the PLS functions similarly in different CcO species. The PLS is a cluster rather than a single residue, as different structures show 1-4 residues load and release protons. However, the proton affinity of the heme a3 propionic acids primarily determines the number of protons loaded into the PLS; if their proton affinity is too low, less than one proton is loaded.

  2. Proton transfer roles of lysine 64 and glutamic acid 64 replacing histidine 64 in the active site of human carbonic anhydrase II.

    PubMed

    Engstrand, C; Forsman, C; Liang, Z; Lindskog, S

    1992-08-21

    The CO2 hydration activities of cloned human carbonic anhydrase II (carbonate hydro-lyase, EC 4.2.1.1) and variants with Lys, Glu, Gln or Ala replacing His at sequence position 64 have been measured in a variety of different buffers in the pH range 6-9. The variants with Lys-64, Gln-64 and Ala-64 showed non-Michaelis-Menten behavior under some conditions, apparent substrate inhibition being prominent near pH 9. However, asymptotic Michaelis-Menten parameters could be estimated for the limit of low substrate concentrations. All variants show distinct buffer specificities, and imidazole derivatives, Ches and phosphate buffers yield higher kcat values that Bicine, Taps and Mops buffers under otherwise similar conditions. These results are interpreted in terms of different pathways for a rate-limiting proton transfer. In unmodified enzyme, the very high catalytic activity depends on His-64 functioning as an efficient proton transfer group, but this pathway is not available in the variants with Gln-64 and Ala-64. Imidazoles, Ches and phosphate are thought to participate in a metal center-to-buffer proton transfer pathway, whereas Bicine, Taps, Mops and Mes appear to lack this capacity, so that the rate-limiting proton transfer occurs in a metal center-to-bulk water pathway for these variants. The Lys-64 and Glu-64 variants give significantly higher kcat values in Taps, Mops and Mes buffers than the Ala-64 and Gln-64 variants. The pH dependencies of these kcat values are compatible with the hypothesis that Lys-64 and Glu-64 can function as proton transfer groups. Thus, at pH near 9, Lys-64 appears to be only 5-times less efficient than His-64, while Glu-64 is inefficient. At pH 6, Lys-64 is an inefficient proton transfer group, but Glu-64 is only 2-3-times less efficient than His-64. The data indicate that Lys-64 and Glu-64 have pKa values near 8 and below 6, respectively.

  3. Probing the Protonation State and the Redox-Active Sites of Pendant Base Iron(II) and Zinc(II) Pyridinediimine Complexes.

    PubMed

    Delgado, Mayra; Sommer, Samantha K; Swanson, Seth P; Berger, Robert F; Seda, Takele; Zakharov, Lev N; Gilbertson, John D

    2015-08-01

    Utilizing the pyridinediimine ligand [(2,6-(i)PrC6H3)N═CMe)(N((i)Pr)2C2H4)N═CMe)C5H3N] (didpa), the zinc(II) and iron(II) complexes Zn(didpa)Cl2 (1), Fe(didpa)Cl2 (2), [Zn(Hdidpa)Cl2][PF6] (3), [Fe(Hdidpa)Cl2][PF6] (4), Zn(didpa)Br2 (5), and [Zn(Hdidpa)Br2][PF6] (6), Fe(didpa)(CO)2 (7), and [Fe(Hdidpa)(CO)2][PF6] (8) were synthesized and characterized. These complexes allowed for the study of the secondary coordination sphere pendant base and the redox-activity of the didpa ligand scaffold. The protonated didpa ligand is capable of forming metal halogen hydrogen bonds (MHHBs) in complexes 3, 4, and 6. The solution behavior of the MHHBs was probed via pKa measurements and (1)H NMR titrations of 3 and 6 with solvents of varying H-bond accepting strength. The H-bond strength in 3 and 6 was calculated in silico to be 5.9 and 4.9 kcal/mol, respectively. The relationship between the protonation state and the ligand-based redox activity was probed utilizing 7 and 8, where the reduction potential of the didpa scaffold was found to shift by 105 mV upon protonation of the reduced ligand in Fe(didpa)(CO)2.

  4. pH-Differential Spectroscopy of Dissolved Organic Matter: Contributions of Protonation-Active Functionalities and Their Site-Specificity (Invited)

    NASA Astrophysics Data System (ADS)

    Korshin, G. V.; Dryer, D. J.; Benedetti, M. F.; Fabbricino, M.

    2009-12-01

    This presentation will address effects of pH on the absorbance and fluorescence emission spectra of dissolved organic matter (DOM) originating from several fluvial systems, notably the Rio Negro basin, Suwannee River and South Platte River. Changes in the absorbance spectra of DOM were quantified based on calculations of differential spectra. These are defined as the difference between the absorbance of DOM at any specified reference pH (e.g., that corresponding to nearly-total protonation of all functionalities) and that at which DOM molecules are deprotonated. A similar technique was applied to process fluorescence emission spectra. This approach significantly increased the ability to discern the presence of multiple spectroscopic bands that emerge in different pH ranges. Analysis of the evolution of the pH-differential absorbance spectra of DOM showed that they have distinct features associated with contributions of operationally defined carboxylic and phenolic chromophores, as well as those of inter-chromophore interactions. The spectroscopic manifestations of the engagement of these functionalities were site-specific, indicating effects of the size and charge of NOM molecules and prevalence of specific chemical functionalities whose nature remains to be ascertained. Comparison of the evolution of the pH-differential spectra of NOM with prediction made based on modified NICA-Donnan theory showed the general applicability of that theory and also the need to account for presence of minor functionalities that are active in the circumneutral range of pHs. This comparison also showed that the contributions of the phenolic groups in the observed changes of the absorbance and emission spectra significantly exceeded their prevalence determined via potentiometric titrations. The spectroscopic data were interpreted to indicate the pH-differential approach applied to absorbance and fluorescence measurements is a novel and effective techniques that allows examining the

  5. A delocalized proton-binding site within a membrane protein.

    PubMed

    Wolf, Steffen; Freier, Erik; Gerwert, Klaus

    2014-07-01

    The role of protein-bound water molecules in protein function and catalysis is an emerging topic. Here, we studied the solvation of an excess proton by protein-bound water molecules and the contribution of the surrounding amino acid residues at the proton release site of the membrane protein bacteriorhodopsin. It hosts an excess proton within a protein-bound water cluster, which is hydrogen bonded to several surrounding amino acids. Indicative of delocalization is a broad continuum absorbance experimentally observed by time-resolved Fourier transform infrared spectroscopy. In combination with site-directed mutagenesis, the involvement of several amino acids (especially Glu-194 and Glu-204) in the delocalization was elaborated. Details regarding the contributions of the glutamates and water molecules to the delocalization mode in biomolecular simulations are controversial. We carried out quantum mechanics/molecular mechanics (QM/MM) self-consistent charge density functional tight-binding simulations for all amino acids that have been experimentally shown to be involved in solvation of the excess proton, and systematically investigated the influence of the quantum box size. We compared calculated theoretical infrared spectra with experimental ones as a measure for the correct description of excess proton delocalization. A continuum absorbance can only be observed for small quantum boxes containing few amino acids and/or water molecules. Larger quantum boxes, including all experimentally shown involved amino acids, resulted in narrow absorbance bands, indicating protonation of a single binding site in contradiction to experimental results. We conclude that small quantum boxes seem to reproduce representative extreme cases of proton delocalization modes: proton delocalization only on water molecules or only between Glu-194 and Glu-204. Extending the experimental spectral region to lower wave numbers, a water-delocalized proton reproduces the observed continuum

  6. Long-Range Electrostatics-Induced Two-Proton Transfer Captured by Neutron Crystallography in an Enzyme Catalytic Site.

    PubMed

    Gerlits, Oksana; Wymore, Troy; Das, Amit; Shen, Chen-Hsiang; Parks, Jerry M; Smith, Jeremy C; Weiss, Kevin L; Keen, David A; Blakeley, Matthew P; Louis, John M; Langan, Paul; Weber, Irene T; Kovalevsky, Andrey

    2016-04-11

    Neutron crystallography was used to directly locate two protons before and after a pH-induced two-proton transfer between catalytic aspartic acid residues and the hydroxy group of the bound clinical drug darunavir, located in the catalytic site of enzyme HIV-1 protease. The two-proton transfer is triggered by electrostatic effects arising from protonation state changes of surface residues far from the active site. The mechanism and pH effect are supported by quantum mechanics/molecular mechanics (QM/MM) calculations. The low-pH proton configuration in the catalytic site is deemed critical for the catalytic action of this enzyme and may apply more generally to other aspartic proteases. Neutrons therefore represent a superb probe to obtain structural details for proton transfer reactions in biological systems at a truly atomic level.

  7. Long-range electrostatics-induced two-proton transfer captured by neutron crystallography in an enzyme catalytic site

    DOE PAGES

    Gerlits, Oksana; Wymore, Troy; Das, Amit; Shen, Chen -Hsiang; Parks, Jerry M.; Smith, Jeremy C.; Weiss, Kevin L.; Keen, David A.; Blakeley, Matthew P.; Louis, John M.; et al

    2016-03-09

    Neutron crystallography was used to directly locate two protons before and after a pH-induced two-proton transfer between catalytic aspartic acid residues and the hydroxy group of the bound clinical drug darunavir, located in the catalytic site of enzyme HIV-1 protease. The two-proton transfer is triggered by electrostatic effects arising from protonation state changes of surface residues far from the active site. The mechanism and pH effect are supported by quantum mechanics/molecular mechanics (QM/MM) calculations. The low-pH proton configuration in the catalytic site is deemed critical for the catalytic action of this enzyme and may apply more generally to other asparticmore » proteases. Neutrons therefore represent a superb probe to obtain structural details for proton transfer reactions in biological systems at a truly atomic level.« less

  8. Long-Range Electrostatics-Induced Two-Proton Transfer Captured by Neutron Crystallography in an Enzyme Catalytic Site.

    PubMed

    Gerlits, Oksana; Wymore, Troy; Das, Amit; Shen, Chen-Hsiang; Parks, Jerry M; Smith, Jeremy C; Weiss, Kevin L; Keen, David A; Blakeley, Matthew P; Louis, John M; Langan, Paul; Weber, Irene T; Kovalevsky, Andrey

    2016-04-11

    Neutron crystallography was used to directly locate two protons before and after a pH-induced two-proton transfer between catalytic aspartic acid residues and the hydroxy group of the bound clinical drug darunavir, located in the catalytic site of enzyme HIV-1 protease. The two-proton transfer is triggered by electrostatic effects arising from protonation state changes of surface residues far from the active site. The mechanism and pH effect are supported by quantum mechanics/molecular mechanics (QM/MM) calculations. The low-pH proton configuration in the catalytic site is deemed critical for the catalytic action of this enzyme and may apply more generally to other aspartic proteases. Neutrons therefore represent a superb probe to obtain structural details for proton transfer reactions in biological systems at a truly atomic level. PMID:26958828

  9. Activation and proton transport mechanism in influenza A M2 channel.

    PubMed

    Wei, Chenyu; Pohorille, Andrew

    2013-11-01

    Molecular dynamics trajectories 2 μs in length have been generated for the pH-activated, tetrameric M2 proton channel of the influenza A virus in all protonation states of the pH sensor located at the His(37) tetrad. All simulated structures are in very good agreement with high-resolution structures. Changes in the channel caused by progressive protonation of His(37) provide insight into the mechanism of proton transport. The channel is closed at both His(37) and Trp(41) sites in the singly and doubly protonated states, but it opens at Trp(41) upon further protonation. Anions access the charged His(37) and by doing so stabilize the protonated states of the channel. The narrow opening at the His(37) site, further blocked by anions, is inconsistent with the water-wire mechanism of proton transport. Instead, conformational interconversions of His(37) correlated with hydrogen bonding to water molecules indicate that these residues shuttle protons in high-protonation states. Hydrogen bonds between charged and uncharged histidines are rare. The valve at Val(27) remains on average quite narrow in all protonation states but fluctuates sufficiently to support water and proton transport. A proton transport mechanism in which the channel, depending on pH, opens at either the histidine or valine gate is only partially supported by the simulations. PMID:24209848

  10. X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O2-reduction states.

    PubMed

    Serrano-Posada, Hugo; Centeno-Leija, Sara; Rojas-Trejo, Sonia Patricia; Rodríguez-Almazán, Claudia; Stojanoff, Vivian; Rudiño-Piñera, Enrique

    2015-12-01

    During X-ray data collection from a multicopper oxidase (MCO) crystal, electrons and protons are mainly released into the system by the radiolysis of water molecules, leading to the X-ray-induced reduction of O2 to 2H2O at the trinuclear copper cluster (TNC) of the enzyme. In this work, 12 crystallographic structures of Thermus thermophilus HB27 multicopper oxidase (Tth-MCO) in holo, apo and Hg-bound forms and with different X-ray absorbed doses have been determined. In holo Tth-MCO structures with four Cu atoms, the proton-donor residue Glu451 involved in O2 reduction was found in a double conformation: Glu451a (∼7 Å from the TNC) and Glu451b (∼4.5 Å from the TNC). A positive peak of electron density above 3.5σ in an Fo - Fc map for Glu451a O(ℇ2) indicates the presence of a carboxyl functional group at the side chain, while its significant absence in Glu451b strongly suggests a carboxylate functional group. In contrast, for apo Tth-MCO and in Hg-bound structures neither the positive peak nor double conformations were observed. Together, these observations provide the first structural evidence for a proton-relay mechanism in the MCO family and also support previous studies indicating that Asp106 does not provide protons for this mechanism. In addition, eight composite structures (Tth-MCO-C1-8) with different X-ray-absorbed doses allowed the observation of different O2-reduction states, and a total depletion of T2Cu at doses higher than 0.2 MGy showed the high susceptibility of this Cu atom to radiation damage, highlighting the importance of taking radiation effects into account in biochemical interpretations of an MCO structure.

  11. Neutron and proton activation measurements from Skylab

    NASA Technical Reports Server (NTRS)

    Fishman, G. J.

    1974-01-01

    Radioactivity induced by high-energy protons and secondary neutrons (from nuclear interactions) in various samples returned from different locations in Skylab was measured directly by gamma-ray spectroscopy measurements of decay gamma rays from the samples. Incident fluxes were derived from the activation measurements, using known nuclear cross-section. Neutron and proton flux values were found to range from 0.2 to 5 particles/sq cm-sec, depending on the energy range and location in Skylab. The thermal neutron flux was less than 0.07 neutrons/sq cm-sec. The results are useful for data analysis and planning of future high-energy astronomy experiments.

  12. Electron transfer activation of a second water channel for proton transport in [FeFe]-hydrogenase

    SciTech Connect

    Sode, Olaseni; Voth, Gregory A.

    2014-12-14

    Hydrogenase enzymes are important because they can reversibly catalyze the production of molecular hydrogen. Proton transport mechanisms have been previously studied in residue pathways that lead to the active site of the enzyme via residues Cys299 and Ser319. The importance of this pathway and these residues has been previously exhibited through site-specific mutations, which were shown to interrupt the enzyme activity. It has been shown recently that a separate water channel (WC2) is coupled with electron transport to the active site of the [FeFe]-hydrogenase. The water-mediated proton transport mechanisms of the enzyme in different electronic states have been studied using the multistate empirical valence bond reactive molecular dynamics method, in order to understand any role WC2 may have in facilitating the residue pathway in bringing an additional proton to the enzyme active site. In a single electronic state A{sup 2−}, a water wire was formed through which protons can be transported with a low free energy barrier. The remaining electronic states were shown, however, to be highly unfavorable to proton transport in WC2. A double amino acid substitution is predicted to obstruct proton transport in electronic state A{sup 2-} by closing a cavity that could otherwise fill with water near the proximal Fe of the active site.

  13. Proton and cation transport activity of the M2 proton channel from influenza A virus.

    PubMed

    Leiding, Thom; Wang, Jun; Martinsson, Jonas; DeGrado, William F; Arsköld, Sindra Peterson

    2010-08-31

    The M2 protein is a small, single-span transmembrane (TM) protein from the influenza A virus. This virus enters cells via endosomes; as the endosomes mature and become more acidic M2 facilitates proton transport into the viral interior, thereby disrupting matrix protein/RNA interactions required for infectivity. A mystery has been how protons can accumulate in the viral interior without developing a large electrical potential that impedes further inward proton translocation. Progress in addressing this question has been limited by the availability of robust methods of unidirectional insertion of the protein into virus-like vesicles. Using an optimized procedure for reconstitution, we show that M2 has antiporter-like activity, facilitating K(+) or Na(+) efflux when protons flow down a concentration gradient into the vesicles. Cation efflux is very small except under conditions mimicking those encountered by the endosomally entrapped virus, in which protons are flowing through the channel. This proton/cation exchange function is consistent with the known high proton selectivity of the channel. Thus, M2 acts as a proton uniporter that occasionally allows K(+) to flow to maintain electrical neutrality. Remarkably, as the pH inside M2-containing vesicles (pH(in)) decreases, the proton channel activity of M2 is inhibited, but its cation transport activity is activated. This reciprocal inhibition of proton flux and activation of cation flux with decreasing pH(in) first allows accumulation of protons in the early stages of acidification, then trapping of protons within the virus when low pH(in) is achieved.

  14. Equilibrium constants and protonation site for N-methylbenzenesulfonamides

    PubMed Central

    Rosa da Costa, Ana M; García-Río, Luis; Pessêgo, Márcia

    2011-01-01

    Summary The protonation equilibria of four substituted N-methylbenzenesulfonamides, X-MBS: X = 4-MeO (3a), 4-Me (3b), 4-Cl (3c) and 4-NO2 (3d), in aqueous sulfuric acid were studied at 25 °C by UV–vis spectroscopy. As expected, the values for the acidity constants are highly dependent on the electron-donor character of the substituent (the pK BH+ values are −3.5 ± 0.2, −4.2 ± 0.2, −5.2 ± 0.3 and −6.0 ± 0.3 for 3a, 3b, 3c and 3d, respectively). The solvation parameter m* is always higher than 0.5 and points to a decrease in the importance of solvation on the cation stabilization as the electron-donor character of the substituent increases. Hammett plots of the equilibrium constants showed a better correlation with the σ+ substituent parameter than with σ, which indicates that the initial protonation site is the oxygen atom of the sulfonyl group. PMID:22238552

  15. External protons destabilize the activated voltage sensor in hERG channels.

    PubMed

    Shi, Yu Patrick; Cheng, Yen May; Van Slyke, Aaron C; Claydon, Tom W

    2014-03-01

    Extracellular acidosis shifts hERG channel activation to more depolarized potentials and accelerates channel deactivation; however, the mechanisms underlying these effects are unclear. External divalent cations, e.g., Ca(2+) and Cd(2+), mimic these effects and coordinate within a metal ion binding pocket composed of three acidic residues in hERG: D456 and D460 in S2 and D509 in S3. A common mechanism may underlie divalent cation and proton effects on hERG gating. Using two-electrode voltage clamp, we show proton sensitivity of hERG channel activation (pKa = 5.6), but not deactivation, was greatly reduced in the presence of Cd(2+) (0.1 mM), suggesting a common binding site for the Cd(2+) and proton effect on activation and separable effects of protons on activation and deactivation. Mutational analysis confirmed that D509 plays a critical role in the pH dependence of activation, as shown previously, and that cooperative actions involving D456 and D460 are also required. Importantly, neutralization of all three acidic residues abolished the proton-induced shift of activation, suggesting that the metal ion binding pocket alone accounts for the effects of protons on hERG channel activation. Voltage-clamp fluorimetry measurements demonstrated that protons shifted the voltage dependence of S4 movement to more depolarized potentials. The data indicate a site and mechanism of action for protons on hERG activation gating; protonation of D456, D460 and D509 disrupts interactions between these residues and S4 gating charges to destabilize the activated configuration of S4.

  16. The Protonation Site of para-Dimethylaminobenzoic Acid Using Atmospheric Pressure Ionization Methods

    NASA Astrophysics Data System (ADS)

    Chai, Yunfeng; Weng, Guofeng; Shen, Shanshan; Sun, Cuirong; Pan, Yuanjiang

    2015-04-01

    The protonation site of para-dimethylaminobenzoic acid ( p-DMABA) was investigated using atmospheric pressure ionization methods (ESI and APCI) coupled with collision-induced dissociation (CID), nuclear magnetic resonance (NMR), and computational chemistry. Theoretical calculations and NMR experiments indicate that the dimethyl amino group is the preferred site of protonation both in the gas phase and aqueous solution. Protonation of p-DMABA occurs at the nitrogen atom by ESI independent of the solvents and other operation conditions under typical thermodynamic control. However, APCI produces a mixture of the nitrogen- and carbonyl oxygen-protonated p-DMABA when aprotic organic solvents (acetonitrile, acetone, and tetrahydrofuran) are used, exhibiting evident kinetic characteristics of protonation. But using protic organic solvents (methanol, ethanol, and isopropanol) in APCI still leads to the formation of thermodynamically stable N-protonated p-DMABA. These structural assignments were based on the different CID behavior of the N- and O-protonated p-DMABA. The losses of methyl radical and water are the diagnostic fragmentations of the N- and O-protonated p-DMABA, respectively. In addition, the N-protonated p-DMABA is more stable than the O-protonated p-DMABA in CID revealed by energy resolved experiments and theoretical calculations.

  17. Atomistic Characterization of the First Step of Calcium Pump Activation Associated with Proton Countertransport.

    PubMed

    Ramírez-Salinas, G Lizbeth; Espinoza-Fonseca, L Michel

    2015-08-25

    The calcium pump [sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA)] transports Ca(2+) from the cytosol to the SR lumen at the expense of ATP hydrolysis and proton countertransport, thus playing a central role in Ca(2+) homeostasis and muscle contractility. Proton countertransport via deprotonation of transport site residue Glu309 is a critical first step in SERCA activation because it accelerates the E2-E1 structural transition. Previous studies have suggested that flipping of Glu309 toward the cytosol constitutes the primary mechanism for Glu309 deprotonation, but no conclusive data to support this hypothesis have been published. Therefore, we performed three independent 1 μs molecular dynamics simulations of the E2 state protonated at transport site residues Glu309, Glu771, and Glu908. Structural analysis and pKa calculations showed that Glu309 deprotonation occurs by an inward-to-outward side-chain transition. We also found that Glu309 deprotonation and proton countertransport occur through transient (~113 ps) water wires connecting Glu309 with the cytosol. Although both mechanisms are operational, we found that transient water wire formation, and not Glu309 flipping, is the primary mechanism for Glu309 deprotonation and translocation of protons to the cytosol. The outward-to-inward transition of protonated Glu309 and the presence of water wires suggest that protons from the cytosol might be passively transported to the lumen via Glu309. However, structural analysis indicates that passive SR proton leakage into the lumen unlikely occurs through Glu309 in the E2 state. These findings provide a time-resolved visualization of the first step in the molecular mechanism of SERCA activation and proton transport across the SR.

  18. Proton-linked contributions to site-specific interactions of lambda cI repressor and OR.

    PubMed

    Senear, D F; Ackers, G K

    1990-07-17

    The effects of proton activity on the site-specific interactions of cI repressors with operator sites OR were studied by using DNase I footprint titration. Individual-site binding isotherms were obtained for the binding of repressor to each site of wild-type OR and of mutant operators in which binding to some sites is eliminated. The Gibbs energies for binding and for cooperativity (in every operator configuration) were determined at each pH (range 5-8). The proton-linked effects clearly account for a significant fraction of the difference in affinities for the three operator sites. The most dramatic effects on the repressor-operator binding interactions are at acid pH, and therefore do not involve the basic groups in the repressor N-terminal arm known to contact the DNA. Also, the proton-linked effects are different at the three operator sites as indicated by significantly different derivative relationships, partial derivative of ln k versus partial derivative of ln aH = net proton absorption (delta nu bar(H)). These results implicate ionizable repressor groups which may not contact the DNA and conformational differences between the three repressor-operator site complexes as being important components to the mechanism of site specificity. The extensive data base generated by these studies was also used to reevaluate the traditional models used to describe cooperativity in this system. The results confirm the lack of significant cooperative interaction between OR1 and OR3 at all conditions. However, the data for some experimental conditions are clearly inconsistent with the (selection) rule, that cooperative interaction between OR2 and OR3 is eliminated by ligation at OR1.

  19. Proton currents constrain structural models of voltage sensor activation

    PubMed Central

    Randolph, Aaron L; Mokrab, Younes; Bennett, Ashley L; Sansom, Mark SP; Ramsey, Ian Scott

    2016-01-01

    The Hv1 proton channel is evidently unique among voltage sensor domain proteins in mediating an intrinsic ‘aqueous’ H+ conductance (GAQ). Mutation of a highly conserved ‘gating charge’ residue in the S4 helix (R1H) confers a resting-state H+ ‘shuttle’ conductance (GSH) in VGCs and Ci VSP, and we now report that R1H is sufficient to reconstitute GSH in Hv1 without abrogating GAQ. Second-site mutations in S3 (D185A/H) and S4 (N4R) experimentally separate GSH and GAQ gating, which report thermodynamically distinct initial and final steps, respectively, in the Hv1 activation pathway. The effects of Hv1 mutations on GSH and GAQ are used to constrain the positions of key side chains in resting- and activated-state VS model structures, providing new insights into the structural basis of VS activation and H+ transfer mechanisms in Hv1. DOI: http://dx.doi.org/10.7554/eLife.18017.001 PMID:27572256

  20. Site-specific and compensatory mutations imply unexpected pathways for proton delivery to the QB binding site of the photosynthetic reaction center.

    PubMed Central

    Hanson, D K; Tiede, D M; Nance, S L; Chang, C H; Schiffer, M

    1993-01-01

    In photosynthetic reaction centers, a quinone molecule, QB, is the terminal acceptor in light-induced electron transfer. The protonatable residues Glu-L212 and Asp-L213 have been implicated in the binding of QB and in proton transfer to QB anions generated by electron transfer from the primary quinone QA. Here we report the details of the construction of the Ala-L212/Ala-L213 double mutant strain by site-specific mutagenesis and show that its photosynthetic incompetence is due to an inability to deliver protons to the QB anions. We also report the isolation and biophysical characterization of a collection of revertant and suppressor strains that have regained the photosynthetic phenotype. The compensatory mutations that restore function are diverse and show that neither Glu-L212 nor Asp-L213 is essential for efficient light-induced electron or proton transfer in Rhodobacter capsulatus. Second-site mutations, located within the QB binding pocket or at more distant sites, can compensate for mutations at L212 and L213 to restore photocompetence. Acquisition of a single negatively charged residue (at position L213, across the binding pocket at position L225, or outside the pocket at M43) or loss of a positively charged residue (at position M231) is sufficient to restore proton transfer activity to the complex. The proton transport pathways in the suppressor strains cannot, in principle, be identical to that of the wild type. The apparent mutability of this pathway suggests that the reaction center can serve as a model system to study the structural basis of protein-mediated proton transport. PMID:8105468

  1. Copper and protons directly activate the zinc-activated channel.

    PubMed

    Trattnig, Sarah M; Gasiorek, Agnes; Deeb, Tarek Z; Ortiz, Eydith J Comenencia; Moss, Stephen J; Jensen, Anders A; Davies, Paul A

    2016-03-01

    The zinc-activated channel (ZAC) is a cationic ion channel belonging to the superfamily of Cys-loop receptors, which consists of pentameric ligand-gated ion channels. ZAC is the least understood member of this family so in the present study we sought to characterize the properties of this channel further. We demonstrate that not only zinc (Zn(2+)) but also copper (Cu(2+)) and protons (H(+)) are agonists of ZAC, displaying potencies and efficacies in the rank orders of H(+)>Cu(2+)>Zn(2+) and H(+)>Zn(2+)>Cu(2+), respectively. The responses elicited by Zn(2+), Cu(2+) and H(+) through ZAC are all characterized by low degrees of desensitization. In contrast, currents evoked by high concentrations of the three agonists comprise distinctly different activation and decay components, with transitions to and from an open state being significantly faster for H(+) than for the two metal ions. The permeabilities of ZAC for Na(+) and K(+) relative to Cs(+) are indistinguishable, whereas replacing all of extracellular Na(+) and K(+) with the divalent cations Ca(2+) or Mg(2+) results in complete elimination of Zn(2+)-activated currents at both negative and positive holding potentials. This indicates that ZAC is non-selectively permeable to monovalent cations, whereas Ca(2+) and Mg(2+) inhibit the channel. In conclusion, this is the first report of a Cys-loop receptor being gated by Zn(2+), Cu(2+) and H(+). ZAC could be an important mediator of some of the wide range of physiological functions regulated by or involving Zn(2+), Cu(2+) and H(+).

  2. Redox-induced activation of the proton pump in the respiratory complex I

    PubMed Central

    Sharma, Vivek; Belevich, Galina; Gamiz-Hernandez, Ana P.; Róg, Tomasz; Vattulainen, Ilpo; Verkhovskaya, Marina L.; Wikström, Mårten; Hummer, Gerhard; Kaila, Ville R. I.

    2015-01-01

    Complex I functions as a redox-linked proton pump in the respiratory chains of mitochondria and bacteria, driven by the reduction of quinone (Q) by NADH. Remarkably, the distance between the Q reduction site and the most distant proton channels extends nearly 200 Å. To elucidate the molecular origin of this long-range coupling, we apply a combination of large-scale molecular simulations and a site-directed mutagenesis experiment of a key residue. In hybrid quantum mechanics/molecular mechanics simulations, we observe that reduction of Q is coupled to its local protonation by the His-38/Asp-139 ion pair and Tyr-87 of subunit Nqo4. Atomistic classical molecular dynamics simulations further suggest that formation of quinol (QH2) triggers rapid dissociation of the anionic Asp-139 toward the membrane domain that couples to conformational changes in a network of conserved charged residues. Site-directed mutagenesis data confirm the importance of Asp-139; upon mutation to asparagine the Q reductase activity is inhibited by 75%. The current results, together with earlier biochemical data, suggest that the proton pumping in complex I is activated by a unique combination of electrostatic and conformational transitions. PMID:26330610

  3. A voltage-activated proton current in human cardiac fibroblasts

    SciTech Connect

    El Chemaly, Antoun; Guinamard, Romain; Demion, Marie; Fares, Nassim; Jebara, Victor; Faivre, Jean-Francois; Bois, Patrick . E-mail: patrick.bois@univ-poitiers.fr

    2006-02-10

    A voltage-activated proton current in human cardiac fibroblasts, measured using the whole-cell recording configuration of the patch-clamp technique, is reported. Increasing the pH of the bathing solution shifted the current activation threshold to more negative potentials and increased both the current amplitude and its rate of activation. Changing the pH gradient by one unit caused a 51 mV shift in the reversal potential of the current, demonstrating a high selectivity for protons of the channel carrying the current. Extracellularly applied Zn{sup 2+} reversibly inhibited the current. Activation of the current contributes to the resting membrane conductance under conditions of intracellular acidosis. It is proposed that this current in cardiac fibroblasts is involved in the regulation of the intracellular pH and the membrane potential under physiological conditions as well as in response to pathological conditions such as ischemia.

  4. Salt site performance assessment activities

    SciTech Connect

    Kircher, J.F.; Gupta, S.K.

    1983-01-01

    During this year the first selection of the tools (codes) for performance assessments of potential salt sites have been tentatively selected and documented; the emphasis has shifted from code development to applications. During this period prior to detailed characterization of a salt site, the focus is on bounding calculations, sensitivity and with the data available. The development and application of improved methods for sensitivity and uncertainty analysis is a focus for the coming years activities and the subject of a following paper in these proceedings. Although the assessments to date are preliminary and based on admittedly scant data, the results indicate that suitable salt sites can be identified and repository subsystems designed which will meet the established criteria for protecting the health and safety of the public. 36 references, 5 figures, 2 tables.

  5. Mapping of sites facing aqueous environment of voltage-gated proton channel at resting state: a study with PEGylation protection.

    PubMed

    Kurokawa, Tatsuki; Okamura, Yasushi

    2014-01-01

    Hv1 (also named, voltage-sensor only protein, VSOP) lacks an authentic pore domain, and its voltage sensor domain plays both roles in voltage sensing and proton permeation. The activities of a proton channel are intrinsic to protomers of Hv1, while Hv1 is dimeric in biological membranes; cooperative gating is exerted by interaction between two protomers. As the signature pattern conserved among voltage-gated channels and voltage-sensing phosphatase, Hv1 has multiple arginines intervened by two hydrophobic residues on the fourth transmembrane segment, S4. S4 moves upward relative to other helices upon depolarization, causing conformational change possibly leading to the formation of a proton-selective conduction pathway. However, detailed mechanisms of proton-selectivity and gating of Hv1 are unknown. Here we took an approach of PEGylation protection assay to define residues facing the aqueous environment of mouse Hv1 (mHv1). Accessibilities of two maleimide molecules, N-ethylmaleimide (NEM) and 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), were examined on cysteine introduced into individual sites. Only the first arginine on S4 (R1: R201) was inaccessible by NEM and AMS in mHv1. This is consistent with previous results of electrophysiology on the resting state channel, suggesting that the accessibility profile represents the resting state of mHv1. D108, critical for proton selectivity, was accessible by AMS and NEM, suggesting that D108 faces the vestibule. F146, a site critical for blocking by a guanidinium-reagent, was accessible by NEM, suggesting that F146 also faces the inner vestibule. These findings suggest an inner vestibule lined by several residues on S2 including F146, D108 on S1, and the C-terminal half of S4.

  6. First fusion proton measurements in TEXTOR plasmas using activation technique

    SciTech Connect

    Bonheure, G.; Wassenhove, G. Van; Mlynar, J.; Hult, M.; Gonzalez de Orduna, R.; Lutter, G.; Vermaercke, P.; Huber, A.; Schweer, B.; Esser, G.; Biel, W.

    2012-10-15

    MeV particle loss measurements from fusion plasmas, in particular alpha particles, remain difficult in large fusion devices and further R and D is needed for ITER. This paper describes the first attempt to measure 3 MeV escaping fusion protons emitted from TEXTOR tokamak plasmas using activation technique. This technique was successfully demonstrated, initially, in 2006 on the JET tokamak. An ion camera equipped with a collimator and several types of activation detectors was installed inside the TEXTOR vacuum vessel to perform these measurements. After irradiation, the detectors were analyzed using ultra low level gamma-ray spectrometry at the HADES underground laboratory. 3 MeV escaping fusion protons were detected in larger number -{approx}6 times more - compared to earlier measurements using this technique on JET. Another major progress was the reduction of the cooling time by a factor of 50, which made possible to detect radionuclides with half-life of less than 90 min.

  7. Mechanism of Oxygen Reduction in Cytochrome c Oxidase and the Role of the Active Site Tyrosine.

    PubMed

    Blomberg, Margareta R A

    2016-01-26

    Cytochrome c oxidase, the terminal enzyme in the respiratory chain, reduces molecular oxygen to water and stores the released energy through electrogenic chemistry and proton pumping across the membrane. Apart from the heme-copper binuclear center, there is a conserved tyrosine residue in the active site (BNC). The tyrosine delivers both an electron and a proton during the O-O bond cleavage step, forming a tyrosyl radical. The catalytic cycle then occurs in four reduction steps, each taking up one proton for the chemistry (water formation) and one proton to be pumped. It is here suggested that in three of the reduction steps the chemical proton enters the center of the BNC, leaving the tyrosine unprotonated with radical character. The reproprotonation of the tyrosine occurs first in the final reduction step before binding the next oxygen molecule. It is also suggested that this reduction mechanism and the presence of the tyrosine are essential for the proton pumping. Density functional theory calculations on large cluster models of the active site show that only the intermediates with the proton in the center of the BNC and with an unprotonated tyrosyl radical have a high electron affinity of similar size as the electron donor, which is essential for the ability to take up two protons per electron and thus for the proton pumping. This type of reduction mechanism is also the only one that gives a free energy profile in accordance with experimental observations for the amount of proton pumping in the working enzyme.

  8. Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling.

    PubMed

    Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

    2015-01-01

    Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system. PMID:26370138

  9. Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling

    PubMed Central

    Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

    2015-01-01

    Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system. PMID:26370138

  10. Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling.

    PubMed

    Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

    2015-09-15

    Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system.

  11. Characterizing proton-activated materials to develop PET-mediated proton range verification markers

    NASA Astrophysics Data System (ADS)

    Cho, Jongmin; Ibbott, Geoffrey S.; Kerr, Matthew D.; Amos, Richard A.; Stingo, Francesco C.; Marom, Edith M.; Truong, Mylene T.; Palacio, Diana M.; Betancourt, Sonia L.; Erasmus, Jeremy J.; DeGroot, Patricia M.; Carter, Brett W.; Gladish, Gregory W.; Sabloff, Bradley S.; Benveniste, Marcelo F.; Godoy, Myrna C.; Patil, Shekhar; Sorensen, James; Mawlawi, Osama R.

    2016-06-01

    Conventional proton beam range verification using positron emission tomography (PET) relies on tissue activation alone and therefore requires particle therapy PET whose installation can represent a large financial burden for many centers. Previously, we showed the feasibility of developing patient implantable markers using high proton cross-section materials (18O, Cu, and 68Zn) for in vivo proton range verification using conventional PET scanners. In this technical note, we characterize those materials to test their usability in more clinically relevant conditions. Two phantoms made of low-density balsa wood (~0.1 g cm‑3) and beef (~1.0 g cm‑3) were embedded with Cu or 68Zn foils of several volumes (10–50 mm3). The metal foils were positioned at several depths in the dose fall-off region, which had been determined from our previous study. The phantoms were then irradiated with different proton doses (1–5 Gy). After irradiation, the phantoms with the embedded foils were moved to a diagnostic PET scanner and imaged. The acquired data were reconstructed with 20–40 min of scan time using various delay times (30–150 min) to determine the maximum contrast-to-noise ratio. The resultant PET/computed tomography (CT) fusion images of the activated foils were then examined and the foils’ PET signal strength/visibility was scored on a 5 point scale by 13 radiologists experienced in nuclear medicine. For both phantoms, the visibility of activated foils increased in proportion to the foil volume, dose, and PET scan time. A linear model was constructed with visibility scores as the response variable and all other factors (marker material, phantom material, dose, and PET scan time) as covariates. Using the linear model, volumes of foils that provided adequate visibility (score 3) were determined for each dose and PET scan time. The foil volumes that were determined will be used as a guideline in developing practical implantable markers.

  12. Characterizing proton-activated materials to develop PET-mediated proton range verification markers.

    PubMed

    Cho, Jongmin; Ibbott, Geoffrey S; Kerr, Matthew D; Amos, Richard A; Stingo, Francesco C; Marom, Edith M; Truong, Mylene T; Palacio, Diana M; Betancourt, Sonia L; Erasmus, Jeremy J; DeGroot, Patricia M; Carter, Brett W; Gladish, Gregory W; Sabloff, Bradley S; Benveniste, Marcelo F; Godoy, Myrna C; Patil, Shekhar; Sorensen, James; Mawlawi, Osama R

    2016-06-01

    Conventional proton beam range verification using positron emission tomography (PET) relies on tissue activation alone and therefore requires particle therapy PET whose installation can represent a large financial burden for many centers. Previously, we showed the feasibility of developing patient implantable markers using high proton cross-section materials ((18)O, Cu, and (68)Zn) for in vivo proton range verification using conventional PET scanners. In this technical note, we characterize those materials to test their usability in more clinically relevant conditions. Two phantoms made of low-density balsa wood (~0.1 g cm(-3)) and beef (~1.0 g cm(-3)) were embedded with Cu or (68)Zn foils of several volumes (10-50 mm(3)). The metal foils were positioned at several depths in the dose fall-off region, which had been determined from our previous study. The phantoms were then irradiated with different proton doses (1-5 Gy). After irradiation, the phantoms with the embedded foils were moved to a diagnostic PET scanner and imaged. The acquired data were reconstructed with 20-40 min of scan time using various delay times (30-150 min) to determine the maximum contrast-to-noise ratio. The resultant PET/computed tomography (CT) fusion images of the activated foils were then examined and the foils' PET signal strength/visibility was scored on a 5 point scale by 13 radiologists experienced in nuclear medicine. For both phantoms, the visibility of activated foils increased in proportion to the foil volume, dose, and PET scan time. A linear model was constructed with visibility scores as the response variable and all other factors (marker material, phantom material, dose, and PET scan time) as covariates. Using the linear model, volumes of foils that provided adequate visibility (score 3) were determined for each dose and PET scan time. The foil volumes that were determined will be used as a guideline in developing practical implantable markers. PMID:27203621

  13. Characterizing proton-activated materials to develop PET-mediated proton range verification markers

    NASA Astrophysics Data System (ADS)

    Cho, Jongmin; Ibbott, Geoffrey S.; Kerr, Matthew D.; Amos, Richard A.; Stingo, Francesco C.; Marom, Edith M.; Truong, Mylene T.; Palacio, Diana M.; Betancourt, Sonia L.; Erasmus, Jeremy J.; DeGroot, Patricia M.; Carter, Brett W.; Gladish, Gregory W.; Sabloff, Bradley S.; Benveniste, Marcelo F.; Godoy, Myrna C.; Patil, Shekhar; Sorensen, James; Mawlawi, Osama R.

    2016-06-01

    Conventional proton beam range verification using positron emission tomography (PET) relies on tissue activation alone and therefore requires particle therapy PET whose installation can represent a large financial burden for many centers. Previously, we showed the feasibility of developing patient implantable markers using high proton cross-section materials (18O, Cu, and 68Zn) for in vivo proton range verification using conventional PET scanners. In this technical note, we characterize those materials to test their usability in more clinically relevant conditions. Two phantoms made of low-density balsa wood (~0.1 g cm-3) and beef (~1.0 g cm-3) were embedded with Cu or 68Zn foils of several volumes (10-50 mm3). The metal foils were positioned at several depths in the dose fall-off region, which had been determined from our previous study. The phantoms were then irradiated with different proton doses (1-5 Gy). After irradiation, the phantoms with the embedded foils were moved to a diagnostic PET scanner and imaged. The acquired data were reconstructed with 20-40 min of scan time using various delay times (30-150 min) to determine the maximum contrast-to-noise ratio. The resultant PET/computed tomography (CT) fusion images of the activated foils were then examined and the foils’ PET signal strength/visibility was scored on a 5 point scale by 13 radiologists experienced in nuclear medicine. For both phantoms, the visibility of activated foils increased in proportion to the foil volume, dose, and PET scan time. A linear model was constructed with visibility scores as the response variable and all other factors (marker material, phantom material, dose, and PET scan time) as covariates. Using the linear model, volumes of foils that provided adequate visibility (score 3) were determined for each dose and PET scan time. The foil volumes that were determined will be used as a guideline in developing practical implantable markers.

  14. Protonation Sites, Tandem Mass Spectrometry and Computational Calculations of o-Carbonyl Carbazolequinone Derivatives.

    PubMed

    Martínez-Cifuentes, Maximiliano; Clavijo-Allancan, Graciela; Zuñiga-Hormazabal, Pamela; Aranda, Braulio; Barriga, Andrés; Weiss-López, Boris; Araya-Maturana, Ramiro

    2016-01-01

    A series of a new type of tetracyclic carbazolequinones incorporating a carbonyl group at the ortho position relative to the quinone moiety was synthesized and analyzed by tandem electrospray ionization mass spectrometry (ESI/MS-MS), using Collision-Induced Dissociation (CID) to dissociate the protonated species. Theoretical parameters such as molecular electrostatic potential (MEP), local Fukui functions and local Parr function for electrophilic attack as well as proton affinity (PA) and gas phase basicity (GB), were used to explain the preferred protonation sites. Transition states of some main fragmentation routes were obtained and the energies calculated at density functional theory (DFT) B3LYP level were compared with the obtained by ab initio quadratic configuration interaction with single and double excitation (QCISD). The results are in accordance with the observed distribution of ions. The nature of the substituents in the aromatic ring has a notable impact on the fragmentation routes of the molecules. PMID:27399676

  15. Protonation Sites, Tandem Mass Spectrometry and Computational Calculations of o-Carbonyl Carbazolequinone Derivatives

    PubMed Central

    Martínez-Cifuentes, Maximiliano; Clavijo-Allancan, Graciela; Zuñiga-Hormazabal, Pamela; Aranda, Braulio; Barriga, Andrés; Weiss-López, Boris; Araya-Maturana, Ramiro

    2016-01-01

    A series of a new type of tetracyclic carbazolequinones incorporating a carbonyl group at the ortho position relative to the quinone moiety was synthesized and analyzed by tandem electrospray ionization mass spectrometry (ESI/MS-MS), using Collision-Induced Dissociation (CID) to dissociate the protonated species. Theoretical parameters such as molecular electrostatic potential (MEP), local Fukui functions and local Parr function for electrophilic attack as well as proton affinity (PA) and gas phase basicity (GB), were used to explain the preferred protonation sites. Transition states of some main fragmentation routes were obtained and the energies calculated at density functional theory (DFT) B3LYP level were compared with the obtained by ab initio quadratic configuration interaction with single and double excitation (QCISD). The results are in accordance with the observed distribution of ions. The nature of the substituents in the aromatic ring has a notable impact on the fragmentation routes of the molecules. PMID:27399676

  16. Protonation Sites, Tandem Mass Spectrometry and Computational Calculations of o-Carbonyl Carbazolequinone Derivatives.

    PubMed

    Martínez-Cifuentes, Maximiliano; Clavijo-Allancan, Graciela; Zuñiga-Hormazabal, Pamela; Aranda, Braulio; Barriga, Andrés; Weiss-López, Boris; Araya-Maturana, Ramiro

    2016-07-05

    A series of a new type of tetracyclic carbazolequinones incorporating a carbonyl group at the ortho position relative to the quinone moiety was synthesized and analyzed by tandem electrospray ionization mass spectrometry (ESI/MS-MS), using Collision-Induced Dissociation (CID) to dissociate the protonated species. Theoretical parameters such as molecular electrostatic potential (MEP), local Fukui functions and local Parr function for electrophilic attack as well as proton affinity (PA) and gas phase basicity (GB), were used to explain the preferred protonation sites. Transition states of some main fragmentation routes were obtained and the energies calculated at density functional theory (DFT) B3LYP level were compared with the obtained by ab initio quadratic configuration interaction with single and double excitation (QCISD). The results are in accordance with the observed distribution of ions. The nature of the substituents in the aromatic ring has a notable impact on the fragmentation routes of the molecules.

  17. Diagnosing the Protonation Site of b 2 Peptide Fragment Ions using IRMPD in the X-H (X = O, N, and C) Stretching Region

    NASA Astrophysics Data System (ADS)

    Sinha, Rajeev K.; Erlekam, Undine; Bythell, Benjamin J.; Paizs, Béla; Maître, Philippe

    2011-09-01

    Charge-directed fragmentation has been shown to be the prevalent dissociation step for protonated peptides under the low-energy activation (eV) regime. Thus, the determination of the ion structure and, in particular, the characterization of the protonation site(s) of peptides and their fragments is a key approach to substantiate and refine peptide fragmentation mechanisms. Here we report on the characterization of the protonation site of oxazolone b 2 ions formed in collision-induced dissociation (CID) of the doubly protonated tryptic model-peptide YIGSR. In support of earlier work, here we provide complementary IR spectra in the 2800-3800 cm-1 range acquired on a table-top laser system. Combining this tunable laser with a high power CO2 laser to improve spectroscopic sensitivity, well resolved bands are observed, with an excellent correspondence to the IR absorption bands of the ring-protonated oxazolone isomer as predicted by quantum chemical calculations. In particular, it is shown that a band at 3445 cm-1, corresponding to the asymmetric N-H stretch of the (nonprotonated) N-terminal NH2 group, is a distinct vibrational signature of the ring-protonated oxazolone structure.

  18. Mutation of a single residue in the ba3 oxidase specifically impairs protonation of the pump site.

    PubMed

    von Ballmoos, Christoph; Gonska, Nathalie; Lachmann, Peter; Gennis, Robert B; Ädelroth, Pia; Brzezinski, Peter

    2015-03-17

    The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative "pump site" was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.

  19. Correlation of the antimicrobial activity of salicylaldehydes with broadening of the NMR signal of the hydroxyl proton. Possible involvement of proton exchange processes in the antimicrobial activity.

    PubMed

    Elo, Hannu; Kuure, Matti; Pelttari, Eila

    2015-03-01

    Certain substituted salicylaldehydes are potent antibacterial and antifungal agents and some of them merit consideration as potential chemotherapeutic agents against Candida infections, but their mechanism of action has remained obscure. We report here a distinct correlation between broadening of the NMR signal of the hydroxyl proton of salicylaldehydes and their activity against several types of bacteria and fungi. When proton NMR spectra of the compounds were determined using hexadeuterodimethylsulfoxide as solvent and the height of the OH proton signal was measured, using the signal of the aldehyde proton as an internal standard, it was discovered that a prerequisite of potent antimicrobial activity is that the proton signal is either unobservable or relatively very low, i.e. that it is extremely broadened. Thus, none of the congeners whose OH proton signal was high were potent antimicrobial agents. Some congeners that gave a very low OH signal were, however, essentially inactive against the microbes, indicating that although drastic broadening of the OH signal appears to be a prerequisite, also other (so far unknown) factors are needed for high antimicrobial activity. Because broadening of the hydroxyl proton signal is related to the speed of the proton exchange process(es) involving that proton, proton exchange may be involved in the mechanism of action of the compounds. Further studies are needed to analyze the relative importance of different factors (such as electronic effects, strength of the internal hydrogen bond, co-planarity of the ring and the formyl group) that determine the rates of those processes. PMID:25621992

  20. The Use of Chromium(III) to Supercharge Peptides by Protonation at Low Basicity Sites

    PubMed Central

    Feng, Changgeng; Commodore, Juliette J.; Cassady, Carolyn J.

    2014-01-01

    The addition of chromium(III) nitrate to solutions of peptides with seven or more residues greatly increases the formation of doubly protonated peptides, [M+2H]2+, by electrospray ionization. The test compound heptaalanine has only one highly basic site (the N-terminal amino group) and undergoes almost exclusive single protonation using standard solvents. When Cr(III) is added to the solution, abundant [M+2H]2+ forms, which involves protonation of the peptide backbone or the C-terminus. Salts of Al(III), Mn(II), Fe(III), Fe(II), Cu(II), Zn (II), Rh(III), La(III), Ce(IV), and Eu(III) were also studied. While several metal ions slightly enhance protonation, Cr(III) has by far the greatest ability to generate [M+2H]2+. Cr(III) does not supercharge peptide methyl esters, which suggests that the mechanism involves interaction of Cr(III) with a carboxylic acid group. Other factors may include the high acidity of hexaaquochromium(III) and the resistance of Cr(III) to reduction. Nitrate salts enhance protonation more than chloride salts and a molar ratio of 10:1 Cr(III):peptide produces the most intense [M+2H]2+. Cr(III) also supercharges numerous other small peptides, including highly acidic species. For basic peptides, Cr(III) increases the charge state (2+ versus 1+) and causes the number of peptide molecules being protonated to double or triple. Chromium(III) does not supercharge the proteins cytochrome c and myoglobin. The ability of Cr(III) to enhance [M+2H]2+ intensity may prove useful in tandem mass spectrometry because of the resulting overall increase in signal-to-noise ratio, the fact that [M+2H]2+ generally dissociate more readily than [M+H]+, and the ability to produce [M+2H]2+ precursors for electron-based dissociation techniques. PMID:25395012

  1. The Use of Chromium(III) to Supercharge Peptides by Protonation at Low Basicity Sites

    NASA Astrophysics Data System (ADS)

    Feng, Changgeng; Commodore, Juliette J.; Cassady, Carolyn J.

    2015-02-01

    The addition of chromium(III) nitrate to solutions of peptides with seven or more residues greatly increases the formation of doubly protonated peptides, [M + 2H]2+, by electrospray ionization. The test compound heptaalanine has only one highly basic site (the N-terminal amino group) and undergoes almost exclusive single protonation using standard solvents. When Cr(III) is added to the solution, abundant [M + 2H]2+ forms, which involves protonation of the peptide backbone or the C-terminus. Salts of Al(III), Mn(II), Fe(III), Fe(II), Cu(II), Zn (II), Rh(III), La(III), Ce(IV), and Eu(III) were also studied. Although several metal ions slightly enhance protonation, Cr(III) has by far the greatest ability to generate [M + 2H]2+. Cr(III) does not supercharge peptide methyl esters, which suggests that the mechanism involves interaction of Cr(III) with a carboxylic acid group. Other factors may include the high acidity of hexa-aquochromium(III) and the resistance of Cr(III) to reduction. Nitrate salts enhance protonation more than chloride salts and a molar ratio of 10:1 Cr(III):peptide produces the most intense [M + 2H]2+. Cr(III) also supercharges numerous other small peptides, including highly acidic species. For basic peptides, Cr(III) increases the charge state (2+ versus 1+) and causes the number of peptide molecules being protonated to double or triple. Chromium(III) does not supercharge the proteins cytochrome c and myoglobin. The ability of Cr(III) to enhance [M + 2H]2+ intensity may prove useful in tandem mass spectrometry because of the resulting overall increase in signal-to-noise ratio, the fact that [M + 2H]2+ generally dissociate more readily than [M + H]+, and the ability to produce [M + 2H]2+ precursors for electron-based dissociation techniques.

  2. Gaugement of the inner space of the apomyoglobin's heme binding site by a single free diffusing proton. I. Proton in the cavity.

    PubMed Central

    Shimoni, E; Tsfadia, Y; Nachliel, E; Gutman, M

    1993-01-01

    Time resolved fluorimetry was employed to monitor the geminate recombination between proton and excited pyranine anion locked, together with less than 30 water molecules, inside the heme binding site of Apomyoglobin (sperm whale). The results were analyzed by a numerical reconstruction of the differential rate equation for time-dependent diffusion controlled reaction with radiating boundaries using N. Agmon's procedure (Huppert, Pines, and Agmon, 1990, J. Opt. Soc. Am. B., 7:1541-1550). The analysis of the curve provided the effective dielectric constant of the proton permeable space in the cavity and the diffusion coefficient of the proton. The electrostatic potential within the cavity was investigated by the equations given by Gilson et al. (1985, J. Mol. Biol., 183:503-516). According to this analysis the dielectric constant of the protein surrounding the site is epsilon prot < or = 6.5. The diffusion coefficient of the proton in the heme binding site of Apomyoglobin-pyranine complex is D = 4 x 10(-5) cm2/s. This value is approximately 50% of the diffusion coefficient of proton in water. The lower value indicates enhanced ordering of water in the cavity, a finding which is corroborated by a large negative enthropy of binding delta S0 = -46.6 cal.mole-1 deg-1. The capacity of a small cavity in a protein to retain a proton had been investigated through the mathematical reconstruction of the dynamics. It has been demonstrated that Coulombic attraction, as large as delta psi of energy coupling membrane, is insufficient to delay a free proton for a time frame comparable to the turnover time of protogenic sites. PMID:8384501

  3. Protonation Equilibria of Biologically Active Ligands in Mixed Aqueous Organic Solvents

    PubMed Central

    El-Sherif, Ahmed A.; Shoukry, Mohamed M.; Abd Elkarim, Abeer T.; Barakat, Mohammad H.

    2014-01-01

    The review is mainly concerned with the protonation equilibria of biologically active ligands like amino acids, peptides, DNA constituents, and amino acid esters in nonaqueous media. Equilibrium concentrations of proton-ligand formation as a function of pH were investigated. Also, thermodynamics associated with protonation equilibria were also discussed. PMID:25197267

  4. Interactions of protons with single open L-type calcium channels. Location of protonation site and dependence of proton-induced current fluctuations on concentration and species of permeant ion

    PubMed Central

    1989-01-01

    We further investigated the rapid fluctuations between two different conductance levels promoted by protons when monovalent ions carry current through single L-type Ca channels. We tested for voltage dependence of the proton-induced current fluctuations and for accessibility of the protonation site from both sides of the membrane patch. The results strongly suggest an extracellular location of the protonation site. We also studied the dependence of the kinetics of the fluctuations and of the two conductance levels on the concentration of permeant ion and on external ionic strength. We find that saturation curves of channel conductance vs. [K] are similar for the two conductance levels. This provides evidence that protonation does not appreciably change the surface potential near the entry of the permeation pathway. The proton-induced conduction change must therefore result from an indirect interaction between the protonation site and the ion-conducting pathway. Concentration of permeant ion and ionic strength also affect the kinetics of the current fluctuations, in a manner consistent with our previous hypothesis that channel occupancy destabilizes the low conductance channel conformation. We show that the absence of measurable fluctuations with Li and Ba as charge carriers can be explained by significantly higher affinities of these ions for permeation sites. Low concentrations of Li reduce the Na conductance and abbreviate the lifetimes of the low conductance level seen in the presence of Na. We use whole-cell recordings to extrapolate our findings to the physiological conditions of Ca channel permeation and conclude that in the presence of 1.8 mM Ca no proton-induced fluctuations occur between pH 7.5 and 6.5. Finally, we propose a possible physical interpretation of the formal model of the protonation cycle introduced in the companion paper. PMID:2553858

  5. Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants.

    PubMed

    Meyer, Thomas; Melin, Frédéric; Richter, Oliver-M H; Ludwig, Bernd; Kannt, Aimo; Müller, Hanne; Michel, Hartmut; Hellwig, Petra

    2015-02-27

    Two different pathways through which protons access cytochrome c oxidase operate during oxygen reduction from the mitochondrial matrix, or the bacterial cytoplasm. Here, we use electrocatalytic current measurements to follow oxygen reduction coupled to proton uptake in cytochrome c oxidase isolated from Paracoccus denitrificans. Wild type enzyme and site-specific variants with defects in both proton uptake pathways (K354M, D124N and K354M/D124N) were immobilized on gold nanoparticles, and oxygen reduction was probed electrochemically in the presence of varying concentrations of Zn(2+) ions, which are known to inhibit both the entry and the exit proton pathways in the enzyme. Our data suggest that under these conditions substrate protons gain access to the oxygen reduction site via the exit pathway.

  6. Proton Transfers in a Channelrhodopsin-1 Studied by Fourier Transform Infrared (FTIR) Difference Spectroscopy and Site-directed Mutagenesis*

    PubMed Central

    Ogren, John I.; Yi, Adrian; Mamaev, Sergey; Li, Hai; Spudich, John L.; Rothschild, Kenneth J.

    2015-01-01

    Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2380 state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2380 formation. The unusual charge neutrality of both Schiff base counterions in the P2380 conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs. PMID:25802337

  7. Proton transfers in a channelrhodopsin-1 studied by Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis.

    PubMed

    Ogren, John I; Yi, Adrian; Mamaev, Sergey; Li, Hai; Spudich, John L; Rothschild, Kenneth J

    2015-05-15

    Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs. PMID:25802337

  8. Comparison of short-lived medical isotopes activation by laser thin target induced protons and conventional cyclotron proton beams

    NASA Astrophysics Data System (ADS)

    Murray, Joseph; Dudnikova, Galina; Liu, Tung-Chang; Papadopoulos, Dennis; Sagdeev, Roald; Su, J. J.; UMD MicroPET Team

    2014-10-01

    Production diagnostic or therapeutic nuclear medicines are either by nuclear reactors or by ion accelerators. In general, diagnostic nuclear radioisotopes have a very short half-life varying from tens of minutes for PET tracers and few hours for SPECT tracers. Thus supplies of PET and SPECT radiotracers are limited by regional production facilities. For example 18F-fluorodeoxyglucose (FDG) is the most desired tracer for positron emission tomography because its 110 minutes half-life is sufficient long for transport from production facilities to nearby users. From nuclear activation to completing image taking must be done within 4 hours. Decentralized production of diagnostic radioisotopes will be idea to make high specific activity radiotracers available to researches and clinicians. 11 C, 13 N, 15 O and 18 F can be produced in the energy range from 10-20 MeV by protons. Protons of energies up to tens of MeV generated by intense laser interacting with hydrogen containing targets have been demonstrated by many groups in the past decade. We use 2D PIC code for proton acceleration, Geant4 Monte Carlo code for nuclei activation to compare the yields and specific activities of short-lived isotopes produced by cyclotron proton beams and laser driven protons.

  9. Experimental verification of proton beam monitoring in a human body by use of activity image of positron-emitting nuclei generated by nuclear fragmentation reaction.

    PubMed

    Nishio, Teiji; Miyatake, Aya; Inoue, Kazumasa; Gomi-Miyagishi, Tomoko; Kohno, Ryosuke; Kameoka, Satoru; Nakagawa, Keiichi; Ogino, Takashi

    2008-01-01

    Proton therapy is a form of radiotherapy that enables concentration of dose on a tumor by use of a scanned or modulated Bragg peak. Therefore, it is very important to evaluate the proton-irradiated volume accurately. The proton-irradiated volume can be confirmed by detection of pair-annihilation gamma rays from positron-emitting nuclei generated by the nuclear fragmentation reaction of the incident protons on target nuclei using a PET apparatus. The activity of the positron-emitting nuclei generated in a patient was measured with a PET-CT apparatus after proton beam irradiation of the patient. Activity measurement was performed in patients with tumors of the brain, head and neck, liver, lungs, and sacrum. The 3-D PET image obtained on the CT image showed the visual correspondence with the irradiation area of the proton beam. Moreover, it was confirmed that there were differences in the strength of activity from the PET-CT images obtained at each irradiation site. The values of activity obtained from both measurement and calculation based on the reaction cross section were compared, and it was confirmed that the intensity and the distribution of the activity changed with the start time of the PET imaging after proton beam irradiation. The clinical use of this information about the positron-emitting nuclei will be important for promoting proton treatment with higher accuracy in the future.

  10. Characterization of the proton binding sites of extracellular polymeric substances in an anaerobic membrane bioreactor.

    PubMed

    Liu, Yi; Chang, Sheng; Defersha, Fantahun M

    2015-07-01

    This paper focuses on the characterization of the chemical compositions and acidic constants of the extracellular polymeric substances (EPSs) in an anaerobic membrane bioreactor treating synthetic brewery wastewater by using chemical analysis, linear programming analysis (LPA) of titration data, and FT-IR analysis. The linear programming analysis of titration data revealed that the EPSs have proton binding sites with pKa values from pKa ≤ 6, between 6 and 7, and approximately 9.8. The strong acidic sites (pKa ≤ 6) and some weak acidic sites (7.5 < pKa < 9.0) were found to be readily removed by 0.45-μm membrane filtration. In addition, the FT-IR analysis confirmed the presence of proteins, carbohydrates, nucleic acids, and lipids in the EPS samples. Based on the FT-IR analysis and the main chemical functional groups at the bacterial cell surfaces, the identified proton binding sites were related to carboxyl, phosphate, and hydroxyl/amine groups with pKa values of 4.6 ± 0.7, 6.6 ± 0.01, and 9.7 ± 0.1, respectively, with the corresponding respective intensities of 0.31 ± 0.05, 0.96 ± 0.3, and 1.53 ± 0.3 mmole/g-EPS. The pKa values and intensities of the proton binding sites are the fundamental molecular properties of EPSs that affect the EPS charge, molecular interactions, and metal complexation characteristics. Determination of such properties can advance Derjaguin-Landau-Verwey-Overbeek (DLVO)-based concentration polarization modeling, facilitate the estimation of the osmotic pressure of the EPS concentration polarization layers, and lead to a deeper understanding of the role of metal complexation in membrane fouling. PMID:25935368

  11. Proton transfer in organic scaffolds

    NASA Astrophysics Data System (ADS)

    Basak, Dipankar

    This dissertation focuses on the fundamental understanding of the proton transfer process and translating the knowledge into design/development of new organic materials for efficient non-aqueous proton transport. For example, what controls the shuttling of a proton between two basic sites? a) Distance between two groups? or b) the basicity? c) What is the impact of protonation on molecular conformation when the basic sites are attached to rigid scaffolds? For this purpose, we developed several tunable proton sponges and studied proton transfer in these scaffolds theoretically as well as experimentally. Next we moved our attention to understand long-range proton conduction or proton transport. We introduced liquid crystalline (LC) proton conductor based on triphenylene molecule and established that activation energy barrier for proton transport is lower in the LC phase compared to the crystalline phase. Furthermore, we investigated the impact of several critical factors: the choice of the proton transferring groups, mobility of the charge carriers, intrinsic vs. extrinsic charge carrier concentrations and the molecular architectures on long-range proton transport. The outcome of this research will lead to a deeper understanding of non-aqueous proton transfer process and aid the design of next generation proton exchange membrane (PEM) for fuel cell.

  12. Crystal Structure of Phosphatidylglycerophosphatase (PGPase), a Putative Membrane-Bound Lipid Phosphatase, Reveals a Novel Binuclear Metal Binding Site and Two Proton Wires

    SciTech Connect

    Kumaran,D.; Bonnano, J.; Burley, S.; Swaminathan, S.

    2006-01-01

    Phosphatidylglycerophosphatase (PGPase), an enzyme involved in lipid metabolism, catalyzes formation of phosphatidylglycerol from phosphatidylglycerophosphate. Phosphatidylglycerol is a multifunctional phospholipid, found in the biological membranes of many organisms. Here, we report the crystal structure of Listeria monocytogenes PGPase at 1.8 Angstroms resolution. PGPase, an all-helical molecule, forms a homotetramer. Each protomer contains an independent active site with two metal ions, Ca{sup 2+} and Mg{sup 2+}, forming a hetero-binuclear center located in a hydrophilic cavity near the surface of the molecule. The binuclear center, conserved ligands, metal-bound water molecules, and an Asp-His dyad form the active site. The catalytic mechanism of this enzyme is likely to proceed via binuclear metal activated nucleophilic water. The binuclear metal-binding active-site environment of this structure should provide insights into substrate binding and metal-dependent catalysis. A long channel with inter-linked linear water chains, termed 'proton wires', is observed at the tetramer interface. Comparison of similar water chain structures in photosynthetic reaction centers (RCs), Cytochrome f, gramicidin, and bacteriorhodopsin, suggests that PGPase may conduct protons via proton wires.

  13. Activation of lactoperoxidase by heme-linked protonation and heme-independent iodide binding.

    PubMed

    Toyama, Akira; Tominaga, Aya; Inoue, Tatsuo; Takeuchi, Hideo

    2010-01-01

    Lactoperoxidase (LPO), a mammalian secretory heme peroxidase, catalyzes the oxidation of thiocyanate by hydrogen peroxide to produce hypothiocyanate, an antibacterial agent. Although LPO is known to be activated at acidic pH and in the presence of iodide, the structural basis of the activation is not well understood. We have examined the effects of pH and iodide concentration on the catalytic activity and the structure of LPO. Electrochemical and colorimetric assays have shown that the catalytic activity is maximized at pH 4.5. The heme Soret absorption band exhibits a small red-shift at pH 5.0 upon acidification, which is ascribable to a structural transition from a neutral to an acidic form. Resonance Raman spectra suggest that the heme porphyrin core is slightly contracted and the Fe-His bond is strengthened in the acidic form compared to the neutral form. The structural change of LPO upon activation at acidic pH is similar to that observed for myeloperoxidase, another mammalian heme peroxidase, upon activation at neutral pH. Binding of iodide enhances the catalytic activity of LPO without affecting either the optimum pH of activity or the heme structure, implying that the iodide binding occurs at a protein site away from the heme-linked protonation site.

  14. Site-specific range uncertainties caused by dose calculation algorithms for proton therapy

    NASA Astrophysics Data System (ADS)

    Schuemann, J.; Dowdell, S.; Grassberger, C.; Min, C. H.; Paganetti, H.

    2014-08-01

    The purpose of this study was to assess the possibility of introducing site-specific range margins to replace current generic margins in proton therapy. Further, the goal was to study the potential of reducing margins with current analytical dose calculations methods. For this purpose we investigate the impact of complex patient geometries on the capability of analytical dose calculation algorithms to accurately predict the range of proton fields. Dose distributions predicted by an analytical pencil-beam algorithm were compared with those obtained using Monte Carlo (MC) simulations (TOPAS). A total of 508 passively scattered treatment fields were analyzed for seven disease sites (liver, prostate, breast, medulloblastoma-spine, medulloblastoma-whole brain, lung and head and neck). Voxel-by-voxel comparisons were performed on two-dimensional distal dose surfaces calculated by pencil-beam and MC algorithms to obtain the average range differences and root mean square deviation for each field for the distal position of the 90% dose level (R90) and the 50% dose level (R50). The average dose degradation of the distal falloff region, defined as the distance between the distal position of the 80% and 20% dose levels (R80-R20), was also analyzed. All ranges were calculated in water-equivalent distances. Considering total range uncertainties and uncertainties from dose calculation alone, we were able to deduce site-specific estimations. For liver, prostate and whole brain fields our results demonstrate that a reduction of currently used uncertainty margins is feasible even without introducing MC dose calculations. We recommend range margins of 2.8% + 1.2 mm for liver and prostate treatments and 3.1% + 1.2 mm for whole brain treatments, respectively. On the other hand, current margins seem to be insufficient for some breast, lung and head and neck patients, at least if used generically. If no case specific adjustments are applied, a generic margin of 6.3% + 1.2 mm would be

  15. Drastic Enhancement of Photocatalytic Activities over Phosphoric Acid Protonated Porous g-C3 N4 Nanosheets under Visible Light.

    PubMed

    Shi, Li; Chang, Kun; Zhang, Huabin; Hai, Xiao; Yang, Liuqing; Wang, Tao; Ye, Jinhua

    2016-08-01

    A simple method is developed to fabricate protonated porous graphitic carbon nitride nanosheets (P-PCNNS) by protonation-exfoliation of bulk graphitic carbon nitride (BCN) with phosphoric acid (H3 PO4 ). The H3 PO4 treatment not only helps to exfoliate the BCN into 2D ultrathin nanosheets with abundant micro- and mesopores, endowing P-PCNNS with more exposed active catalytic sites and cross-plane diffusion channels to facilitate the mass and charge transport, but also induces the protonation of carbon nitride polymer, leading to the moderate removal of the impurities of carbon species in BCN for the optimization of the aromatic π-conjugated system for better charge separation without changing its chemical structure. As a result, the P-PCNNS show much higher photocatalytic performance for hydrogen evolution and CO2 conversion than bare BCN and graphitic carbon nitride nanosheets. PMID:27410192

  16. Characterization of the active site of chloroperoxidase using physical techniques

    SciTech Connect

    Hall, K.S.

    1986-01-01

    Chloroperoxidase (CPO) and Cytochrome P-450, two very different hemeproteins, have been shown to have similar active sites by several techniques. Recent work has demonstrated thiolate ligation from a cysteine residue to the iron in P-450. A major portion of this research has been devoted to obtaining direct evidence that CPO also has a thiolate 5th ligand from a cysteine residue. This information will provide the framework for a detailed analysis of the structure-function relationships between peroxidases, catalase and cytochrome P-450 hemeproteins. To determine whether the 5th ligand is a cysteine, methionine or a unique amino acid, specific isotope enrichment experiments were used. Preliminary /sup 1/H-NMR studies show that the carbon monoxide-CPO complex has a peak in the upfield region corresponding to alpha-protons of a thiolate amino acid. C. fumago was grown on 95% D/sub 2/O media with a small amount of /sup 1/H-cysteine added. Under these conditions C. fumago slows down the biosynthesis of cysteine by at least 50% and utilizes the exogenous cysteine in the media. GC-MS was able to show that the methylene protons next to the sulfur atom in cysteine are 80-90% protonated while these positions in methionine are approximately 73% deuterated. Comparison of the /sup 1/H-NMR spectra of CO-CPO and CO-CPO indicate the presence of a cysteine ligand in chloroperoxidase.

  17. Complex active regions as the main source of extreme and large solar proton events

    NASA Astrophysics Data System (ADS)

    Ishkov, V. N.

    2013-12-01

    A study of solar proton sources indicated that solar flare events responsible for ≥2000 pfu proton fluxes mostly occur in complex active regions (CARs), i.e., in transition structures between active regions and activity complexes. Different classes of similar structures and their relation to solar proton events (SPEs) and evolution, depending on the origination conditions, are considered. Arguments in favor of the fact that sunspot groups with extreme dimensions are CARs are presented. An analysis of the flare activity in a CAR resulted in the detection of "physical" boundaries, which separate magnetic structures of the same polarity and are responsible for the independent development of each structure.

  18. A pathogenic mutation in cytochrome c oxidase results in impaired proton pumping while retaining O(2)-reduction activity.

    PubMed

    Namslauer, Ida; Lee, Hyun Ju; Gennis, Robert B; Brzezinski, Peter

    2010-05-01

    In this work we have investigated the effect of a pathogenic mitochondrial DNA mutation found in human colon cells, at a functional-molecular level. The mutation results in the amino-acid substitution Tyr19His in subunit I of the human CytcO and it is associated with respiratory deficiency. It was introduced into Rhodobacter sphaeroides, which carries a cytochrome c oxidase (cytochrome aa(3)) that serves as a model of the mitochondrial counterpart. The residue is situated in the middle of a pathway that is used to transfer substrate protons as well as protons that are pumped across the membrane. The Tyr33His (equivalent residue in the bacterial CytcO) structural variant of the enzyme was purified and its function was investigated. The results show that in the structurally altered CytcO the activity decreased due to slowed proton transfer; proton transfer from an internal proton donor, the highly-conserved Glu286, to the catalytic site was slowed by a factor of approximately 5, while reprotonation of the Glu from solution was slowed by a factor of approximately 40. In addition, in the structural variant proton pumping was completely impaired. These results are explained in terms of introduction of a barrier for proton transfer through the D pathway and changes in the coordination of water molecules surrounding the Glu286 residue. The study offers an explanation, at the molecular level, to the link between a specific amino-acid substitution and a pathogenic phenotype identified in human colon cells.

  19. Catalysis: Elusive active site in focus

    NASA Astrophysics Data System (ADS)

    Labinger, Jay A.

    2016-08-01

    The identification of the active site of an iron-containing catalyst raises hopes of designing practically useful catalysts for the room-temperature conversion of methane to methanol, a potential fuel for vehicles. See Letter p.317

  20. Site-specific range uncertainties caused by dose calculation algorithms for proton therapy

    PubMed Central

    Schuemann, J.; Dowdell, S.; Grassberger, C.; Min, C. H.; Paganetti, H.

    2014-01-01

    The purpose of this study was to investigate the impact of complex patient geometries on the capability of analytical dose calculation algorithms to accurately predict the range of proton fields. Dose distributions predicted by an analytical pencil-beam algorithm were compared with those obtained using Monte Carlo simulations (TOPAS). A total of 508 passively scattered treatment fields were analyzed for 7 disease sites (liver, prostate, breast, medulloblastoma-spine, medulloblastoma-whole brain, lung and head & neck). Voxel-by-voxel comparisons were performed on two-dimensional distal dose surfaces calculated by pencil-beam and Monte Carlo algorithms to obtain the average range differences (ARD) and root mean square deviation (RMSD) for each field for the distal position of the 90% dose level (R90) and the 50% dose level (R50). The average dose degradation (ADD) of the distal falloff region, defined as the distance between the distal position of the 80% and 20% dose levels (R80-R20), was also analyzed. All ranges were calculated in water-equivalent distances. Considering total range uncertainties and uncertainties from dose calculation alone, we were able to deduce site-specific estimations. For liver, prostate and whole brain fields our results demonstrate that a reduction of currently used uncertainty margins is feasible even without introducing Monte Carlo dose calculations. We recommend range margins of 2.8% + 1.2 mm for liver and prostate treatments and 3.1% + 1.2 mm for whole brain treatments, respectively. On the other hand, current margins seem to be insufficient for some breast, lung and head & neck patients, at least if used generically. If no case specific adjustments are applied, a generic margin of 6.3% + 1.2 mm would be needed for breast, lung and head & neck treatments. We conclude that currently used generic range uncertainty margins in proton therapy should be redefined site specific and that complex geometries may require a field specific

  1. Feasibility of PET/CT 3-D dosimetry for proton-activated PRESAGE® dosimeters

    NASA Astrophysics Data System (ADS)

    Carroll, M.; Ibbott, G.; Adamovics, J.

    2015-01-01

    A feasibility study is considered using PRESAGE® dosimeters as a direct dosimetry comparison tool to correlate proton activation to dose in a clinical beam. Proton activation measured by PET/CT has been studied as a future tool method for in vivo dosimetry. PRESAGE® is a 3D offline dosimeter that has a material composition giving it a longer activation decay time than similar dosimeters allowing easier measurements. In this study, the PRESAGE® positron emissions were measured by PET and directly compared to dose measurements made by optical-CT to make spatial comparisons of dose and proton activation. Profiles along the central axis of the beam found a shift in the distal-80% resulting in the proton activation profile being 1 cm shallower than the dose profile. Cross beam profiles demonstrated little discrepancy between long (3 hour) and short (30 minute) PET scan times.

  2. Solubilization Site of Organic Perfume Molecules in Sodium Dodecyl Sulfate Micelles: New Insights from Proton NMR Studies.

    PubMed

    Suratkar; Mahapatra

    2000-05-01

    The site of incorporation of solubilizates in sodium dodecyl sulfate (SDS) micellar systems has been investigated by proton NMR spectroscopy. The solubilizate molecules chosen for the present study are phenol, 4-methylphenol, 4-allyl-2-methoxyphenol, anisole, 4-methylanisole, 4-propenylanisole, 1,8-cineole, and limonene. These molecules possess a wide variety of functional groups with different degrees of hydrophilic/hydrophobic character and are thereby solubilized at different micellar locations. Aromatic compounds, especially those having a phenolic-OH group, showed a large upfield shift of SDS methylene protons that are closely linked to the terminal sulfate groups. Additionally, in the case of phenolic compounds, the unresolved signals of the nine straight-chain bulk methylene protons of SDS are split into a broad doublet with uneven intensity. This splitting of methylene protons was found to be dependent on the concentration of the substrate. Based on these observations, probable solubilization sites and orientation of the substrate molecule within the micelles are discussed. Phenolic compounds, being the most hydrophilic among the present set, reside at the hydrophilic/hydrophobic boundary of micelle-water interface and thus influence the resonances of SDS protons the most. Aromatic methoxy and aliphatic compounds, being relatively more hydrophobic in nature, reside inside the micellar core and thereby result in smaller shifts. Copyright 2000 Academic Press.

  3. A comparison of energy-resolved vibrational activation/dissociation characteristics of protonated and sodiated high mannose N-glycopeptides.

    PubMed

    Aboufazeli, Forouzan; Kolli, Venkata; Dodds, Eric D

    2015-04-01

    Fragmentation of glycopeptides in tandem mass spectrometry (MS/MS) plays a pivotal role in site-specific protein glycosylation profiling by allowing specific oligosaccharide compositions and connectivities to be associated with specific loci on the corresponding protein. Although MS/MS analysis of glycopeptides has been successfully performed using a number of distinct ion dissociation methods, relatively little is known regarding the fragmentation characteristics of glycopeptide ions with various charge carriers. In this study, energy-resolved vibrational activation/dissociation was examined via collision-induced dissociation for a group of related high mannose tryptic glycopeptides as their doubly protonated, doubly sodiated, and hybrid protonated sodium adduct ions. The doubly protonated glycopeptide ions with various compositions were found to undergo fragmentation over a relatively low but wide range of collision energies compared with the doubly sodiated and hybrid charged ions, and were found to yield both glycan and peptide fragmentation depending on the applied collision energy. By contrast, the various doubly sodiated glycopeptides were found to dissociate over a significantly higher but narrow range of collision energies, and exhibited only glycan cleavages. Interestingly, the hybrid protonated sodium adduct ions were consistently the most stable of the precursor ions studied, and provided fragmentation information spanning both the glycan and the peptide moieties. Taken together, these findings illustrate the influence of charge carrier over the energy-resolved vibrational activation/dissociation characteristics of glycopeptides, and serve to suggest potential strategies that exploit the analytically useful features uniquely afforded by specific charge carriers or combinations thereof.

  4. Activation calculations for trapped protons below 200 MeV: Appendix

    NASA Technical Reports Server (NTRS)

    Laird, C. E.

    1991-01-01

    Tables are given displaying of the results of the activation calculations of metal samples and other material aboard the Long Duration Exposure Facility-1 (LDEF-1) and Spacelab-2 with the computer program, PTRAP4. The computer printouts give the reaction, the reactant product, the proton reaction cross sections as a function of the energy of the incident protons, and the activation as a function of distance into the sample from the exposed surface.

  5. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

    Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two waters in the Y16S mutant, one water in the Y16F and FFF mutants, and intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of 1H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less

  6. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  7. Site-directed mutation of arginine 282 to glutamate uncouples the movement of peptides and protons by the rabbit proton-peptide cotransporter PepT1.

    PubMed

    Meredith, David

    2004-04-16

    A conserved positive residue in the seventh transmembrane domain of the mammalian proton-coupled di- and tripeptide transporter PepT1 has been shown by site-directed mutagenesis to be a key residue for protein function. Substitution of arginine 282 with a glutamate residue (R282E-PepT1) gave a protein at the plasma membrane of Xenopus laevis oocytes that was able to transport the non-hydrolyzable dipeptide [3H]d-Phe-l-Gln, although unlike the wild type, the rate of transport by R282E-PepT1 was independent of the extracellular pH level, and the substrate could not be accumulated above equilibrium. The binding affinity of the mutant transport protein was unchanged from the wild type. Thus, R282E-Pept1 appears to have been changed from a proton-driven to a facilitated transporter for peptides. In addition, peptide transport by R282E-PepT1 still induced depolarization as measured by microelectrode recordings of membrane potential. A more detailed study by two-electrode voltage clamping revealed that R282E-PepT1 behaved as a peptide-gated non-selective cation channel with the ion selectivity series lithium > sodium > N-methyl-d-glucamine at pH 7.4. There was also a proton conductance (comparing pH 7.4 and 8.4), and at pH 5.5 the predominant conductance was for potassium ions. Therefore, it can be concluded that changing arginine 282 to a glutamate not only uncouples the cotransport of protons and peptides of the wild-type PepT1 but also creates a peptide-gated cation channel in the protein.

  8. Solvent exchangeable protons and the activation of molecular oxygen: the galactose oxidase reaction.

    PubMed

    Kosman, D J; Driscoll, J J

    1988-01-01

    The reduction of O2 to H2O2 requires two protons as well as two electrons. Thus, activation of dioxygen reasonably may involve either general or specific acid catalysis. Consequently, the reduction of O2 to H2O2 could exhibit a kinetic solvent isotope effect (KSIE). The reaction catalyzed by the mononuclear Cu(II) enzyme, galactose oxidase does exhibit a KSIE (+1.55). The pL-rate profile exhibits an alkaline shift in D2O which can be attributed to the differential partitioning of H+ versus D+ between bulk water and a metal-bound H2O (delta pKa = +0.19). A variety of spectral evidence places an equatorial, Cu(II)-liganded water molecule at the active site of galactose oxidase. The analysis of the KSIE data is detailed and the potential generality of the function of such metal-bound H2O at other type 2 Cu(II) sites is discussed.

  9. Studies of neutron and proton nuclear activation in low-Earth orbit

    NASA Technical Reports Server (NTRS)

    Laird, C. E.

    1982-01-01

    The expected induced radioactivity of experimental material in low Earth orbit was studied for characteristics of activating particles such as cosmic rays, high energy Earth albedo neutrons, trapped protons, and secondary protons and neutrons. The activation cross sections for the production of long lived radioisotopes and other existing nuclear data appropriate to the study of these reactions were compiled. Computer codes which are required to calculate the expected activation of orbited materials were developed. The decreased computer code used to predict the activation of trapped protons of materials placed in the expected orbits of LDEF and Spacelab II. Techniques for unfolding the fluxes of activating particles from the measured activation of orbited materials are examined.

  10. Proton Channel Activity of Influenza A Virus Matrix Protein 2 Contributes to Autophagy Arrest

    PubMed Central

    Ren, Yizhong; Feng, Liqiang; Pan, Weiqi; Li, Liang; Wang, Qian; Li, Jiashun; Li, Na; Han, Ling; Zheng, Xuehua; Niu, Xuefeng; Sun, Caijun

    2015-01-01

    Influenza A virus infection can arrest autophagy, as evidenced by autophagosome accumulation in infected cells. Here, we report that this autophagosome accumulation can be inhibited by amantadine, an antiviral proton channel inhibitor, in amantadine-sensitive virus infected cells or cells expressing influenza A virus matrix protein 2 (M2). Thus, M2 proton channel activity plays a role in blocking the fusion of autophagosomes with lysosomes, which might be a key mechanism for arresting autophagy. PMID:26468520

  11. CMOS Active Pixel Sensors as energy-range detectors for proton Computed Tomography

    NASA Astrophysics Data System (ADS)

    Esposito, M.; Anaxagoras, T.; Evans, P. M.; Green, S.; Manolopoulos, S.; Nieto-Camero, J.; Parker, D. J.; Poludniowski, G.; Price, T.; Waltham, C.; Allinson, N. M.

    2015-06-01

    Since the first proof of concept in the early 70s, a number of technologies has been proposed to perform proton CT (pCT), as a means of mapping tissue stopping power for accurate treatment planning in proton therapy. Previous prototypes of energy-range detectors for pCT have been mainly based on the use of scintillator-based calorimeters, to measure proton residual energy after passing through the patient. However, such an approach is limited by the need for only a single proton passing through the energy-range detector in a read-out cycle. A novel approach to this problem could be the use of pixelated detectors, where the independent read-out of each pixel allows to measure simultaneously the residual energy of a number of protons in the same read-out cycle, facilitating a faster and more efficient pCT scan. This paper investigates the suitability of CMOS Active Pixel Sensors (APSs) to track individual protons as they go through a number of CMOS layers, forming an energy-range telescope. Measurements performed at the iThemba Laboratories will be presented and analysed in terms of correlation, to confirm capability of proton tracking for CMOS APSs.

  12. Protons inhibit Cl- conductance by direct or allosteric interaction with the GABA-binding site in the rat recombinant alpha1beta2gamma2L and alpha1beta2 GABAA receptor.

    PubMed

    Wang, Ming-De; Rahman, Mozibur; Zhu, Di

    2005-12-28

    Functional roles of external pH on the Cl- conductance were examined on Xenopus oocytes expressing rat recombinant alpha1beta2gamma2L and alpha1beta2 GABAA receptors. Acidic pH inhibited GABA-response in a reversible and concentration-dependent manner, significantly increasing the EC50 without appreciably changing the slope or maximal currents induced by GABA in the alpha1beta2gamma2L and alpha1beta2 receptors. In contrast, protonation did not influence the pentobarbital-gated currents in the alpha1beta2gamma2L receptors, suggesting that protons do not modulate channel activity by directly affecting the channel gating process. Protons competitively inhibited the bicuculline-induced antagonism on GABA in the alpha1beta2gamma2L receptors. The data support the hypothesis that protons inhibit GABAA receptor function by direct or allosteric interaction with the GABA-binding site.

  13. The molecular basis for species-specific activation of human TRPA1 protein by protons involves poorly conserved residues within transmembrane domains 5 and 6.

    PubMed

    de la Roche, Jeanne; Eberhardt, Mirjam J; Klinger, Alexandra B; Stanslowsky, Nancy; Wegner, Florian; Koppert, Wolfgang; Reeh, Peter W; Lampert, Angelika; Fischer, Michael J M; Leffler, Andreas

    2013-07-12

    The surveillance of acid-base homeostasis is concerted by diverse mechanisms, including an activation of sensory afferents. Proton-evoked activation of rodent sensory neurons is mainly mediated by the capsaicin receptor TRPV1 and acid-sensing ion channels. In this study, we demonstrate that extracellular acidosis activates and sensitizes the human irritant receptor TRPA1 (hTRPA1). Proton-evoked membrane currents and calcium influx through hTRPA1 occurred at physiological acidic pH values, were concentration-dependent, and were blocked by the selective TRPA1 antagonist HC030031. Both rodent and rhesus monkey TRPA1 failed to respond to extracellular acidosis, and protons even inhibited rodent TRPA1. Accordingly, mouse dorsal root ganglion neurons lacking TRPV1 only responded to protons when hTRPA1 was expressed heterologously. This species-specific activation of hTRPA1 by protons was reversed in both mouse and rhesus monkey TRPA1 by exchange of distinct residues within transmembrane domains 5 and 6. Furthermore, protons seem to interact with an extracellular interaction site to gate TRPA1 and not via a modification of intracellular N-terminal cysteines known as important interaction sites for electrophilic TRPA1 agonists. Our data suggest that hTRPA1 acts as a sensor for extracellular acidosis in human sensory neurons and should thus be taken into account as a yet unrecognized transduction molecule for proton-evoked pain and inflammation. The species specificity of this property is unique among known endogenous TRPA1 agonists, possibly indicating that evolutionary pressure enforced TRPA1 to inherit the role as an acid sensor in human sensory neurons.

  14. The Molecular Basis for Species-specific Activation of Human TRPA1 Protein by Protons Involves Poorly Conserved Residues within Transmembrane Domains 5 and 6*

    PubMed Central

    de la Roche, Jeanne; Eberhardt, Mirjam J.; Klinger, Alexandra B.; Stanslowsky, Nancy; Wegner, Florian; Koppert, Wolfgang; Reeh, Peter W.; Lampert, Angelika; Fischer, Michael J. M.; Leffler, Andreas

    2013-01-01

    The surveillance of acid-base homeostasis is concerted by diverse mechanisms, including an activation of sensory afferents. Proton-evoked activation of rodent sensory neurons is mainly mediated by the capsaicin receptor TRPV1 and acid-sensing ion channels. In this study, we demonstrate that extracellular acidosis activates and sensitizes the human irritant receptor TRPA1 (hTRPA1). Proton-evoked membrane currents and calcium influx through hTRPA1 occurred at physiological acidic pH values, were concentration-dependent, and were blocked by the selective TRPA1 antagonist HC030031. Both rodent and rhesus monkey TRPA1 failed to respond to extracellular acidosis, and protons even inhibited rodent TRPA1. Accordingly, mouse dorsal root ganglion neurons lacking TRPV1 only responded to protons when hTRPA1 was expressed heterologously. This species-specific activation of hTRPA1 by protons was reversed in both mouse and rhesus monkey TRPA1 by exchange of distinct residues within transmembrane domains 5 and 6. Furthermore, protons seem to interact with an extracellular interaction site to gate TRPA1 and not via a modification of intracellular N-terminal cysteines known as important interaction sites for electrophilic TRPA1 agonists. Our data suggest that hTRPA1 acts as a sensor for extracellular acidosis in human sensory neurons and should thus be taken into account as a yet unrecognized transduction molecule for proton-evoked pain and inflammation. The species specificity of this property is unique among known endogenous TRPA1 agonists, possibly indicating that evolutionary pressure enforced TRPA1 to inherit the role as an acid sensor in human sensory neurons. PMID:23709225

  15. Changes in active site histidine hydrogen bonding trigger cryptochrome activation.

    PubMed

    Ganguly, Abir; Manahan, Craig C; Top, Deniz; Yee, Estella F; Lin, Changfan; Young, Michael W; Thiel, Walter; Crane, Brian R

    2016-09-01

    Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions. PMID:27551082

  16. ENERGETIC PROTONS, RADIONUCLIDES, AND MAGNETIC ACTIVITY IN PROTOSTELLAR DISKS

    SciTech Connect

    Turner, N. J.; Drake, J. F.

    2009-10-01

    We calculate the location of the magnetically inactive dead zone in the minimum-mass protosolar disk, under ionization scenarios including stellar X-rays, long- or short-lived radionuclide decay, and energetic protons arriving from the general interstellar medium, from a nearby supernova explosion, from the disk corona, or from the corona of the young star. The disk contains a dead zone in all scenarios except those with small dust grains removed and a fraction of the short-lived radionuclides remaining in the gas. All the cases without exception have an 'undead zone' where intermediate resistivities prevent magneto-rotational turbulence while allowing shear-generated large-scale magnetic fields. The mass column in the undead zone is typically greater than the column in the turbulent surface layers. The results support the idea that the dead and undead zones are robust consequences of cold, dusty gas with mass columns exceeding 1000 g cm{sup -2}.

  17. Anti-angiogenic activity in metastasis of human breast cancer cells irradiated by a proton beam

    NASA Astrophysics Data System (ADS)

    Lee, Kyu-Shik; Shin, Jin-Sun; Nam, Kyung-Soo; Shon, Yun-Hee

    2012-07-01

    Angiogenesis is an essential process of metastasis in human breast cancer. We investigated the effects of proton beam irradiation on angiogenic enzyme activities and their expressions in MCF-7 human breast cancer cells. The regulation of angiogenic regulating factors, of transforming growth factor- β (TGF- β) and of vesicular endothelial growth factor (VEGF) expression in breast cancer cells irradiated with a proton beam was studied. Aromatase activity and mRNA expression, which is correlated with metastasis, were significantly decreased by irradiation with a proton beam in a dose-dependent manner. TGF- β and VEGF transcriptions were also diminished by proton beam irradiation. In contrast, transcription of tissue inhibitors of matrix metalloproteinases (TIMPs), also known as biological inhibitors of matrix metalloproteinases (MMPs), was dose-dependently enhanced. Furthermore, an increase in the expression of TIMPs caused th MMP-9 activity to be diminished and the MMP-9 and the MMP-2 expressions to be decreased. These results suggest that inhibition of angiogenesis by proton beam irradiation in breast cancer cells is closely related to inhibitions of aromatase activity and transcription and to down-regulation of TGF- β and VEGF transcription.

  18. Enzymatic catalysis of proton transfer at carbon: activation of triosephosphate isomerase by phosphite dianion.

    PubMed

    Amyes, Tina L; Richard, John P

    2007-05-15

    More than 80% of the rate acceleration for enzymatic catalysis of the aldose-ketose isomerization of (R)-glyceraldehyde 3-phosphate (GAP) by triosephosphate isomerase (TIM) can be attributed to the phosphodianion group of GAP [Amyes, T. L., O'Donoghue, A. C., and Richard, J. P. (2001) J. Am. Chem. Soc. 123, 11325-11326]. We examine here the necessity of the covalent connection between the phosphodianion and triose sugar portions of the substrate by "carving up" GAP into the minimal neutral two-carbon sugar glycolaldehyde and phosphite dianion pieces. This "two-part substrate" preserves both the alpha-hydroxycarbonyl and oxydianion portions of GAP. TIM catalyzes proton transfer from glycolaldehyde in D2O, resulting in deuterium incorporation that can be monitored by 1H NMR spectroscopy, with kcat/Km = 0.26 M-1 s-1. Exogenous phosphite dianion results in a very large increase in the observed second-order rate constant (kcat/Km)obsd for turnover of glycolaldehyde, and the dependence of (kcat/Km)obsd on [HPO32-] exhibits saturation. The data give kcat/Km = 185 M-1 s-1 for turnover of glycolaldehyde by TIM that is saturated with phosphite dianion so that the separate binding of phosphite dianion to TIM results in a 700-fold acceleration of proton transfer from carbon. The binding of phosphite dianion to the free enzyme (Kd = 38 mM) is 700-fold weaker than its binding to the fleeting complex of TIM with the altered substrate in the transition state (Kd = 53 muM); the total intrinsic binding energy of phosphite dianion in the transition state is 5.8 kcal/mol. We propose a physical model for catalysis by TIM in which the intrinsic binding energy of the substrate phosphodianion group is utilized to drive closing of the "mobile loop" and a protein conformational change that leads to formation of an active site environment that is optimally organized for stabilization of the transition state for proton transfer from alpha-carbonyl carbon.

  19. Active Sites Environmental Monitoring Program: Action levels

    SciTech Connect

    Ashwood, J.S.; Ashwood, T.L.

    1991-10-01

    The Active Sites Environmental Monitoring Program (ASEMP) was established at Oak Ridge National Laboratory to provide for early leak detection and to monitor performance of the active low-level waste disposal facilities in Solid Waste Storage Area (SWSA) 6 and the transuranic waste storage areas in SWSA 5 North. Early leak detection is accomplished by sampling runoff, groundwater, and perched water in burial trenches. Sample results are compared to action levels that represent background contamination by naturally occurring and fallout-derived radionuclides. 15 refs., 3 figs., 12 tabs.

  20. [Interaction of surface-active base with fraction of membrane-bound Williams's protons].

    PubMed

    Iaguzhinskiĭ, L S; Motovilov, K A; Volkov, E M; Eremeev, S A

    2013-01-01

    In the process of mitochondrial respiratory H(+)-pumps functioning, the fraction membrane-bound protons (R-protons), which have an excess of free energy is formed. According to R.J. Williams this fraction is included as energy source in the reaction of ATP synthesis. Previously, in our laboratory was found the formation of this fraction was found in the mitochondria and on the outer surface of mitoplast. On the mitoslast model we strictly shown that non-equilibrium R-proton fraction is localized on the surface of the inner mitochondrial membrane. In this paper a surface-active compound--anion of 2,4,6-trichloro-3-pentadecylphenol (TCP-C15) is described, which selectively interacts with the R-protons fraction in mitochondria. A detailed description of the specific interaction of the TCP-C15 with R-protons fraction in mitochondria is presented. Moreover, in this work it was found that phosphate transport system reacts with the R-protons fraction in mitochondria and plays the role of the endogenous volume regulation system of this fraction. The results of experiments are discussed in the terms of a local coupling model of the phosphorylation mechanism.

  1. Mechanism of pH-dependent activation of the sodium-proton antiporter NhaA

    NASA Astrophysics Data System (ADS)

    Huang, Yandong; Chen, Wei; Dotson, David L.; Beckstein, Oliver; Shen, Jana

    2016-10-01

    Escherichia coli NhaA is a prototype sodium-proton antiporter, which has been extensively characterized by X-ray crystallography, biochemical and biophysical experiments. However, the identities of proton carriers and details of pH-regulated mechanism remain controversial. Here we report constant pH molecular dynamics data, which reveal that NhaA activation involves a net charge switch of a pH sensor at the entrance of the cytoplasmic funnel and opening of a hydrophobic gate at the end of the funnel. The latter is triggered by charging of Asp164, the first proton carrier. The second proton carrier Lys300 forms a salt bridge with Asp163 in the inactive state, and releases a proton when a sodium ion binds Asp163. These data reconcile current models and illustrate the power of state-of-the-art molecular dynamics simulations in providing atomic details of proton-coupled transport across membrane which is challenging to elucidate by experimental techniques.

  2. Mechanism of pH-dependent activation of the sodium-proton antiporter NhaA

    PubMed Central

    Huang, Yandong; Chen, Wei; Dotson, David L.; Beckstein, Oliver; Shen, Jana

    2016-01-01

    Escherichia coli NhaA is a prototype sodium-proton antiporter, which has been extensively characterized by X-ray crystallography, biochemical and biophysical experiments. However, the identities of proton carriers and details of pH-regulated mechanism remain controversial. Here we report constant pH molecular dynamics data, which reveal that NhaA activation involves a net charge switch of a pH sensor at the entrance of the cytoplasmic funnel and opening of a hydrophobic gate at the end of the funnel. The latter is triggered by charging of Asp164, the first proton carrier. The second proton carrier Lys300 forms a salt bridge with Asp163 in the inactive state, and releases a proton when a sodium ion binds Asp163. These data reconcile current models and illustrate the power of state-of-the-art molecular dynamics simulations in providing atomic details of proton-coupled transport across membrane which is challenging to elucidate by experimental techniques. PMID:27708266

  3. Characterization of active sites in zeolite catalysts

    SciTech Connect

    Eckert, J.; Bug, A.; Nicol, J.M.

    1997-11-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). Atomic-level details of the interaction of adsorbed molecules with active sites in catalysts are urgently needed to facilitate development of more effective and/or environmentally benign catalysts. To this end the authors have carried out neutron scattering studies combined with theoretical calculations of the dynamics of small molecules inside the cavities of zeolite catalysts. The authors have developed the use of H{sub 2} as a probe of adsorption sites by observing the hindered rotations of the adsorbed H{sub 2} molecule, and they were able to show that an area near the four-rings is the most likely adsorption site for H{sub 2} in zeolite A while adsorption of H{sub 2} near cations located on six-ring sites decreases in strength as Ni {approximately} Co > Ca > Zn {approximately} Na. Vibrational and rotational motions of ethylene and cyclopropane adsorption complexes were used as a measure for zeolite-adsorbate interactions. Preliminary studies of the binding of water, ammonia, and methylamines were carried out in a number of related guest-host materials.

  4. An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

    SciTech Connect

    Guidinger, P.F.; Nowak, T. )

    1991-09-10

    The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 {plus minus} 0.025 M{sup {minus}1} min{sup {minus}1}. Inactivation by pyridoxal 5{prime}-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7,700 {plus minus} 860 m{sup {minus}1} min{sup {minus}1}. Treatment of the enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of k{sub obs} vs pH suggests this active-site lysine has a pK{sub a} of 8.1 and a pH-independent rate constant of inactivation of 47,700 m{sup {minus}1} min{sup {minus}1}. Proton relaxation rate measurements suggest that pyridoxal 5{prime}-phosphate modification alters binding of the phosphate-containing substrates. {sup 31}P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme {center dot}Mn{sup 2+}{center dot}substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5{prime}-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.

  5. Thermodynamic view of activation energies of proton transfer in various gramicidin A channels.

    PubMed

    Chernyshev, Anatoly; Cukierman, Samuel

    2002-01-01

    The temperature dependencies (range: 5-45 degrees C) of single-channel proton conductances (g(H)) in native gramicidin A (gA) and in two diastereoisomers (SS and RR) of the dioxolane-linked gA channels were measured in glycerylmonooleate/decane (GMO) and diphytanoylphosphatidylcholine/decane (DiPhPC) bilayers. Linear Arrhenius plots (ln (g(H)) versus K(-1)) were obtained for the native gA and RR channels in both types of bilayers, and for the SS channel in GMO bilayers only. The Arrhenius plot for proton transfer in the SS channel in DiPhPC bilayers had a break in linearity around 20 degrees C. This break seems to occur only when protons are the permeating cations in the SS channel. The activation energies (E(a)) for proton transfer in various gA channels (approximately 15 kJ/mol) are consistent with the rate-limiting step being in the channel and/or at the membrane-channel/solution interface, and not in bulk solution. E(a) values for proton transfer in gA channels are considerably smaller than for the permeation of nonproton currents in gA as well as in various other ion channels. The E(a) values for proton transfer in native gA channels are nearly the same in both GMO and DiPhPC bilayers. In contrast, for the dioxolane linked gA dimers, E(a) values were strongly modulated by the lipid environment. The Gibbs activation free energies (Delta G(#)(o)) for protons in various gA channels are within the range of 27-29 kJ/mol in GMO bilayers and of 20-22 kJ/mol in DiPhPC bilayers. The largest difference between Delta G(#)(o) for proton currents occurs between native gA (or SS channels) and the RR channel. In general, the activation entropy (Delta S) is mostly responsible for the differences between g(H) values in various gA channels, and also in distinct bilayers. However, significant differences between the activation enthalpies (Delta H(#)(o)) for proton transfer in the SS and RR channels occur in distinct membranes.

  6. Hybrid [FeFe]-hydrogenases with modified active sites show remarkable residual enzymatic activity.

    PubMed

    Siebel, Judith F; Adamska-Venkatesh, Agnieszka; Weber, Katharina; Rumpel, Sigrun; Reijerse, Edward; Lubitz, Wolfgang

    2015-02-24

    [FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S](2-)) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66-70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607-610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN(-) ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN(-) ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme. PMID:25633077

  7. Active Site and Laminarin Binding in Glycoside Hydrolase Family 55*

    PubMed Central

    Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; Udell, Hannah S.; Yik, Eric J.; Bergeman, Lai F.; Fox, Brian G.

    2015-01-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100–10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  8. The role of the K-channel and the active-site tyrosine in the catalytic mechanism of cytochrome c oxidase.

    PubMed

    Sharma, Vivek; Wikström, Mårten

    2016-08-01

    The active site of cytochrome c oxidase (CcO) comprises an oxygen-binding heme, a nearby copper ion (CuB), and a tyrosine residue that is covalently linked to one of the histidine ligands of CuB. Two proton-conducting pathways are observed in CcO, namely the D- and the K-channels, which are used to transfer protons either to the active site of oxygen reduction (substrate protons) or for pumping. Proton transfer through the D-channel is very fast, and its role in efficient transfer of both substrate and pumped protons is well established. However, it has not been fully clear why a separate K-channel is required, apparently for the supply of substrate protons only. In this work, we have analysed the available experimental and computational data, based on which we provide new perspectives on the role of the K-channel. Our analysis suggests that proton transfer in the K-channel may be gated by the protonation state of the active-site tyrosine (Tyr244) and that the neutral radical form of this residue has a more general role in the CcO mechanism than thought previously. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:26898520

  9. Computational Insights into Substrate and Site Specificities, Catalytic Mechanism, and Protonation States of the Catalytic Asp Dyad of β-Secretase

    PubMed Central

    Barman, Arghya; Prabhakar, Rajeev

    2014-01-01

    In this review, information regarding substrate and site specificities, catalytic mechanism, and protonation states of the catalytic Asp dyad of β-secretase (BACE1) derived from computational studies has been discussed. BACE1 catalyzes the rate-limiting step in the generation of Alzheimer amyloid beta peptide through the proteolytic cleavage of the amyloid precursor protein. Due to its biological functioning, this enzyme has been considered as one of the most important targets for finding the cure for Alzheimer's disease. Molecular dynamics (MD) simulations suggested that structural differences in the key regions (inserts A, D, and F and the 10s loop) of the enzyme are responsible for the observed difference in its activities towards the WT- and SW-substrates. The modifications in the flap, third strand, and insert F regions were found to be involved in the alteration in the site specificity of the glycosylphosphatidylinositol bound form of BACE1. Our QM and QM/MM calculations suggested that BACE1 hydrolyzed the SW-substrate more efficiently than the WT-substrate and that cleavage of the peptide bond occurred in the rate-determining step. The results from molecular docking studies showed that the information concerning a single protonation state of the Asp dyad is not enough to run an in silico screening campaign. PMID:25309776

  10. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.; Stringer, C.D.; Milanez, S.; Lee, E.H.

    1985-01-01

    Previous affinity labeling studies and comparative sequence analyses have identified two different lysines at the active site of ribulosebisphosphate carboxylase/oxygenase and have suggested their essentiality to function. The essential lysines occupy positions 166 and 329 in the Rhodospirillum rubrum enzyme and positions 175 and 334 in the spinach enzyme. Based on the pH-dependencies of inactivations of the two enzymes by trinitrobenzene sulfonate, Lys-166 (R. rubrum enzyme) exhibits a pK/sub a/ of 7.9 and Lys-334 (spinach enzyme) exhibits a pK/sub a/ of 9.0. These low pK/sub a/ values as well as the enhanced nucleophilicities of the lysyl residues argue that both are important to catalysis rather than to substrate binding. Lys-166 may correspond to the essential base that initiates catalysis and that displays a pK/sub a/ of 7.5 in the pH-curve for V/sub max//K/sub m/. Cross-linking experiments with 4,4'-diisothiocyano-2,2'-disulfonate stilbene demonstrate that the two active-site lysines are within 12 A. 50 refs., 7 figs., 1 tab.

  11. Superoxide activates a GDP-sensitive proton conductance in skeletal muscle mitochondria from king penguin (Aptenodytes patagonicus).

    PubMed

    Talbot, Darren A; Hanuise, Nicolas; Rey, Benjamin; Rouanet, Jean-Louis; Duchamp, Claude; Brand, Martin D

    2003-12-26

    We present the partial nucleotide sequence of the avian uncoupling protein (avUCP) gene from king penguin (Aptenodytes patagonicus), showing that the protein is 88-92% identical to chicken (Gallus gallus), turkey (Meleagris gallopavo), and hummingbird (Eupetomena macroura). We show that superoxide activates the proton conductance of mitochondria isolated from king penguin skeletal muscle. GDP abolishes the superoxide-activated proton conductance, indicating that it is mediated via avUCP. In the absence of superoxide there is no GDP-sensitive component of the proton conductance from penguin muscle mitochondria demonstrating that avUCP plays no role in the basal proton leak. PMID:14651968

  12. Proton transport by halorhodopsin

    SciTech Connect

    Varo, G.; Brown, L.S.; Needleman, R.

    1996-05-28

    In halorhodopsin from Natronobacterium pharaonis, a light-driven chloride pump, the chloride binding site also binds azide. When azide is bound at this location the retinal Schiff base transiently deprotonates after photoexcitation with light >530 nm, like in the light-driven proton pump bacteriorhodopsin. As in the photocycle of bacteriorhodopsin, pyranine detects the release of protons to the bulk. The subsequent reprotonation of the Schiff base is also dependent on azide, but with different kinetics that suggest a shuttling of protons from the surface as described earlier for halorhodopsin from Halobacterium salinarium. The azide-dependent, bacteriorhodopsin-like photocycle results in active electrogenic proton transport in the cytoplasmic to extracellular direction, detected in cell envelope vesicle suspensions both with a potential-sensitive electrode and by measuring light-dependent pH change. We conclude that in halorhodopsin an azide bound to the extracellular side of the Schiff base, and another azide shuttling between the Schiff base and the cytoplasmic surface, fulfill the functions of Asp-85 and Asp-96, respectively, in bacteriorhodopsin. Thus, although halorhodopsin is normally a chloride ion pump, it evidently contains all structural requirements, except an internal proton acceptor and a donor, of a proton pump. This observation complements our earlier finding that when a chloride binding site was created in bacteriorhodopsin through replacement of Asp-85 with a threonine, that protein became a chloride ion pump. 52 refs., 9 figs.

  13. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  14. Aminoalcohols as Probes of the Two-subsite Active Site of Beta-D-xylosidase from Selenomonas ruminantium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation state of catalytic base (D14, pKa 5.0) and catalytic acid (E186, pKa 7.2) which reside in subsite -1 of the two-subsite active site. Cationic aminoalcohols are shown to bind exclusively to subsite -1 of the ...

  15. Modulation of Active Site Electronic Structure by the Protein Matrix to Control [NiFe] Hydrogenase Reactivity

    SciTech Connect

    Smith, Dayle MA; Raugei, Simone; Squier, Thomas C.

    2014-09-30

    Control of the reactivity of the nickel center of the [NiFe] hydrogenase and other metalloproteins commonly involves outer coordination sphere ligands that act to modify the geometry and physical properties of the active site metal centers. We carried out a combined set of classical molecular dynamics and quantum/classical mechanics calculations to provide quantitative estimates of how dynamic fluctuations of the active site within the protein matrix modulate the electronic structure at the catalytic center. Specifically we focused on the dynamics of the inner and outer coordination spheres of the cysteinate-bound Ni–Fe cluster in the catalytically active Ni-C state. There are correlated movements of the cysteinate ligands and the surrounding hydrogen-bonding network, which modulate the electron affinity at the active site and the proton affinity of a terminal cysteinate. On the basis of these findings, we hypothesize a coupling between protein dynamics and electron and proton transfer reactions critical to dihydrogen production.

  16. Retinoids activate proton transport by the uncoupling proteins UCP1 and UCP2.

    PubMed

    Rial, E; González-Barroso, M; Fleury, C; Iturrizaga, S; Sanchis, D; Jiménez-Jiménez, J; Ricquier, D; Goubern, M; Bouillaud, F

    1999-11-01

    In mammalian brown adipose tissue, thermogenesis is explained by uncoupling mitochondrial respiration from ATP synthesis. Uncoupling protein-1 (UCP1) is responsible for this uncoupled state, because it allows proton re-entry into the matrix and thus dissipates the proton gradient generated by the respiratory chain. Proton transport by UCP1 is regulated negatively by nucleotides and positively by fatty acids. Adrenergic stimulation of brown adipocytes stimulates lipolysis and therefore enhances uncoupling and thermogenesis. Adrenergic stimulation also boosts ucp1 gene transcription. Since retinoic acid also promotes ucp1 gene transcription and its structure makes it a possible activator of UCP1, we hypothesized that retinoic acid, like noradrenaline, could have a dual action and trigger the activity of the protein UCP1 itself. Here we show that retinoic acid strongly increases proton transport by UCP1 in brown adipose tissue mitochondria and that it is much more potent than fatty acids. These data are corroborated with yeast mitochondria where UCP1 was introduced by genetic manipulation. The yeast expression system allows the comparison of the UCP1 with the newly described homologues UCP2 and UCP3. The search for regulators of UCP2 has demonstrated that it is positively regulated by retinoids in a pH-dependent manner.

  17. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    SciTech Connect

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  18. 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.

  19. Intracellular proton-mediated activation of TRPV3 channels accounts for the exfoliation effect of α-hydroxyl acids on keratinocytes.

    PubMed

    Cao, Xu; Yang, Fan; Zheng, Jie; Wang, Kewei

    2012-07-27

    α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders.

  20. Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation

    SciTech Connect

    Marti, T.; Otto, H.; Mogi, T.; Roesselet, S.J.H.; Heyn, M.P.; Khorana, H.G. )

    1991-04-15

    To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.

  1. Biological effects of passive versus active scanning proton beams on human lung epithelial cells.

    PubMed

    Gridley, Daila S; Pecaut, Michael J; Mao, Xiao W; Wroe, Andrew J; Luo-Owen, Xian

    2015-02-01

    The goal was to characterize differences in cell response after exposure to active beam scanning (ABS) protons compared to a passive delivery system. Human lung epithelial (HLE) cells were evaluated at various locations along the proton depth dose profile. The dose delivered at the Bragg peak position was essentially identical (∼4 Gy) with the two techniques, but depth dose data showed that ABS resulted in lower doses at entry and more rapid drop-off after the peak. Average dose rates for the passive and ABS beams were 1.1 Gy/min and 5.1 Gy/min, respectively; instantaneous dose rates were 19.2 Gy/min and 2,300 Gy/min (to a 0.5 × 0.5 mm(2) voxel). Analysis of DNA synthesis was based on (3)H-TdR incorporation. Quantitative real-time polymerase chain reaction (RT-PCR) was done to determine expression of genes related to p53 signaling and DNA damage; a total of 152 genes were assessed. Spectral karyotyping and analyses of the Golgi apparatus and cytokines produced by the HLE cells were also performed. At or near the Bragg peak position, ABS protons resulted in a greater decrease in DNA synthesis compared to passively delivered protons. Genes with >2-fold change (P < 0.05 vs. 0 Gy) after passive proton irradiation at one or more locations within the Bragg curve were BTG2, CDKN1A, IFNB1 and SIAH1. In contrast, many more genes had >2-fold difference with ABS protons: BRCA1, BRCA2, CDC25A, CDC25C, CCNB2, CDK1, DMC1, DNMT1, E2F1, EXO1, FEN1, GADD45A, GTSE1, IL-6, JUN, KRAS, MDM4, PRC1, PTTG1, RAD51, RPA1, TNF, WT1, XRCC2, XRCC3 and XRCC6BP1. Spectral karyotyping revealed numerous differences in chromosomal abnormalities between the two delivery systems, especially at or near the Bragg peak. Percentage of cells staining for the Golgi apparatus was low after exposure to passive and active proton beams. Studies such as this are needed to ensure patient safety and make modifications in ABS delivery, if necessary.

  2. Receptor kinase-mediated control of primary active proton pumping at the plasma membrane.

    PubMed

    Fuglsang, Anja T; Kristensen, Astrid; Cuin, Tracey A; Schulze, Waltraud X; Persson, Jörgen; Thuesen, Kristina H; Ytting, Cecilie K; Oehlenschlæger, Christian B; Mahmood, Khalid; Sondergaard, Teis E; Shabala, Sergey; Palmgren, Michael G

    2014-12-01

    Acidification of the cell wall space outside the plasma membrane is required for plant growth and is the result of proton extrusion by the plasma membrane-localized H+-ATPases. Here we show that the major plasma membrane proton pumps in Arabidopsis, AHA1 and AHA2, interact directly in vitro and in planta with PSY1R, a receptor kinase of the plasma membrane that serves as a receptor for the peptide growth hormone PSY1. The intracellular protein kinase domain of PSY1R phosphorylates AHA2/AHA1 at Thr-881, situated in the autoinhibitory region I of the C-terminal domain. When expressed in a yeast heterologous expression system, the introduction of a negative charge at this position caused pump activation. Application of PSY1 to plant seedlings induced rapid in planta phosphorylation at Thr-881, concomitant with an instantaneous increase in proton efflux from roots. The direct interaction between AHA2 and PSY1R observed might provide a general paradigm for regulation of plasma membrane proton transport by receptor kinases.

  3. The role of proton mobility in determining the energy-resolved vibrational activation/dissociation channels of N-glycopeptide ions.

    PubMed

    Kolli, Venkata; Roth, Heidi A; De La Cruz, Gabriela; Fernando, Ganga S; Dodds, Eric D

    2015-10-01

    Site-specific glycoproteomic analysis largely hinges on the use of tandem mass spectrometry (MS/MS) to identify glycopeptides. Experiments of this type are usually aimed at drawing connections between individual oligosaccharide structures and their specific sites of attachment to the polypeptide chain. These determinations inherently require ion dissociation methods capable of interrogating both the monosaccharide and amino acid connectivity of the glycopeptide. Collision-induced dissociation (CID) shows potential to satisfy this requirement, as the vibrational activation/dissociation of protonated N-glycopeptides has been observed to access cleavage of either glycosidic bonds of the glycan or amide bonds of the peptide in an energy-resolved manner. Nevertheless, the relative energy requirement for these fragmentation pathways varies considerably among analytes. This research addresses the influence of proton mobility on the vibrational energy necessary to achieve either glycan or peptide cleavage in a collection of protonated N-glycopeptide ions. While greater proton mobility of the precursor ion was found to correlate with lower energy requirements for precursor ion depletion and appearance of glycosidic fragments, the vibrational energy deposition necessary for appearance of peptide backbone fragments showed no relation to the precursor ion proton mobility. These results are consistent with observations suggesting that peptide fragments arise from an intermediate fragment which is generally of lower proton mobility than the precursor ion. Such findings have potential to facilitate the rational selection of CID conditions which are best suited to provide either glycan or peptide cleavage products in MS/MS based N-glycoproteomic analysis.

  4. Study of proton and neutron activation of metal samples in low Earth orbit

    NASA Technical Reports Server (NTRS)

    Laird, C. E.

    1984-01-01

    Progress in the following activities has been made: the analysis of the gamma ray spectra taken from samples flown in Spacelab 2; the search for and review of neutron and proton activation cross sections needed to analyze the results of the Long Duration Exposure Facility (LDEF) activation measurements; the consideration given to data analysis of the LDEF and Spacelab 2 samples; the plan to measure relevant cross sections with nuclear accelerator measurements; and the preparation of an extended gamma ray calibration sources continues through planning and direct measurement of gamma ray efficiency for a Ge(Li) as a function of position along the surface of the detector housing.

  5. protonation behavior of histidine during HSF1 activation by physiological acidification.

    PubMed

    Lu, Ming; Park, Jang-Su

    2015-06-01

    The expression of eukaryotic molecular chaperones (heat shock proteins, HSPs) is triggered in response to a wide range of environmental stresses, including: heat shock, hydrogen peroxide, heavy metal, low-pH, or virus infection. Biochemical and genetic studies have clearly shown the fundamental roles of heat shock factor 1 (HSF1) in stress-inducible HSP gene expression, resistance to stress-induced cell death, carcinogenesis, and other biological phenomena. Previous studies show that acidic pH changes within the physiological range directly activate the HSF1 function in vitro. However, the detailed mechanism is unclear. Though computational pKa-predications of the amino acid side-chain, acidic-pH induced protonation of a histidine residue was found to be most-likely involved in this process. The histidine 83 (His83) residue, which could be protonated by mild decrease in pH, causes mild acidic-induced HSF1 activation (including in-vitro trimerization, DNA binding, in-vivo nuclear accumulation, and HSPs expression). His83, which is located in the loop region of the HSF1 DNA binding domain, was suggested to enhance the intermolecular force with Arginine 79, which helps HSF1 form a DNA-binding competent. Therefore, low-pH-induced activation of HSF1 by the protonation of histidine can help us better to understand the HSF1 mechanism and develop more therapeutic applications (particularly in cancer therapy). J. Cell. Biochem. 116: 977-984, 2015. © 2015 Wiley Periodicals, Inc.

  6. Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine 200.

    PubMed

    Kovaleva, Elena G; Rogers, Melanie S; Lipscomb, John D

    2015-09-01

    Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady-state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homoprotocatechuate, 4-sulfonylcatechol, and 4-nitrocatechol) possessing substituents with different inductive capacity. Structures determined at 1.35-1.75 Å resolution show that there is essentially no change in overall active site architecture or substrate binding mode for these variants when compared to the structures of the wild-type enzyme and its analogous complexes. This shows that the maximal 50-fold decrease in kcat for ring cleavage, the dramatic changes in pH dependence, and the switch from ring cleavage to ring oxidation of 4-nitrocatechol by the FeHPCD variants can be attributed specifically to the properties of the altered second-sphere residue and the substrate. The results suggest that proton transfer is necessary for catalysis, and that it occurs most efficiently when the substrate provides the proton and His200 serves as a catalyst. However, in the absence of an available substrate proton, a defined proton-transfer pathway in the protein can be utilized. Changes in the steric bulk and charge of the residue at position 200 appear to be capable of altering the rate-limiting step in catalysis and, perhaps, the nature of the reactive species.

  7. Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine-200

    PubMed Central

    Kovaleva, Elena G.; Rogers, Melanie S.; Lipscomb, John D.

    2015-01-01

    Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homoprotocatechuate, 4-sulfonylcatechol, and 4-nitrocatechol) possessing substituents with different inductive capacity. Structures solved at 1.35 –1.75 Å resolution show that there is essentially no change in overall active site architecture or substrate binding mode for these variants when compared to the structures of the wild type enzyme and its analogous complexes. This shows that the maximal 50-fold decrease in kcat for ring cleavage, the dramatic changes in pH dependence, and the switch from ring cleavage to ring oxidation of 4-nitrocatechol by the FeHPCD variants can be attributed specifically to the properties of the altered second sphere residue and the substrate. The results suggest that proton transfer is necessary for catalysis, and that it occurs most efficiently when the substrate provides the proton and His200 serves as a catalyst. However, in the absence of an available substrate proton, a defined proton-transfer pathway in the protein can be utilized. Changes in steric bulk and charge of the residue at position 200 appear capable of altering the rate-limiting step in catalysis, and perhaps, the nature of the reactive species. PMID:26267790

  8. Control of active sites in flocculation: Concept of equivalent active sites''

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    Flocculation and dispersion of solids are strong functions of the amount and conformation of the adsorbed polymer. Regions of dispersion and flocculation of solids with particular polymer molecules may be deduced from saturation adsorption data. The concept of equivalent active sites'' is proposed to explain flocculation and dispersion behavior irrespective of the amount or conformation of the adsorbed polymer. The concept has been further extended to study the selective flocculation process.

  9. The two active sites in human branched-chain alpha-keto acid dehydrogenase operate independently without an obligatory alternating-site mechanism.

    PubMed

    Li, Jun; Machius, Mischa; Chuang, Jacinta L; Wynn, R Max; Chuang, David T

    2007-04-20

    A long standing controversy is whether an alternating activesite mechanism occurs during catalysis in thiamine diphosphate (ThDP)-dependent enzymes. We address this question by investigating the ThDP-dependent decarboxylase/dehydrogenase (E1b) component of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC). Our crystal structure reveals that conformations of the two active sites in the human E1b heterotetramer harboring the reaction intermediate are identical. Acidic residues in the core of the E1b heterotetramer, which align with the proton-wire residues proposed to participate in active-site communication in the related pyruvate dehydrogenase from Bacillus stearothermophilus, are mutated. Enzyme kinetic data show that, except in a few cases because of protein misfolding, these alterations are largely without effect on overall activity of BCKDC, ruling out the requirement of a proton-relay mechanism in E1b. BCKDC overall activity is nullified at 50% phosphorylation of E1b, but it is restored to nearly half of the pre-phosphorylation level after dissociation and reconstitution of BCKDC with the same phosphorylated E1b. The results suggest that the abolition of overall activity likely results from the specific geometry of the half-phosphorylated E1b in the BCKDC assembly and not due to a disruption of the alternating active-site mechanism. Finally, we show that a mutant E1b containing only one functional active site exhibits half of the wild-type BCKDC activity, which directly argues against the obligatory communication between active sites. The above results provide evidence that the two active sites in the E1b heterotetramer operate independently during the ThDP-dependent decarboxylation reaction. PMID:17329260

  10. A Threonine on the Active Site Loop Controls Transition State Formation in Escherichia Coli Respiratory Complex II

    SciTech Connect

    Tomasiak, T.M.; Maklashina, E.; Cecchini, G.; Iverson, T.M.

    2009-05-26

    In Escherichia coli, the complex II superfamily members succinate:ubiquinone oxidoreductase (SQR) and quinol:fumarate reductase (QFR) participate in aerobic and anaerobic respiration, respectively. Complex II enzymes catalyze succinate and fumarate interconversion at the interface of two domains of the soluble flavoprotein subunit, the FAD binding domain and the capping domain. An 11-amino acid loop in the capping domain (Thr-A234 to Thr-A244 in quinol:fumarate reductase) begins at the interdomain hinge and covers the active site. Amino acids of this loop interact with both the substrate and a proton shuttle, potentially coordinating substrate binding and the proton shuttle protonation state. To assess the loop's role in catalysis, two threonine residues were mutated to alanine: QFR Thr-A244 (act-T; Thr-A254 in SQR), which hydrogen-bonds to the substrate at the active site, and QFR Thr-A234 (hinge-T; Thr-A244 in SQR), which is located at the hinge and hydrogen-bonds the proton shuttle. Both mutations impair catalysis and decrease substrate binding. The crystal structure of the hinge-T mutation reveals a reorientation between the FAD-binding and capping domains that accompanies proton shuttle alteration. Taken together, hydrogen bonding from act-T to substrate may coordinate with interdomain motions to twist the double bond of fumarate and introduce the strain important for attaining the transition state.

  11. Sustained activation of proton channels and NADPH oxidase in human eosinophils and murine granulocytes requires PKC but not cPLA2α activity

    PubMed Central

    Morgan, Deri; Cherny, Vladimir V; Finnegan, Alison; Bollinger, James; Gelb, Michael H; DeCoursey, Thomas E

    2007-01-01

    The prevailing hypothesis that a signalling pathway involving cPLA2α is required to enhance the gating of the voltage-gated proton channel associated with NADPH oxidase was tested in human eosinophils and murine granulocytes. This hypothesis invokes arachidonic acid (AA) liberated by cPLA2α as a final activator of proton channels. In human eosinophils studied in the perforated-patch configuration, phorbol myristate acetate (PMA) stimulation elicited NADPH oxidase-generated electron current (Ie) and enhanced proton channel gating identically in the presence or absence of three specific cPLA2α inhibitors, Wyeth-1, pyrrolidine-2 and AACOCF3 (arachidonyl trifluoromethyl ketone). In contrast, PKC inhibitors GFX (GF109203X) or staurosporine prevented the activation of either proton channels or NADPH oxidase. PKC inhibition during the respiratory burst reversed the activation of both molecules, suggesting that ongoing phosphorylation is required. This effect of GFX was inhibited by okadaic acid, implicating phosphatases in proton channel deactivation. Proton channel activation by AA was partially reversed by GFX or staurosporine, indicating that AA effects are due in part to activation of PKC. In granulocytes from mice with the cPLA2α gene disrupted (knockout mice), PMA or fMetLeuPhe activated NADPH oxidase and proton channels in a manner indistinguishable from the responses of control cells. Thus, cPLA2α is not essential to activate the proton conductance or for a normal respiratory burst. Instead, phosphorylation of the proton channel or an activating molecule converts the channel to its activated gating mode. The existing paradigm for regulation of the concerted activity of proton channels and NADPH oxidase must be revised. PMID:17185330

  12. Activation cross sections of proton induced nuclear reactions on gold up to 65MeV.

    PubMed

    Ditrói, F; Tárkányi, F; Takács, S; Hermanne, A

    2016-07-01

    Activation cross sections of proton induced reactions on gold for production of (197m,197g,195m,195g, 193m,193g,192)Hg, (196m,196g(cum),195g(cum),194,191(cum))Au, (191(cum))Pt and (192)Ir were measured up to 65MeV proton energy, some of them for the first time. The new data are in acceptably good agreement with the recently published earlier experimental data in the overlapping energy region. The experimental data are compared with the predictions of the TALYS 1.6 (results in TENDL-2015 on-line library) and EMPIRE 3.2 code. PMID:27156194

  13. Structural and energetic determinants of proton transfer in bacteriorhodopsin

    SciTech Connect

    Bondar, A.N.; Smith, Jeremy C; Fischer, S.

    2006-05-01

    In the light-driven bacteriorhodopsin proton pump, the first proton transfer step is from the retinal Schiff base to a nearby carboxylate group. The mechanism of this transfer step is highly controversial, in particular whether a direct proton jump is allowed. Here, we review the structural and energetic determinants of the direct proton transfer path computed by using a combined quantum mechanical/molecular mechanical approach. Both protein flexibility and electrostatic interactions play an important role in shaping the proton transfer energy profile. Detailed analysis of the energetics of putative transitions in the first half of the photocycle focuses on two elements that determine the likelihood that a given configuration of the active site is populated during the proton-pumping cycle. First, the rate-limiting barrier for proton transfer must be consistent with the kinetics of the photocycle. Second, the active-site configuration must be compatible with a productive overall pumping cycle.

  14. Modelling active sites for the Beckmann rearrangement reaction in boron-containing zeolites and their interaction with probe molecules.

    PubMed

    Lezcano-González, Inés; Vidal-Moya, Alejandro; Boronat, Mercedes; Blasco, Teresa; Corma, Avelino

    2010-06-28

    Theoretical calculations and in situ solid state NMR spectroscopy have been combined to get insight on the nature of the active sites for the Beckmann rearrangement reaction in borosilicate zeolites. The interaction of a B site in zeolite Beta with a series of probe molecules (ammonia, pyridine, acetone and water) has been modelled and the (15)N and (11)B NMR isotropic chemical shift of the resulting complexes calculated and compared with experimental in situ NMR results. This approach has allowed validation of the methodology to model the adsorption on a zeolite boron site of molecules of varying basicity which are either protonated or non-protonated. The limitation is that theoretical calculations overestimate the effect of molecular adsorption through hydrogen bonds on the calculated isotropic (11)B NMR chemical shift.Theoretical and experimental results on the adsorption of acetophenone and cyclohexanone oximes on zeolite B-Beta indicate that Brønsted acid sites protonate the oximes, changing the boron coordination from trigonal to tetrahedral. Comparison of theoretical and experimental (15)N NMR chemical shifts of the adsorbed amides (acetanilide and epsilon-caprolactam) indicates that they are non-protonated, and the (11)B NMR spectra show that, as expected, boron remains in trigonal coordination with an isotropic delta(11)B(exp) which differs from the calculated value delta(11)B(calc).

  15. Modelling active sites for the Beckmann rearrangement reaction in boron-containing zeolites and their interaction with probe molecules.

    PubMed

    Lezcano-González, Inés; Vidal-Moya, Alejandro; Boronat, Mercedes; Blasco, Teresa; Corma, Avelino

    2010-06-28

    Theoretical calculations and in situ solid state NMR spectroscopy have been combined to get insight on the nature of the active sites for the Beckmann rearrangement reaction in borosilicate zeolites. The interaction of a B site in zeolite Beta with a series of probe molecules (ammonia, pyridine, acetone and water) has been modelled and the (15)N and (11)B NMR isotropic chemical shift of the resulting complexes calculated and compared with experimental in situ NMR results. This approach has allowed validation of the methodology to model the adsorption on a zeolite boron site of molecules of varying basicity which are either protonated or non-protonated. The limitation is that theoretical calculations overestimate the effect of molecular adsorption through hydrogen bonds on the calculated isotropic (11)B NMR chemical shift.Theoretical and experimental results on the adsorption of acetophenone and cyclohexanone oximes on zeolite B-Beta indicate that Brønsted acid sites protonate the oximes, changing the boron coordination from trigonal to tetrahedral. Comparison of theoretical and experimental (15)N NMR chemical shifts of the adsorbed amides (acetanilide and epsilon-caprolactam) indicates that they are non-protonated, and the (11)B NMR spectra show that, as expected, boron remains in trigonal coordination with an isotropic delta(11)B(exp) which differs from the calculated value delta(11)B(calc). PMID:20454729

  16. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  17. Research activities at the Loma Linda University and Proton Treatment Facility--an overview

    NASA Technical Reports Server (NTRS)

    Nelson, G. A.; Green, L. M.; Gridley, D. S.; Archambeau, J. O.; Slater, J. M.

    2001-01-01

    The Loma Linda University (LLU) Radiobiology Program coordinates basic research and proton beam service activities for the university and extramural communities. The current focus of the program is on the biological and physical properties of protons and the operation of radiobiology facilities for NASA-sponsored projects. The current accelerator, supporting facilities and operations are described along with a brief review of extramural research projects supported by the program. These include space craft electronic parts and shielding testing as well as tumorigenesis and animal behavior experiments. An overview of research projects currently underway at LLU is also described. These include: 1) acute responses of the C57Bl/6 mouse immune system, 2) modulation of gene expression in the nematode C. elegans and rat thyroid cells, 3) quantitation of dose tolerance in rat CNS microvasculature, 4) behavioral screening of whole body proton and iron ion-irradiated C57Bl/6 mice, and 5) investigation of the role of cell integration into epithelial structures on responses to radiation.

  18. SU-E-J-49: Distal Edge Activity Fall Off Of Proton Therapy Beams

    SciTech Connect

    Elmekawy, A; Ewell, L; Butuceanu, C; Zhu, L

    2014-06-01

    Purpose: To characterize and quantify the distal edge activity fall off, created in a phantom by a proton therapy beam Method and Materials: A 30x30x10cm polymethylmethacrylate phantom was irradiated with a proton therapy beam using different ranges and beams. The irradiation volume is approximated by a right circular cylinder of diameter 7.6cm and varying lengths. After irradiation, the phantom was scanned via a Philips Gemini Big Bore™ PET-CT for isotope activation. Varian Eclipse™ treatment planning system as well as ImageJ™ were used to analyze the resulting PET and CT scans. The region of activity within the phantom was longitudinally measured as a function of PET slice number. Dose estimations were made via Monte Carlo (GATE) simulation. Results: For both the spread out Bragg peak (SOBP) and the mono-energetic pristine Bragg peak proton beams, the proximal activation rise was steep: average slope −0.735 (average intensity/slice number) ± 0.091 (standard deviation) for the pristine beams and −1.149 ± 0.117 for the SOBP beams. In contrast, the distal fall offs were dissimilar. The distal fall off in activity for the pristine beams was fit well by a linear curve: R{sup 2} (Pierson Product) was 0.9968, 0.9955 and 0.9909 for the 13.5, 17.0 and 21.0cm range beams respectively. The good fit allows for a slope comparison between the different ranges. The slope varied as a function of range from 1.021 for the 13.5cm beam to 0.8407 (average intensity/slice number) for the 21.0cm beam. This dependence can be characterized: −0.0234(average intensity/slice number/cm range). For the SOBP beams, the slopes were significantly less and were also less linear: average slope 0.2628 ± 0.0474, average R{sup 2}=0.9236. Conclusion: The distal activation fall off edge for pristine proton beams was linear and steep. The corresponding quantities for SOBP beams were shallower and less linear. Philips has provided support for this work.

  19. Release of halide ions from the buried active site of the haloalkane dehalogenase LinB revealed by stopped-flow fluorescence analysis and free energy calculations.

    PubMed

    Hladilkova, Jana; Prokop, Zbynek; Chaloupkova, Radka; Damborsky, Jiri; Jungwirth, Pavel

    2013-11-21

    Release of halide ions is an essential step of the catalytic cycle of haloalkane dehalogenases. Here we describe experimentally and computationally the process of release of a halide anion from the buried active site of the haloalkane dehalogenase LinB. Using stopped-flow fluorescence analysis and umbrella sampling free energy calculations, we show that the anion binding is ion-specific and follows the ordering I(-) > Br(-) > Cl(-). We also address the issue of the protonation state of the catalytic His272 residue and its effect on the process of halide release. While deprotonation of His272 increases binding of anions in the access tunnel, we show that the anionic ordering does not change with the switch of the protonation state. We also demonstrate that a sodium cation could relatively easily enter the active site, provided the His272 residue is singly protonated, and replace thus the missing proton. In contrast, Na(+) is strongly repelled from the active site containing the doubly protonated His272 residue. Our study contributes toward understanding of the reaction mechanism of haloalkane dehalogenase enzyme family. Determination of the protonation state of the catalytic histidine throughout the catalytic cycle remains a challenge for future studies.

  20. The selectivity of the Na+/K+-pump is controlled by binding site protonation and self-correcting occlusion

    PubMed Central

    Rui, Huan; Artigas, Pablo; Roux, Benoît

    2016-01-01

    The Na+/K+-pump maintains the physiological K+ and Na+ electrochemical gradients across the cell membrane. It operates via an 'alternating-access' mechanism, making iterative transitions between inward-facing (E1) and outward-facing (E2) conformations. Although the general features of the transport cycle are known, the detailed physicochemical factors governing the binding site selectivity remain mysterious. Free energy molecular dynamics simulations show that the ion binding sites switch their binding specificity in E1 and E2. This is accompanied by small structural arrangements and changes in protonation states of the coordinating residues. Additional computations on structural models of the intermediate states along the conformational transition pathway reveal that the free energy barrier toward the occlusion step is considerably increased when the wrong type of ion is loaded into the binding pocket, prohibiting the pump cycle from proceeding forward. This self-correcting mechanism strengthens the overall transport selectivity and protects the stoichiometry of the pump cycle. DOI: http://dx.doi.org/10.7554/eLife.16616.001 PMID:27490484

  1. The selectivity of the Na(+)/K(+)-pump is controlled by binding site protonation and self-correcting occlusion.

    PubMed

    Rui, Huan; Artigas, Pablo; Roux, Benoît

    2016-01-01

    The Na(+)/K(+)-pump maintains the physiological K(+) and Na(+) electrochemical gradients across the cell membrane. It operates via an 'alternating-access' mechanism, making iterative transitions between inward-facing (E1) and outward-facing (E2) conformations. Although the general features of the transport cycle are known, the detailed physicochemical factors governing the binding site selectivity remain mysterious. Free energy molecular dynamics simulations show that the ion binding sites switch their binding specificity in E1 and E2. This is accompanied by small structural arrangements and changes in protonation states of the coordinating residues. Additional computations on structural models of the intermediate states along the conformational transition pathway reveal that the free energy barrier toward the occlusion step is considerably increased when the wrong type of ion is loaded into the binding pocket, prohibiting the pump cycle from proceeding forward. This self-correcting mechanism strengthens the overall transport selectivity and protects the stoichiometry of the pump cycle. PMID:27490484

  2. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, Virginia C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program --now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history The missions will develop technology and acquire data necessary for eventual human Exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines be opportunities for the Mars community to provide input into the landing site selection process.

  3. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, V. C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program -- now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history. The missions will develop technology and acquire data necessary for eventual human exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines the opportunities for the Mars community to provide input into the landing site selection process.

  4. Proton interrogation

    SciTech Connect

    Morris, Christopher L

    2008-01-01

    Energetic proton beams may provide an attractive alternative when compared to electromagnetic and neutron beams for active interrogation of nuclear threats because: they have large fission cross sections, long mean free paths and high penetration, and proton beams can be manipulated with magnetic optics. We have measured time-dependent cross sections for delayed neutrons and gamma-rays using the 800 MeV proton beam from the Los Alamos Neutron Science Center for a set of bare and shielded targets. The results show significant signals from both unshielded and shielded nuclear materials. Results will be presented.

  5. Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site*S

    PubMed Central

    Maresso, Anthony W.; Wu, Ruiying; Kern, Justin W.; Zhang, Rongguang; Janik, Dorota; Missiakas, Dominique M.; Duban, Mark-Eugene; Joachimiak, Andrzej; Schneewind, Olaf

    2011-01-01

    Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of sortase leads to large reductions in virulence, which identifies sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (β-amino)ethyl ketones inhibit sortase enzymes from staphylococci and bacilli. Inhibition of sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via β-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery. PMID:17545669

  6. The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

    S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the

  7. Site-specific protonation kinetics of acidic side chains in proteins determined by pH-dependent carboxyl (13)C NMR relaxation.

    PubMed

    Wallerstein, Johan; Weininger, Ulrich; Khan, M Ashhar I; Linse, Sara; Akke, Mikael

    2015-03-01

    Proton-transfer dynamics plays a critical role in many biochemical processes, such as proton pumping across membranes and enzyme catalysis. The large majority of enzymes utilize acid-base catalysis and proton-transfer mechanisms, where the rates of proton transfer can be rate limiting for the overall reaction. However, measurement of proton-exchange kinetics for individual side-chain carboxyl groups in proteins has been achieved in only a handful of cases, which typically have involved comparative analysis of mutant proteins in the context of reaction network modeling. Here we describe an approach to determine site-specific protonation and deprotonation rate constants (kon and koff, respectively) of carboxyl side chains, based on (13)C NMR relaxation measurements as a function of pH. We validated the method using an extensively studied model system, the B1 domain of protein G, for which we measured rate constants koff in the range (0.1-3) × 10(6) s(-1) and kon in the range (0.6-300) × 10(9) M(-1) s(-1), which correspond to acid-base equilibrium dissociation constants (Ka) in excellent agreement with previous results determined by chemical shift titrations. Our results further reveal a linear free-energy relationship between log kon and pKa, which provides information on the free-energy landscape of the protonation reaction, showing that the variability among residues in these parameters arises primarily from the extent of charge stabilization of the deprotonated state by the protein environment. We find that side-chain carboxyls with extreme values of koff or kon are involved in hydrogen bonding, thus providing a mechanistic explanation for the observed stabilization of the protonated or deprotonated state.

  8. Study of proton and neutron activation of metal samples in low Earth orbit

    NASA Technical Reports Server (NTRS)

    Laird, C. E.

    1984-01-01

    The analysis of the gamma-ray spectra taken from samples flown in Spacelab 1; the search for and review of neutron and proton activation cross-sections needed to analyze the results of the Long Duration Exposure Facility (LDEF) activation measurements; additional calculations of neutron induced activation for the LDEF samples; the data analysis plan for the LDEF and Spacelab 2 samples; the measurement of relevant cross-sections with activation of samples of V, Co, In, and Ta at the Indiana University Cyclotron Facility; and the preparation of an extended gamma-ray calibration source through the development of a proper technique to accurately deposit equal quantities of radioactive material onto a large number of point on the source are discussed.

  9. The active site of oxidative phosphorylation and the origin of hyperhomocysteinemia in aging and dementia.

    PubMed

    McCully, Kilmer S

    2015-01-01

    The active site of oxidative phosphorylation and adenosine triphosphate (ATP) synthesis in mitochondria is proposed to consist of two molecules of thioretinamide bound to cobalamin, forming thioretinaco, complexed with ozone, oxygen, nicotinamide adenine dinucleotide. and inorganic phosphate, TR2CoO3O2NAD(+)H2PO4(-). Reduction of the pyridinium nitrogen of the nicotinamide group by an electron from electron transport complexes initiates polymerization of phosphate with adenosine diphosphate, yielding nicotinamide riboside and ATP bound to thioretinaco ozonide oxygen. A second electron reduces oxygen to hydroperoxyl radical, releasing ATP from the active site. A proton gradient is created within F1F0 ATPase complexes of mitochondria by reaction of protons with reduced nicotinamide riboside and with hydroperoxyl radical, yielding reduced nicotinamide riboside and hydroperoxide. The hyperhomocysteinemia of aging and dementia is attributed to decreased synthesis of adenosyl methionine by thioretinaco ozonide and ATP, causing decreased allosteric activation of cystathionine synthase and decreased allosteric inhibition of methylenetetrahydrofolate reductase and resulting in dysregulation of methionine metabolism. PMID:25887881

  10. Effect of an uncE ribosome-binding site mutation on the synthesis and assembly of the Escherichia coli proton-translocating ATPase.

    PubMed

    Solomon, K A; Brusilow, W S

    1988-04-15

    Plasmid pRPG54, which carries the genes for the eight subunits of the proton-translocating ATPase of Escherichia coli, has been found to carry a single base change of a G to an A in the ribosome-binding site for uncE, the gene which codes for the N,N'-dicyclohexylcarbodiimide-binding subunit c of the Fo. This noncoding region mutation both lowers expression of uncE by a factor of 2-3 and affects the function of the ATPase, specifically of the Fo sector. The presence of the mutation results in a decrease in the proton permeability of the Fo or of the entire F1Fo-ATPase complex when either is synthesized from genes on a multicopy plasmid. Expression of uncE from an F1Fo plasmid carrying the wild type ribosome binding site results in increased membrane proton permeability and decreased ability of the resultant ATPase to couple a transmembrane proton gradient to ATP synthesis both in vitro and in vivo. Also, although an Fo plasmid carrying the correct ribosome-binding site causes harmful, F1-dependent proton permeability in unc+ cells (Brusilow, W. S. S. (1987) J. Bacteriol. 169, 4984-4990), an identical plasmid carrying the mutation does not, even though it still codes for a functional reconstitutable Fo. The results show a relationship between the relative level of expression of uncE from a multicopy plasmid and the assembly pathway, proton permeability, and energy-coupling characteristics of the ATPase. PMID:2895768

  11. Water oxidation by amorphous cobalt-based oxides: volume activity and proton transfer to electrolyte bases.

    PubMed

    Klingan, Katharina; Ringleb, Franziska; Zaharieva, Ivelina; Heidkamp, Jonathan; Chernev, Petko; Gonzalez-Flores, Diego; Risch, Marcel; Fischer, Anna; Dau, Holger

    2014-05-01

    Water oxidation in the neutral pH regime catalyzed by amorphous transition-metal oxides is of high interest in energy science. Crucial determinants of electrocatalytic activity were investigated for a cobalt-based oxide film electrodeposited at various thicknesses on inert electrodes. For water oxidation at low current densities, the turnover frequency (TOF) per cobalt ion of the bulk material stayed fully constant for variation of the thickness of the oxide film by a factor of 100 (from about 15 nm to 1.5 μm). Thickness variation changed neither the nanostructure of the outer film surface nor the atomic structure of the oxide catalyst significantly. These findings imply catalytic activity of the bulk hydrated oxide material. Nonclassical dependence on pH was observed. For buffered electrolytes with pKa values of the buffer base ranging from 4.7 (acetate) to 10.3 (hydrogen carbonate), the catalytic activity reflected the protonation state of the buffer base in the electrolyte solution directly and not the intrinsic catalytic properties of the oxide itself. It is proposed that catalysis of water oxidation occurs within the bulk hydrated oxide film at the margins of cobalt oxide fragments of molecular dimensions. At high current densities, the availability of a proton-accepting base at the catalyst-electrolyte interface controls the rate of water oxidation. The reported findings may be of general relevance for water oxidation catalyzed at moderate pH by amorphous transition-metal oxides.

  12. The active site of ribulose-bisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.

    1991-01-01

    The active site of ribulose-bisphosphate carboxylase/oxygenase requires interacting domains of adjacent, identical subunits. Most active-site residues are located within the loop regions of an eight-stranded {beta}/{alpha}-barrel which constitutes the larger C-terminal domain; additional key residues are located within a segment of the smaller N-terminal domain which partially covers the mouth of the barrel. Site-directed mutagenesis of the gene encoding the enzyme from Rhodospirillum rubrum has been used to delineate functions of active-site residues. 6 refs., 2 figs.

  13. Savannah River Site prioritization of transition activities

    SciTech Connect

    Finley, R.H.

    1993-11-01

    Effective management of SRS conversion from primarily a production facility to other missions (or Decontamination and Decommissioning (D&D)) requires a systematic and consistent method of prioritizing the transition activities. This report discusses the design of a prioritizing method developed to achieve systematic and consistent methods of prioritizing these activities.

  14. DOE site performance assessment activities. Radioactive Waste Technical Support Program

    SciTech Connect

    Not Available

    1990-07-01

    Information on performance assessment capabilities and activities was collected from eight DOE sites. All eight sites either currently dispose of low-level radioactive waste (LLW) or plan to dispose of LLW in the near future. A survey questionnaire was developed and sent to key individuals involved in DOE Order 5820.2A performance assessment activities at each site. The sites surveyed included: Hanford Site (Hanford), Idaho National Engineering Laboratory (INEL), Los Alamos National Laboratory (LANL), Nevada Test Site (NTS), Oak Ridge National Laboratory (ORNL), Paducah Gaseous Diffusion Plant (Paducah), Portsmouth Gaseous Diffusion Plant (Portsmouth), and Savannah River Site (SRS). The questionnaire addressed all aspects of the performance assessment process; from waste source term to dose conversion factors. This report presents the information developed from the site questionnaire and provides a comparison of site-specific performance assessment approaches, data needs, and ongoing and planned activities. All sites are engaged in completing the radioactive waste disposal facility performance assessment required by DOE Order 5820.2A. Each site has achieved various degrees of progress and have identified a set of critical needs. Within several areas, however, the sites identified common needs and questions.

  15. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

    The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive material contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites.

  16. Comparing the quality of passively-scattered proton and photon tomotherapy plans for brain and head and neck disease sites.

    PubMed

    Kainz, Kristofer; Firat, Selim; Wilson, J Frank; Schultz, Christopher; Siker, Malika; Wang, Andrew; Olson, Dan; Li, X Allen

    2015-03-21

    We compare the quality of photon IMRT (helical tomotherapy) with classic proton plans for brain, head and neck tumors, in terms of target dose uniformity and conformity along with organ-at-risk (OAR) sparing. Plans were created for twelve target volumes among eight cases. All patients were originally planned and treated using helical tomotherapy. Proton plans were generated using a passively-scattered beam model with a maximum range of 32 g cm(-2) (225 MeV), range modulation in 0.5 g cm(-2) increments and range compensators with 4.8 mm milling tool diameters. All proton plans were limited to two to four beams. Plan quality was compared using uniformity index (UI), conformation number (CN) and a EUD-based plan quality index (fEUD). For 11 of the 12 targets, UI was improved for the proton plan; on average, UI was 1.05 for protons versus 1.08 for tomotherapy. For 7 of the 12 targets, the tomotherapy plan exhibited more favorable CN. For proximal OARs, the improved dose conformity to the target volume from tomotherapy led to a lower maximum dose. For distal OARs, the maximum dose was much lower for proton plans. For 6 of the 8 cases, near-total avoidance for distal OARs provided by protons leads to improved fEUD. However, if distal OARs are excluded in the fEUD calculation, the proton plans exhibit better fEUD in only 3 of the 8 cases. The distal OAR sparing and target dose uniformity are generally better with passive-scatter proton planning than with photon tomotherapy; proton therapy may be preferred if the clinician deems those attributes critical. However, tomotherapy may serve equally as well as protons for cases where superior target dose conformity from tomotherapy leads to plan quality nearly identical to or better than protons and for cases where distal OAR sparing is not concerning.

  17. Pi-interaction tuning of the active site properties of metalloproteins.

    PubMed

    Yanagisawa, Sachiko; Crowley, Peter B; Firbank, Susan J; Lawler, Anne T; Hunter, David M; McFarlane, William; Li, Chan; Kohzuma, Takamitsu; Banfield, Mark J; Dennison, Christopher

    2008-11-19

    The influence of pi-interactions with a His ligand have been investigated in a family of copper-containing redox metalloproteins. The Met16Phe and Met16Trp pseudoazurin, and Leu12Phe spinach and Leu14Phe Phormidium laminosum plastocyanin variants possess active-site pi-contacts between the introduced residue and His81 and His87/92 respectively. The striking overlap of the side chain of Phe16 in the Met16Phe variant and that of Met16 in wild type pseudoazurin identifies that this position provides an important second coordination sphere interaction in both cases. His-ligand protonation and dissociation from Cu(I) occurs in the wild type proteins resulting in diminished redox activity, providing a [H(+)]-driven switch for regulating electron transfer. The introduced pi-interaction has opposing effects on the pKa for the His ligand in pseudoazurin and plastocyanin due to subtle differences in the pi-contact, stabilizing the coordinated form of pseudoazurin whereas in plastocyanin protonation and dissociation is favored. Replacement of Pro36, a residue that has been suggested to facilitate structural changes upon His ligand protonation, with a Gly, has little effect on the pKa of His87 in spinach plastocyanin. The mutations at Met16 have a significant influence on the reduction potential of pseudoazurin. Electron self-exchange is enhanced, whereas association with the physiological partner, nitrite reductase, is only affected by the Met16Phe mutation, but kcat is halved in both the Met16Phe and Met16Trp variants. Protonation of the His ligand is the feature most affected by the introduction of a pi-interaction.

  18. Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site.

    PubMed Central

    Weaver, T.; Lees, M.; Banaszak, L.

    1997-01-01

    Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect. From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation. PMID:9098893

  19. Ligand binding and proton exchange dynamics in site-specific mutants of human myoglobin

    SciTech Connect

    Lambright, D.G.

    1992-01-01

    Site specific mutagenesis was used to make substitutions of four residues in the distal heme pocket of human myoglobin: Val68, His64, Lys45, and Asp60. Strongly diffracting crystals of the conservative mutation K45R in the met aquo form were grown in the trigonal space group P3[sub 2]21 and the X-ray crystal structure determined at 1.6 [angstrom] resolution. The overall structure is similar to that of sperm whale met aquo myoglobin. Several of the mutant proteins were characterized by 2-D NMR spectroscopy. The NMR data suggest the structural changes are localized to the region of the mutation. The dynamics of ligand binding to myoglobin mutants were studied by transient absorption spectroscopy following photolysis of the CO complexes. Transient absorption kinetics and spectra on the ns to ms timescale were measured in aqueous solution from 280 K to 310 K and in 75% glycerol: water from 250 K to 310 K. Two significant basis spectra were obtained from singular value decomposition of the matrix of time dependent spectra. The information was used to obtain approximations for the extent of ligand rebinding and the kinetics of conformational relaxation. Except for K45R, substitutions at Lys45 or Asp60 produce changes in the kinetics for ligand rebinding. Replacement of Lys45 with Arg increases the rate of ligand rebinding from the protein matrix by a factor of 2, but does not alter the rates for ligand escape or entry into the protein or the dynamics of the conformational relaxation. Substitutions at His64 and Val68 influence the kinetics of ligand rebinding and the dynamics of conformational relaxation. The results do not support the hypothesis that ligand migration between the heme pocket and solvent is determined solely by fluctuations of Arg45 and His64 between open and closed conformations of the heme pocket but can be rationalized if ligand diffusion through the protein matrix involves multiple competing pathways.

  20. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

  1. Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors.

    PubMed

    Swaney, Danielle L; McAlister, Graeme C; Wirtala, Matthew; Schwartz, Jae C; Syka, John E P; Coon, Joshua J

    2007-01-15

    Electron-transfer dissociation (ETD) delivers the unique attributes of electron capture dissociation to mass spectrometers that utilize radio frequency trapping-type devices (e.g., quadrupole ion traps). The method has generated significant interest because of its compatibility with chromatography and its ability to: (1) preserve traditionally labile post-translational modifications (PTMs) and (2) randomly cleave the backbone bonds of highly charged peptide and protein precursor ions. ETD, however, has shown limited applicability to doubly protonated peptide precursors, [M + 2H]2+, the charge and type of peptide most frequently encountered in "bottom-up" proteomics. Here we describe a supplemental collisional activation (CAD) method that targets the nondissociated (intact) electron-transfer (ET) product species ([M + 2H]+*) to improve ETD efficiency for doubly protonated peptides (ETcaD). A systematic study of supplementary activation conditions revealed that low-energy CAD of the ET product population leads to the near-exclusive generation of c- and z-type fragment ions with relatively high efficiency (77 +/- 8%). Compared to those formed directly via ETD, the fragment ions were found to comprise increased relative amounts of the odd-electron c-type ions (c+*) and the even-electron z-type ions (z+). A large-scale analysis of 755 doubly charged tryptic peptides was conducted to compare the method (ETcaD) to ion trap CAD and ETD. ETcaD produced a median sequence coverage of 89%-a significant improvement over ETD (63%) and ion trap CAD (77%).

  2. Ulysses observations of electron and proton components in a magnetic cloud and related wave activity

    NASA Technical Reports Server (NTRS)

    Osherovich, V. A.; Fainberg, J.; Stone, R. G.; MacDowall, R. J.; Phillips, J. L.; Balogh, A.

    1995-01-01

    In addition to a smooth rotation of the magnetic field vector, magnetic clouds have a low proton temperature T(sub p). Their expansion in the solar wind leads to depletion and therefore the ion component cools down. It has been shown recently that the electron component in magnetic clouds behaves differently: when the cloud expands, electron temperature Te anti correlates with density and therefore Te increases in the cloud, creating favorable conditions for the rise of ion-acoustic waves. For the magnetic cloud observed by Ulysses on June 10 - 12, 1993 at 4.64 AU at S 32.5 deg, we present observations for both electron and proton components and related plasma wave activity. Our results confirm the anti correlation between T(sub e) and electron density and also exhibit a high ratio of T(sub e)/T(sub P) in the cloud. Since Landau damping is not effective for T(sub e)/T(sub p) much greater than 1, Doppler shifted ion acoustic waves are expected in the cloud. Calculation of ion acoustic wave frequencies in the cloud and comparison with observed wave activity confirm this expectation. As in our previous work, we show that the electron component in the cloud obeys a polytropic law with gamma is less than 1 (gamma approximately equals 0.3-0.4). The dynamics of the magnetic cloud are determined to a large degree by the dominating electron pressure.

  3. Single-site copper(II) water oxidation electrocatalysis: rate enhancements with HPO₄²⁻ as a proton acceptor at pH 8.

    PubMed

    Coggins, Michael K; Zhang, Ming-Tian; Chen, Zuofeng; Song, Na; Meyer, Thomas J

    2014-11-01

    The complex Cu(II)(Py3P) (1) is an electrocatalyst for water oxidation to dioxygen in H2PO4(-)/HPO4(2-) buffered aqueous solutions. Controlled potential electrolysis experiments with 1 at pH 8.0 at an applied potential of 1.40 V versus the normal hydrogen electrode resulted in the formation of dioxygen (84% Faradaic yield) through multiple catalyst turnovers with minimal catalyst deactivation. The results of an electrochemical kinetics study point to a single-site mechanism for water oxidation catalysis with involvement of phosphate buffer anions either through atom-proton transfer in a rate-limiting O-O bond-forming step with HPO4(2-) as the acceptor base or by concerted electron-proton transfer with electron transfer to the electrode and proton transfer to the HPO4(2-) base. PMID:25243584

  4. Distribution of the Effect of Solar Proton Flux And Geomagnetic Activity on the Stratospheric Ozone Profile

    NASA Astrophysics Data System (ADS)

    Velinov, P. I. Y.; Tassev, Y.; Yanev, T.; Tomova, D.

    Two-way MANOVA was used to examine the impact of two factors: 1) the proton flux intensity and 2) the geomagnetic activity on the dependant variable "ozone mixing ratio" which characterizes the stratospheric ozone profiles. The examination was carried out with fixed levels of two other factors: a) the heights at which the "ozone mixing ratio" was recorded, i,e, 35 km, 30.2 km, 24.5 km, 18.4 km, 15.6 km and b) the energetic intervals within which the proton flux was measured, i.e. =0,6-4,2 MeV; 4,2-8,7 MeV; 8,7-14,5 MeV; 15-44 MeV; 39-82 MeV; 84-200 MeV; 110-500 MeV. The analysis was performed for all combinations of levels of the factors a) and b) for which data was available. It was aimed at revealing which of the factors 1) and 2) were dominating with different combinations of the factors a) and b) with fixed levels. For this purpose a post hoc analysis was performed as well. The main results are as follows: factors 1) and 2) exert statistically significant impact on the dependant variable at all of the heights examined, but not for all of energetic intervals; increase of the ozone mixing ratio was observed as a main effect of the proton flux intensity at heights 24.5 km, 18.4 km, 15.6 km, but the analysis of the simultaneous acting of factors 1) and 2) revealed a decrease of the dependant variable at these heights; these effects possibly indicate the existence of two different mechanisms of impact on the ozone mixing ratio; the afore- discussed effects decrease with the height and therefore their graphical image was named "Christmas tree".

  5. Kinetic and theoretical studies on the protonation of [Ni(2-SC6H4N){PhP(CH2CH2PPh2)2}]+: nitrogen versus sulfur as the protonation site.

    PubMed

    Petrou, Athinoula L; Koutselos, Andreas D; Wahab, Hilal S; Clegg, William; Harrington, Ross W; Henderson, Richard A

    2011-02-01

    The complexes [Ni(4-Spy)(triphos)]BPh(4) and [Ni(2-Spy)(triphos)]BPh(4) {triphos = PhP(CH(2)CH(2)PPh(2))(2), 4-Spy = 4-pyridinethiolate, 2-Spy = 2-pyridinethiolate} have been prepared and characterized both spectroscopically and using X-ray crystallography. In both complexes the triphos is a tridentate ligand. However, [Ni(4-Spy)(triphos)](+) comprises a 4-coordinate, square-planar nickel with the 4-Spy ligand bound to the nickel through the sulfur while [Ni(2-Spy)(triphos)](+) contains a 5-coordinate, trigonal-bipyramidal nickel with a bidentate 2-Spy ligand bound to the nickel through both sulfur and nitrogen. The kinetics of the reactions of [Ni(4-Spy)(triphos)](+) and [Ni(2-Spy)(triphos)](+) with lutH(+) (lut = 2,6-dimethylpyridine) in MeCN have been studied using stopped-flow spectrophotometry, and the two complexes show very different reactivities. The reaction of [Ni(4-Spy)(triphos)](+) with lutH(+) is complete within the deadtime of the stopped-flow apparatus (2 ms) and corresponds to protonation of the nitrogen. However, upon mixing [Ni(2-Spy)(triphos)](+) and lutH(+) a reaction is observed (on the seconds time scale) to produce an equilibrium mixture. The mechanistic interpretation of the rate law has been aided by the application of MSINDO semiempirical and ADF calculations. The kinetics and calculations are consistent with the reaction between [Ni(2-Spy)(triphos)](+) and lutH(+) involving initial protonation of the sulfur followed by dissociation of the nitrogen and subsequent transfer of the proton from sulfur to nitrogen. The factors affecting the position of protonation and the coupling of the coordination state of the 2-pyridinethiolate ligand to the site of protonation are discussed.

  6. Measurement of the underlying event activity using charged-particle jets in proton-proton collisions at $\\sqrt{s}$ = 2.76 TeV

    SciTech Connect

    Khachatryan, V.

    2015-09-21

    A measurement of the underlying event (UE) activity in proton-proton collisions is performed using events with charged-particle jets produced in the central pseudorapidity region (|η jet| < 2) and with transverse momentum 1 ≤ pT jet < 100 GeV. The analysis uses a data sample collected at a centre-of-mass energy of 2.76 TeV with the CMS experiment at the LHC. The UE activity is measured as a function of pT jet in terms of the average multiplicity and scalar sum of transverse momenta (pT) of charged particles, with |η| < 2 and pT > 0.5 GeV, in the azimuthal region transverse to the highest pT jet direction. By further dividing the transverse region into two regions of smaller and larger activity, various components of the UE activity are separated. As a result, the measurements are compared to previous results at 0.9 and 7 TeV, and to predictions of several Monte Carlo event generators, providing constraints on the modelling of the UE dynamics

  7. Measurement of the underlying event activity using charged-particle jets in proton-proton collisions at $$\\sqrt{s}$$ = 2.76 TeV

    DOE PAGES

    Khachatryan, V.

    2015-09-21

    A measurement of the underlying event (UE) activity in proton-proton collisions is performed using events with charged-particle jets produced in the central pseudorapidity region (|η jet| < 2) and with transverse momentum 1 ≤ pT jet < 100 GeV. The analysis uses a data sample collected at a centre-of-mass energy of 2.76 TeV with the CMS experiment at the LHC. The UE activity is measured as a function of pT jet in terms of the average multiplicity and scalar sum of transverse momenta (pT) of charged particles, with |η| < 2 and pT > 0.5 GeV, in the azimuthal regionmore » transverse to the highest pT jet direction. By further dividing the transverse region into two regions of smaller and larger activity, various components of the UE activity are separated. As a result, the measurements are compared to previous results at 0.9 and 7 TeV, and to predictions of several Monte Carlo event generators, providing constraints on the modelling of the UE dynamics« less

  8. Spherically shaped active transducer based on proton-irradiated vinylidene fluoride-trifluoroethylene 70/30 mol % copolymer

    SciTech Connect

    Lau, S.T.; Chan, H.L.W.; Choy, C.L.; Cheung, W.Y.; Wong, S.P.

    2006-05-15

    Spherically shaped active transducers using proton-irradiated vinylidene fluoride-trifluoroethylene 70/30 mol % copolymer films as the active elements are described. The copolymer films prepared by hot compression molding were irradiated with a high energy proton over a broad dose range (20-250 Mrad). The electrostrictive and piezoelectric responses of the copolymer have been characterized before subsequent transducer fabrication. The performances of the focused transducers constructed with a 4 mm aperture size and epoxy backing were evaluated under dc bias voltages. The transducers with focal lengths of 17.4-19.0 mm and a center frequency of 19 MHz display a broad bandwidth up to 94%. Besides, the transmitting output of the transducers increases with the dc bias voltage. For the copolymer active element irradiated at a proton dose of 107 Mrad, the transducer shows the highest transmitting voltage response of 1.34 kPa/V.

  9. Tuning the activity of glutathione peroxidase mimics through intramolecular Se···N,O interactions: a DFT study incorporating solvent-assisted proton exchange (SAPE).

    PubMed

    Bayse, Craig A; Pavlou, Andrea

    2011-12-01

    Diaryl diselenide mimics of the antioxidant selenoprotein glutathione peroxidase (GPx) often incorporate intramolecular Se···N,O interactions to enhance their GPx-like activity. Although the strength of the interaction is defined by the Lewis basicity of the donating group and the strength of the Se-X bond, there is not a clear relationship between the interaction and the GPx-like activity. Density-functional theory and natural bond orbital (NBO) calculations are used to show the range of Se···N,O interactions for various functional groups. The strongest interactions are found for groups which stabilize the donor-acceptor interaction through aromatic stabilization. The activation barriers for the GPx-like mechanism of activity of several substituted areneselenols are calculated using DFT and solvent-assisted proton exchange (SAPE), a technique that incorporates networks of solvent molecules into the theoretical model to facilitate proton transfer between sites in the reactant and product. DFT-SAPE models show that, in addition to decreasing the barrier to oxidation of the selenol, Se···N,O interactions generally increase the barriers for selenenic acid reduction and selenol regeneration because the Se···N,O interaction must be broken for the reaction to proceed. Calculated activation barriers for the rate-determining step are consistent with the relative experimental GPx-like activities of a series of diaryl diselenides.

  10. Effects of Extrinsic and Intrinsic Proton Activity on The Mechanism of Oxygen Reduction in Ionic Liquids

    NASA Astrophysics Data System (ADS)

    Zeller, Robert August

    Mechanisms for oxygen reduction are proposed for three distinct cases covering two ionic liquids of fundamentally different archetypes and almost thirty orders of magnitude of proton activity. Proton activity is treated both extrinsically by varying the concentration and intrinsically by selecting proton donors with a wide range of aqueous pKa values. The mechanism of oxygen reduction in ionic liquids is introduced by way of the protic ionic liquid (pIL) triethylammonium triflate (TEATf) which shares some similarities with aqueous acid solutions. Oxygen reduction in TEATf begins as the one electron rate limited step to form superoxide, O2 *-, which is then rapidly protonated by the pIL cation forming the perhydroxyl radical, HO2*. The perhydroxyl radical is further reduced to peroxidate (HO2-) and hydrogen peroxide in proportions in accordance with their pKa. The reaction does not proceed beyond this point due to the adsorption of the conjugate base triethylammine interfering with the disproportionation of hydrogen peroxide. This work demonstrates that this mechanism is consistent across Pt, Au, Pd, and Ag electrodes. Two related sets of experiments were performed in the inherently aprotic ionic liquid 1-butyl-2,3-dimethylimidazolium triflate (C4dMImTf). The first involved the titration of acidic species of varying aqueous pKa into the IL while monitoring the extent of oxygen reduction as a function of pKa and potential on Pt and glassy carbon (GC) electrodes. These experiments confirmed the greater propensity of Pt to reduce oxygen by its immediate and abrupt transition from one electron reduction to four electron reduction, while oxygen reduction on GC gradually approaches four electron reduction as the potentials were driven more cathodic. The potential at which oxygen reduction initiates shows general agreement with the Nernst equation and the acid's tabulated aqueous pKa value, however at the extremely acidic end, a small deviation is observed. The second set

  11. SU-E-T-557: Measuring Neutron Activation of Cardiac Devices Irradiated During Proton Therapy Using Indium Foils

    SciTech Connect

    Avery, S; Christodouleas, J; Delaney, K; Diffenderfer, E; Brown, K

    2014-06-01

    Purpose: Measuring Neutron Activation of Cardiac devices Irradiated during Proton Therapy using Indium Foils Methods: The foils had dimensions of 25mm x 25mm x 1mm. After being activated, the foils were placed in a Canberra Industries well chamber utilizing a NaI(Tl) scintillation detector. The resulting gamma spectrum was acquired and analyzed using Genie 2000 spectroscopy software. One activation foil was placed over the upper, left chest of RANDO where a pacemaker would be. The rest of the foils were placed over the midline of the patient at different distances, providing a spatial distribution over the phantom. Using lasers and BBs to align the patient, 200 MU square fields were delivered to various treatment sites: the brain, the pancreas, and the prostate. Each field was shot at least a day apart, giving more than enough time for activity of the foil to decay (t1=2 = 54.12 min). Results: The net counts (minus background) of the three aforementioned peaks were used for our measurements. These counts were adjusted to account for detector efficiency, relative photon yields from decay, and the natural abundance of 115-In. The average neutron flux for the closed multi-leaf collimator irradiation was measured to be 1.62 x 106 - 0.18 x 106 cm2 s-1. An order of magnitude estimate of the flux for neutrons up to 1 keV from Diffenderfer et al. gives 3 x 106 cm2 s-1 which does agree on the order of magnitude. Conclusion: Lower energy neutrons have higher interaction cross-sections and are more likely to damage pacemakers. The thermal/slow neutron component may be enough to estimate the overall risk. The true test of the applicability of activation foils is whether or not measurements are capable of predicting cardiac device malfunction. For that, additional studies are needed to provide clinical evidence one way or the other.

  12. Hydrogen production by the naked active site of the di-iron hydrogenases in water.

    PubMed

    Zipoli, Federico; Car, Roberto; Cohen, Morrel H; Selloni, Annabella

    2009-10-01

    We explored the reactivity of the active center of the [FeFe]-hydrogenases detached from the enzyme and immersed in acidified water by first-principles Car-Parrinello molecular-dynamics simulations. We focused on the identification of the structures that are stable and metastable in acidified water and on their activity for hydrogen production. Our calculations revealed that the naked active center could be an efficient catalyst provided that electrons are transferred to the cluster. We found that both bridging and terminal isomers are present at equilibrium and that the bridging configuration is essential for efficient hydrogen production. The formation of the hydrogen molecule occurs via sequential protonations of the distal iron and of the N-atom of the S-CH(2)-NH-CH(2)-S chelating group. H(2) desorption does not involve a significant energy barrier, making the process very efficient at room temperature. We established that the bottleneck in the reaction is the direct proton transfer from water to the vacant site of the distal iron. Moreover, we found that even if the terminal isomer is present at the equilibrium, its strong local hydrophobicity prevents poisoning of the cluster. PMID:19737003

  13. Hydrogen production by the naked active site of the di-iron hydrogenases in water.

    PubMed

    Zipoli, Federico; Car, Roberto; Cohen, Morrel H; Selloni, Annabella

    2009-10-01

    We explored the reactivity of the active center of the [FeFe]-hydrogenases detached from the enzyme and immersed in acidified water by first-principles Car-Parrinello molecular-dynamics simulations. We focused on the identification of the structures that are stable and metastable in acidified water and on their activity for hydrogen production. Our calculations revealed that the naked active center could be an efficient catalyst provided that electrons are transferred to the cluster. We found that both bridging and terminal isomers are present at equilibrium and that the bridging configuration is essential for efficient hydrogen production. The formation of the hydrogen molecule occurs via sequential protonations of the distal iron and of the N-atom of the S-CH(2)-NH-CH(2)-S chelating group. H(2) desorption does not involve a significant energy barrier, making the process very efficient at room temperature. We established that the bottleneck in the reaction is the direct proton transfer from water to the vacant site of the distal iron. Moreover, we found that even if the terminal isomer is present at the equilibrium, its strong local hydrophobicity prevents poisoning of the cluster.

  14. Proton Pathways in Green Fluorescence Protein

    PubMed Central

    Agmon, Noam

    2005-01-01

    Proton pathways in green fluorescent protein (GFP) are more extended than previously reported. In the x-ray data of wild-type GFP, a two-step exit pathway exists from the active site to the protein surface, controlled by a threonine switch. A proton entry pathway begins at a glutamate-lysine cluster around Glu-5, and extends all the way to the buried Glu-222 near the active site. This structural evidence suggests that GFP may function as a portable light-driven proton-pump, with proton emitted in the excited state through the switchable exit pathway, and replenished from Glu-222 and the Glu-5 entry pathway in the ground state. PMID:15681647

  15. Water structural changes at the proton uptake site (the Thr46-Asp96 domain) in the L intermediate of bacteriorhodopsin

    SciTech Connect

    Yamazaki, Yoichi; Hatanka, Minoru; Kandori, Hideki

    1995-05-30

    Fourier transform infrared spectra of the L intermediate of light-adapted bacteriorhodopsin were examined for recombinant proteins with amino acid substitutions at Thr46 and Asp96. Two O-H stretching vibrational bands of water, at 3607 and 3577 cm{sup -1}, change into stronger H-bonding states in L of the wild type. Thr46{r_arrow}Val substitution abolished these bands in spite of the fact that [3-{sup 18}O]threonine-labeling did not shift with them indicating that they correspond to coordination of the water with Thr46. The two water bands were restored, although with changed frequencies, by an additional Asp96{r_arrow}Asn substitution in Thr46{r_arrow}Val/Asp96{r_arrow}Asn. A single Asp96{r_arrow}Asn substitution abolished the 3607 cm{sup -1} band. Thus, Asp96 also takes part in structural changes in water. The perturbations of these water molecules in the L intermediate displayed a weak correlation with the ratio of intensity change in the two of vibrational bands of the Schiff base mode at 1312 and 1301 cm{sup -1} and the rate for the deprotonation of the Schiff base at the L-to-M reaction of the photocycle. We find, therefore, that the water molecule sin the cytoplasmic Asp96-Thr46 domain, which comprises the site of proton uptake after formation of the M intermediate, undergo structural changes in the L intermediate already. These changes are transmitted to the extracellular domain and the affect interaction of the Schiff base with Asp 85, that is far removed from this region. 32 refs., 7 figs.

  16. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    SciTech Connect

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-03-20

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme.

  17. Active damping of the e-p instability at the Los Alamos Proton Storage Ring

    SciTech Connect

    Macek, R. J.; Assadi, S.; Byrd, J. M.; Deibele, C. E.; Henderson, S. D.; Lee, S. Y.; McCrady, R. C.; Pivi, M. F. T.; Plum, M. A.; Walbridge, S. B.; Zaugg, T. J.

    2007-12-15

    A prototype of an analog, transverse (vertical) feedback system for active damping of the two-stream (e-p) instability has been developed and successfully tested at the Los Alamos Proton Storage Ring (PSR). This system was able to improve the instability threshold by approximately 30% (as measured by the change in RF buncher voltage at instability threshold). The feedback system configuration, setup procedures, and optimization of performance are described. Results of several experimental tests of system performance are presented including observations of instability threshold improvement and grow-damp experiments, which yield estimates of instability growth and damping rates. A major effort was undertaken to identify and study several factors limiting system performance. Evidence obtained from these tests suggests that performance of the prototype was limited by higher instability growth rates arising from beam leakage into the gap at lower RF buncher voltage and the onset of instability in the horizontal plane, which had no feedback.

  18. Active Damping of the E-P Instability at the Los Alamos Proton Storage Ring

    SciTech Connect

    Macek, R.J.; Assadi, S.; Byrd, J.M.; Deibele, C.E.; Henderson, S.D.; Lee, S.Y.; McCrady, R.C.; Pivi, M.F.T.; Plum, M.A.; Walbridge, S.B.; Zaugg, T.J.; /Los Alamos

    2008-03-17

    A prototype of an analog, transverse (vertical) feedback system for active damping of the two-stream (e-p) instability has been developed and successfully tested at the Los Alamos Proton Storage Ring (PSR). This system was able to improve the instability threshold by approximately 30% (as measured by the change in RF buncher voltage at instability threshold). The feedback system configuration, setup procedures, and optimization of performance are described. Results of several experimental tests of system performance are presented including observations of instability threshold improvement and grow-damp experiments, which yield estimates of instability growth and damping rates. A major effort was undertaken to identify and study several factors limiting system performance. Evidence obtained from these tests suggests that performance of the prototype was limited by higher instability growth rates arising from beam leakage into the gap at lower RF buncher voltage and the onset of instability in the horizontal plane, which had no feedback.

  19. In-growth of an electrically active defect in high-purity silicon after proton irradiation

    SciTech Connect

    Nylandsted Larsen, A.; Juul Pedersen, H.; Christian Petersen, M.; Privitera, V.; Gurimskaya, Y.; Mesli, A.

    2013-12-14

    Defect-related energy levels in the lower half of the band gap of silicon have been studied with transient-capacitance techniques in high-purity, carbon and oxygen lean, plasma-enhanced chemical-vapor deposition grown, n-and p-type silicon layers after 2-MeV proton irradiations at temperatures at or just below room temperature. The in-growth of a distinct line in deep-level transient spectroscopy spectra, corresponding to a level in the band gap at E{sub V} + 0.357 eV where E{sub V} is the energy of the valence band edge, takes place for anneal temperatures at around room temperature with an activation energy of 0.95 ± 0.08 eV. The line disappears at an anneal temperature of around 450 K. The corresponding defect is demonstrated not to contain boron, carbon, oxygen, or phosphorus. Possible defect candidates are discussed.

  20. Energetic protons associated with interplanetary active regions 1-5 AU from the sun

    NASA Technical Reports Server (NTRS)

    Pesses, M. E.; Van Allen, J. A.; Goertz, C. K.

    1978-01-01

    Pioneer 11 has yielded data on approximately 100 energetic proton events at heliocentric distances between 1 and 2 AU. Measurements of absolute intensities, anisotropies, and crude energy spectra are studied in connection with interplanetary active regions (IAR's). It is found that in close vicinity to IAR's, the number of events observed per unit time interval is 10 times greater than in other areas of interplanetary space, and that the frequency of events has a maximum at plus or minus 5 hours of the time IAR edges are crossed. It is also noted that events in IAR vicinity have greater particle densities, softer energy spectra, and smaller time widths than other events. For many events associated with IAR's, particle anisotropies correspond to the net flow of particles along the interplanetary magnetic field toward the sun. This suggests that a mechanism in MHD shocks is responsible for local acceleration in the interplanetary medium.

  1. Activation cross sections of proton induced nuclear reactions on palladium up to 80MeV.

    PubMed

    Tárkányi, F; Ditrói, F; Takács, S; Csikai, J; Hermanne, A; Uddin, M S; Baba, M

    2016-08-01

    Activation cross sections of proton induced nuclear reactions on palladium were measured up to 80MeV by using the stacked foil irradiation technique and gamma ray spectrometry. The beam intensity, the incident energy and the energy degradation were controlled by a method based on flux constancy via normalization to the excitation functions of monitor reactions measured in parallel. Excitation functions for direct and cumulative cross-sections were measured for the production of (104m,104g,105g,106m,110m)Ag, (100,101)Pd, (99m,99g,100,101m,101g,102m,102g,105)Rh and (103,97)Ru radioisotopes. The cross section data were compared with the theoretical predictions of TENDL-2014 and -2015 libraries. For practical applications thick target yields were derived from the measured excitation functions. Application in the field of medical radionuclide production is shortly discussed. PMID:27235887

  2. A novel approach to predict active sites of enzyme molecules.

    PubMed

    Chou, Kuo-Chen; Cai, Yu-dong

    2004-04-01

    Enzymes are critical in many cellular signaling cascades. With many enzyme structures being solved, there is an increasing need to develop an automated method for identifying their active sites. However, given the atomic coordinates of an enzyme molecule, how can we predict its active site? This is a vitally important problem because the core of an enzyme molecule is its active site from the viewpoints of both pure scientific research and industrial application. In this article, a topological entity was introduced to characterize the enzymatic active site. Based on such a concept, the covariant discriminant algorithm was formulated for identifying the active site. As a paradigm, the serine hydrolase family was demonstrated. The overall success rate by jackknife test for a data set of 88 enzyme molecules was 99.92%, and that for a data set of 50 independent enzyme molecules was 99.91%. Meanwhile, it was shown through an example that the prediction algorithm can also be used to find any typographic error of a PDB file in annotating the constituent amino acids of catalytic triad and to suggest a possible correction. The very high success rates are due to the introduction of a covariance matrix in the prediction algorithm that makes allowance for taking into account the coupling effects among the key constituent atoms of active site. It is anticipated that the novel approach is quite promising and may become a useful high throughput tool in enzymology, proteomics, and structural bioinformatics. PMID:14997541

  3. NMR crystallography of enzyme active sites: probing chemically detailed, three-dimensional structure in tryptophan synthase.

    PubMed

    Mueller, Leonard J; Dunn, Michael F

    2013-09-17

    NMR crystallography--the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry--offers unprecedented insight into three-dimensional, chemically detailed structure. Initially, researchers used NMR crystallography to refine diffraction data from organic and inorganic solids. Now we are applying this technique to explore active sites in biomolecules, where it reveals chemically rich detail concerning the interactions between enzyme site residues and the reacting substrate. Researchers cannot achieve this level of detail from X-ray, NMR,or computational methodologies in isolation. For example, typical X-ray crystal structures (1.5-2.5 Å resolution) of enzyme-bound intermediates identify possible hydrogen-bonding interactions between site residues and substrate but do not directly identify the protonation states. Solid-state NMR can provide chemical shifts for selected atoms of enzyme-substrate complexes, but without a larger structural framework in which to interpret them only empirical correlations with local chemical structure are possible. Ab initio calculations and molecular mechanics can build models for enzymatic processes, but they rely on researcher-specified chemical details. Together, however, X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry can provide consistent and testable models for structure and function of enzyme active sites: X-ray crystallography provides a coarse framework upon which scientists can develop models of the active site using computational chemistry; they can then distinguish these models by comparing calculated NMR chemical shifts with the results of solid-state NMR spectroscopy experiments. Conceptually, each technique is a puzzle piece offering a generous view of the big picture. Only when correctly pieced together, however, can they reveal the big picture at the highest possible resolution. In this Account, we detail our first steps in the development of

  4. Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase

    SciTech Connect

    Yip, Wing-Kin; Dong, Jian-Guo; Yang, S.F. ); Kenny, J.W.; Thompson, G.A. )

    1990-10-01

    The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB{sup 3}H{sub 4} or Ado({sup 14}C)Met. Peptide sequencing of both {sup 3}H- and {sup 14}C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the {sup 3}H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the {sup 14}C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado ({sup 14}C)Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6.

  5. Growth exponents in surface models with non-active sites

    NASA Astrophysics Data System (ADS)

    Santos, M.; Figueiredo, W.; Aarão Reis, F. D. A.

    2006-11-01

    In this work, we studied the role played by the inactive sites present on the substrate of a growing surface. In our model, one particle sticks at the surface if the site where it falls is an active site. However, we allow the deposited particle to diffuse along the surface in accordance with some mechanism previously defined. Using Monte Carlo simulations, and some analytical results, we have investigated the model in (1+1) and (2+1) dimensions considering different relaxation mechanisms. We show that the consideration of non-active sites is a crucial point in the model. In fact, we have seen that the saturation regime is not observed for any value of the density of inactive sites. Besides, the growth exponent β turns to be one, at long times, whatever the mechanism of diffusion we consider in one and two dimensions.

  6. A small ribozyme with dual-site kinase activity

    PubMed Central

    Biondi, Elisa; Maxwell, Adam W.R.; Burke, Donald H.

    2012-01-01

    Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids. We find that kinase ribozyme K28 possesses an unusually complex active site that promotes (thio)phosphorylation of two residues widely separated in primary sequence. After allowing the ribozyme to radiolabel itself by phosphoryl transfer from [γ-32P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating phosphorylation sites within each of the two cleavage fragments. These sites were mapped by alkaline digestion and primer extension pausing. Enzymatic digestion and mutational analysis identified nucleotides important for activity and established the active structure as being a constrained pseudoknot with unusual connectivity that may juxtapose the two reactive sites. Nuclease sensitivities for nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. The 5′ target site was more strongly favored in full-length ribozyme K28 (128 nt) than in truncated RNAs (58 nt). Electrophoretic mobilities of self-thiophosphorylated products on organomercurial gels are distinct from the 5′ mono-thiophosphorylated product produced by reaction with polynucleotide kinase, potentially indicating simultaneous labeling of both sites within individual RNA strands. Our evidence supports a single, compact structure with local dynamics, rather than global rearrangement, as being responsible for dual-site phosphorylation. PMID:22618879

  7. SENSITIVE TO PROTON RHIZOTOXICITY1, CALMODULIN BINDING TRANSCRIPTION ACTIVATOR2, and other transcription factors are involved in ALUMINUM-ACTIVATED MALATE TRANSPORTER1 expression.

    PubMed

    Tokizawa, Mutsutomo; Kobayashi, Yuriko; Saito, Tatsunori; Kobayashi, Masatomo; Iuchi, Satoshi; Nomoto, Mika; Tada, Yasuomi; Yamamoto, Yoshiharu Y; Koyama, Hiroyuki

    2015-03-01

    In Arabidopsis (Arabidopsis thaliana) the root apex is protected from aluminum (Al) rhizotoxicity by excretion of malate, an Al chelator, by ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (AtALMT1). AtALMT1 expression is fundamentally regulated by the SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) zinc finger protein, but other transcription factors have roles that enable Al-inducible expression with a broad dynamic range. In this study, we characterized multiple cis-elements in the AtALMT1 promoter that interact with transcription factors. In planta complementation assays of AtALMT1 driven by 5' truncated promoters of different lengths showed that the promoter region between -540 and 0 (the first ATG) restored the Al-sensitive phenotype of atalm1 and thus contains cis-elements essential for AtALMT1 expression for Al tolerance. Computation of overrepresented octamers showed that eight regions in this promoter region contained potential cis-elements involved in Al induction and STOP1 regulation. Mutation in a position around -297 from the first ATG completely inactivated AtALMT1 expression and Al response. In vitro binding assays showed that this region contained the STOP1 binding site, which accounted for the recognition by four zinc finger domains of the protein. Other positions were characterized as cis-elements that regulated expression by repressors and activators and a transcription factor that determines root tip expression of AtALMT1. From the consensus of known cis-elements, we identified CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR2 to be an activator of AtALMT1 expression. Al-inducible expression of AtALMT1 changed transcription starting sites, which increased the abundance of transcripts with a shortened 5' untranslated region. The present analyses identified multiple mechanisms that regulate AtALMT1 expression.

  8. Interplay between excited-state intramolecular proton transfer and charge transfer in flavonols and their use as protein-binding-site fluorescence probes

    SciTech Connect

    Sytnik, A.; Gormin, D.; Kasha, M. )

    1994-12-06

    A comparative study is presented of competitive fluorescences of three flavonols, 3-hydroxyflavone, 3,3[prime],4[prime],7-tetrahydroxyflavone (fisetin), and 4[prime]-diethylamino-3-hydroxyflavone (DHF). The normal fluorescence S[sub 1] [yields] S[sub 0] (400-nm region) is largely replaced by the proton-transfer tautomer fluorescence S[prime][sub 1] [yields] S[prime][sub 0] in the 550-nm region for all three of the flavonols in aprotic solvents at room temperature. For DHF in polar solvents the normal fluorescence becomes a charge-transfer fluorescence (460-500 nm) which competes strongly with the still dominant proton-transfer fluorescence (at 570 nm). In protic solvents, and at 77 K, the interference with intramolecular hydrogen bonding gives rise to greatly enhanced normal fluorescence, lowering the quantum yield of proton-transfer fluorescence. The utility of DHF as a discriminating fluorescence probe for protein binding sites is suggested by the strong dependence of the charge-transfer fluorescence on polarity of the environment and by various static and dynamic parameters of the charge-transfer and proton-transfer fluorescence which can be determined. 49 refs., 6 figs., 1 tab.

  9. Simple thermodynamic model of unassisted proton shuttle uncoupling and prediction of activity from calculated speciation, lipophilicity, and molecular geometry.

    PubMed

    Martineau, Louis C

    2012-06-21

    A mechanistic model of uncoupling of oxidative phosphorylation by lipophilic weak acids (i.e. proton shuttles) was developed for the purposes of predicting the relative activity of xenobiotics of widely varying structure and of guiding the design of optimized derivatives. The model is based on thermodynamic premises not formulated elsewhere that allow for the calculation of steady-state conditions and of rate of energy dissipation on the basis of acid-dissociation and permeability behavior, the later estimated from partitioning behavior and geometric considerations. Moreover, permeability of either the neutral or of the ionized species is proposed to be effectively enhanced under conditions of asymmetrical molecular distribution. Finally, special considerations were developed to accommodate multi-protic compounds. The comparison of predicted to measured activity for a diverse testset of 48 compounds of natural origin spanning a wide range of activity yielded a Spearman's rho of 0.90. The model was used to tentatively identify several novel proton shuttles, as well as to elucidate core structures particularly conducive to proton shuttle activity from which optimized derivatives can be designed. Principles of design were formulated and examples of derivatives projected to be active at concentrations on the order of 10(-7)M are proposed. Among these are di-protic compounds predicted to shuttle two protons per cycle iteration and proposed to maximally exploit the proton shuttle mechanism. This work promotes the design of highly active, yet easily-metabolized uncouplers for therapeutic applications, namely the indirect activation of AMP-kinase, as well as for various industrial applications where low persistence is desirable.

  10. Probing Oxygen Activation Sites in Two Flavoprotein Oxidases Using Chloride as an Oxygen Surrogate

    SciTech Connect

    Kommoju, Phaneeswara-Rao; Chen, Zhi-wei; Bruckner, Robert C.; Mathews, F. Scott; Jorns, Marilyn Schuman

    2011-08-16

    A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a preorganized binding site for superoxide anion, an obligatory intermediate in the two-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. First, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX-chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX-chloride complex and a ternary MSOX-chloride-MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is an effective probe of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.

  11. A proton-activated, outwardly rectifying chloride channel in human umbilical vein endothelial cells

    SciTech Connect

    Ma Zhiyong; Zhang Wei; Chen Liang; Wang Rong; Kan Xiaohong; Sun Guizhen; Liu Chunxi; Li Li Zhang Yun

    2008-07-04

    Extracellular acidic pH-activated chloride channel I{sub Cl,acid}, has been characterized in HEK 293 cells and mammalian cardiac myocytes. This study was designed to characterize I{sub Cl,acid} in human umbilical vein endothelial cells(HUVECs). The activation and deactivation of the current rapidly and repeatedly follows the change of the extracellular solution at pH 4.3, with the threshold pH 5.3. In addition, at very positive potentials, the current displays a time-dependent facilitation. pH-response relationship for I{sub Cl,acid} revealed that EC{sub 50} is pH 4.764 with a threshold pH value of pH 5.3 and nH of 14.545. The current can be blocked by the Cl{sup -} channel inhibitor DIDS (100 {mu}M). In summary, for the first time we report the presence of proton-activated, outwardly rectifying chloride channel in HUVECs. Because an acidic environment can develop in local myocardium under pathological conditions such as myocardial ischemia, I{sub Cl,acid} would play a role in regulation of EC function under these pathological conditions.

  12. Association of Proton Pump Inhibitor (PPI) Use with Energy Intake, Physical Activity, and Weight Gain

    PubMed Central

    Czwornog, Jennifer L.; Austin, Gregory L.

    2015-01-01

    Studies suggest proton pump inhibitor (PPI) use impacts body weight regulation, though the effect of PPIs on energy intake, energy extraction, and energy expenditure is unknown. We used data on 3073 eligible adults from the National Health and Nutrition Examination Survey (NHANES). Medication use, energy intake, diet composition, and physical activity were extracted from NHANES. Multivariate regression models included confounding variables. Daily energy intake was similar between PPI users and non-users (p = 0.41). Diet composition was similar between the two groups, except that PPI users consumed a slightly greater proportion of calories from fat (34.5% vs. 33.2%; p = 0.02). PPI users rated themselves as being as physically active as their age/gender-matched peers and reported similar frequencies of walking or biking. However, PPI users were less likely to have participated in muscle-strengthening activities (OR: 0.53; 95% CI: 0.30–0.95). PPI users reported similar sedentary behaviors to non-users. Male PPI users had an increase in weight (of 1.52 ± 0.59 kg; p = 0.021) over the previous year compared to non-users, while female PPI users had a non-significant increase in weight. The potential mechanisms for PPI-associated weight gain are unclear as we did not find evidence for significant differences in energy intake or markers of energy expenditure. PMID:26492268

  13. Architecture and active site of particulate methane monooxygenase

    PubMed Central

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2012-01-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria, organisms that live on methane gas as their sole carbon source. Understanding pMMO function has important implications for bioremediation applications and for the development of new, environmentally friendly catalysts for the direct conversion of methane to methanol. Crystal structures of pMMOs from three different methanotrophs reveal a trimeric architecture, consisting of three copies each of the pmoB, pmoA, and pmoC subunits. There are three distinct metal centers in each protomer of the trimer, mononuclear and dinuclear copper sites in the periplasmic regions of pmoB and a mononuclear site within the membrane that can be occupied by copper or zinc. Various models for the pMMO active site have been proposed within these structural constraints, including dicopper, tricopper, and diiron centers. Biochemical and spectroscopic data on pMMO and recombinant soluble fragments, denoted spmoB proteins, indicate that the active site involves copper and is located at the site of the dicopper center in the pmoB subunit. Initial spectroscopic evidence for O2 binding at this site has been obtained. Despite these findings, questions remain about the active site identity and nuclearity and will be the focus of future studies. PMID:22725967

  14. Proton Therapy

    MedlinePlus

    ... nucleus is surrounded by electrons. In proton therapy, beams of fast-moving protons are used to destroy ... atoms to release proton, neutron, and helium ion beams. In this highly specialized form of radiosurgery , proton ...

  15. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site.

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  16. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  17. Kinetic Steering of Quinol Oxidation by ‘Proton Stripping’ at the Cytochrome bc1 Complex Qo Site

    SciTech Connect

    Kramer, David M.; Cape, Jonathan L.; Forquer, Isaac P.; Bowman, Michael K.

    2005-05-13

    The cyt bc1 complex and the related cyt b6f and bc-type complexes are key components of chemiosmotic energy conversion in mitochondria, chloroplasts and many bacteria. These complexes oxidize a range of different quinol or hydroquinone (QH2) molecules—ubihydroquinone (UQH2) in the case of mitochondria and many bacteria, plastohydroquinone (PQH2) in chloroplasts and cyanobacteria, and menahydroquinone in many bacteria—to the corresponding quinone (Q), and reduce a range of soluble electron carriers, e.g. cyt c in purple bacteria and mitochondria, plastocyanin in chloroplasts. The redox reactions are coupled to the translocation of protons across the energetic membrane to store energy in an electrochemical proton gradient, or proton motive force (pmf) which drives ATP synthesis.

  18. SU-E-T-590: An Activation Study of Materials and Devices Present in a Proton Treatment Room

    SciTech Connect

    Spitznagel, D

    2014-06-01

    Purpose: The use of protons for radiation therapy is growing rapidly. One consequence of protons interacting with different media is activation. These nuclear reactions induced by the protons, scattered neutrons, and gamma rays, activate different materials encountered, particularly by the therapists. The purpose of this study was to examine the derived nuclides from the activation, and also the decay rate. Methods: The study was conducted in our proton therapy facility. Protons are derived from a synchrocyclotron and pass through field shipping systems, apertures, and range compensators to define the beam within the patient.Included materials of concerns measured; the patient support couch, sheet rock in the wall, solid plastics used for quality assurance and dosimetry, and the passive scattering system itself, which includes brass apertures, and Lucite or blue wax compensators. All devices were studied post irradiation using gamma spectroscopy to determine the nuclides, and a sodium iodine scintillation detector to measure decay, particularly when the dose rate fell to background levels. Results: We have also determined from the measurements we will maintain brass apertures for three months before sending them for scrap. Some of the radionuclides arrived from these measurements included Na-22 for the blue wax compensator, C1-34m for the sheetrock, and Sc-44 and Co-60 for the brass apertures. We found compensators made out of Lucite or wax decayed to background in 2 hours. The patient support couch decayed to background in approximately 40 minutes, and sheet rock decayed in 80 minutes. In terms of the aperture layers, the most proximal aperture slab had much higher activity than the distal slab. Also the proximal sides of the slabs were much more activate than the distal. Conclusion: We have given proper instruction to therapists performing quality assurance in terms of the handled plastics, and to handle apertures rapidly as possible.

  19. Tautomerization lowers the activation barriers for N-glycosidic bond cleavage of protonated uridine and 2'-deoxyuridine.

    PubMed

    Wu, R R; Rodgers, M T

    2016-09-21

    The gas-phase conformations of protonated uridine, [Urd+H](+), and its 2'-deoxy form, protonated 2'-deoxyuridine, [dUrd+H](+), have been examined in detail previously by infrared multiple photon dissociation action spectroscopy techniques. Both 2,4-dihydroxy tautomers and O4 protonated conformers of [Urd+H](+) and [dUrd+H](+) were found to coexist in the experiments with the 2,4-dihydroxy tautomers dominating the population. In the present study, the kinetic energy dependence of the collision-induced dissociation behavior of [Urd+H](+) and [dUrd+H](+) are examined using a guided ion beam tandem mass spectrometer to probe the mechanisms and energetics for activated dissociation of these protonated nucleosides. The primary dissociation pathways observed involve N-glycosidic bond cleavage leading to competitive elimination of protonated or neutral uracil. The potential energy surfaces (PESs) for these N-glycosidic bond cleavage pathways are mapped out via electronic structure calculations for the mixture of 2,4-dihydroxy tautomers and O4 protonated conformers of [Urd+H](+) and [dUrd+H](+) populated in the experiments. The calculated activation energies (AEs) and heats of reaction (ΔHrxns) for N-glycosidic bond cleavage at both the B3LYP and MP2(full) levels of theory are compared to the measured values. The agreement between experiment and theory indicates that B3LYP provides better estimates of the energetics of the species along the PESs for N-glycosidic bond cleavage than MP2, and that the 2,4-dihydroxy tautomers, which are stabilized by strong hydrogen-bonding interactions, predominantly influence the observed threshold dissociation behavior of [Urd+H](+) and [dUrd+H](+). PMID:27536972

  20. The Active Site of Oligogalacturonate Lyase Provides Unique Insights into Cytoplasmic Oligogalacturonate β-Elimination*

    PubMed Central

    Abbott, D. Wade; Gilbert, Harry J.; Boraston, Alisdair B.

    2010-01-01

    Oligogalacturonate lyases (OGLs; now also classified as pectate lyase family 22) are cytoplasmic enzymes found in pectinolytic members of Enterobacteriaceae, such as the enteropathogen Yersinia enterocolitica. OGLs utilize a β-elimination mechanism to preferentially catalyze the conversion of saturated and unsaturated digalacturonate into monogalacturonate and the 4,5-unsaturated monogalacturonate-like molecule, 5-keto-4-deoxyuronate. To provide mechanistic insights into the specificity of this enzyme activity, we have characterized the OGL from Y. enterocolitica, YeOGL, on oligogalacturonides and determined its three-dimensional x-ray structure to 1.65 Å. The model contains a Mn2+ atom in the active site, which is coordinated by three histidines, one glutamine, and an acetate ion. The acetate mimics the binding of the uronate group of galactourono-configured substrates. These findings, in combination with enzyme kinetics and metal supplementation assays, provide a framework for modeling the active site architecture of OGL. This enzyme appears to contain a histidine for the abstraction of the α-proton in the −1 subsite, a residue that is highly conserved throughout the OGL family and represents a unique catalytic base among pectic active lyases. In addition, we present a hypothesis for an emerging relationship observed between the cellular distribution of pectate lyase folding and the distinct metal coordination chemistries of pectate lyases. PMID:20851883

  1. Evidence for nickel/proton antiport activity at the tonoplast of the hyperaccumulator plant Alyssum lesbiacum.

    PubMed

    Ingle, R A; Fricker, M D; Smith, J A C

    2008-11-01

    The mechanism of nickel uptake into vacuoles isolated from leaf tissue of Alyssum lesbiacum was investigated to help understand the ability of this species to hyperaccumulate Ni. An imaging system was designed to monitor Ni uptake by single vacuoles using the metal-sensitive fluorescent dye, Newport Green. Nickel uptake into isolated vacuoles from leaf tissue of A. lesbiacum was enhanced by the presence of Mg/ATP, presumably via energisation of the vacuolar H(+)-ATPase (V-ATPase). This ATP-stimulated Ni uptake was abolished by bafilomycin (a diagnostic inhibitor of the V-ATPase) and by dissipation of the transmembrane pH difference with an uncoupler. These observations are consistent with Ni(2+)/nH(+) antiport activity at the tonoplast driven by a proton electrochemical gradient established by the V-ATPase, which would provide a mechanism for secondary active transport of Ni(2+) into the vacuole. This study provides insights into the molecular basis of Ni tolerance in Alyssum, and may aid in the identification of genes involved in Ni hyperaccumulation. PMID:18950432

  2. Application of activity pencil beam algorithm using measured distribution data of positron emitter nuclei for therapeutic SOBP proton beam

    SciTech Connect

    Miyatake, Aya; Nishio, Teiji

    2013-09-15

    Purpose: Recently, much research on imaging the clinical proton-irradiated volume using positron emitter nuclei based on target nuclear fragment reaction has been carried out. The purpose of this study is to develop an activity pencil beam (APB) algorithm for a simulation system for proton-activated positron-emitting imaging in clinical proton therapy using spread-out Bragg peak (SOBP) beams.Methods: The target nuclei of activity distribution calculations are {sup 12}C nuclei, {sup 16}O nuclei, and {sup 40}Ca nuclei, which are the main elements in a human body. Depth activity distributions with SOBP beam irradiations were obtained from the material information of ridge filter (RF) and depth activity distributions of compounds of the three target nuclei measured by BOLPs-RGp (beam ON-LINE PET system mounted on a rotating gantry port) with mono-energetic Bragg peak (MONO) beam irradiations. The calculated data of depth activity distributions with SOBP beam irradiations were sorted in terms of kind of nucleus, energy of proton beam, SOBP width, and thickness of fine degrader (FD), which were verified. The calculated depth activity distributions with SOBP beam irradiations were compared with the measured ones. APB kernels were made from the calculated depth activity distributions with SOBP beam irradiations to construct a simulation system using the APB algorithm for SOBP beams.Results: The depth activity distributions were prepared using the material information of RF and the measured depth activity distributions with MONO beam irradiations for clinical therapy using SOBP beams. With the SOBP width widening, the distal fall-offs of depth activity distributions and the difference from the depth dose distributions were large. The shapes of the calculated depth activity distributions nearly agreed with those of the measured ones upon comparison between the two. The APB kernels of SOBP beams were prepared by making use of the data on depth activity distributions with SOBP

  3. Active Sites Environmental Monitoring Program: Mid-FY 1991 report

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1991-10-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.

  4. Refining the active site structure of iron-iron hydrogenase using computational infrared spectroscopy.

    PubMed

    Tye, Jesse W; Darensbourg, Marcetta Y; Hall, Michael B

    2008-04-01

    Iron-iron hydrogenases ([FeFe]H2ases) are exceptional natural catalysts for the reduction of protons to dihydrogen. Future biotechnological applications based on these enzymes require a precise understanding of their structures and properties. Although the [FeFe]H2ases have been characterized by single-crystal X-ray crystallography and a range of spectroscopic techniques, ambiguities remain regarding the details of the molecular structures of the spectroscopically observed forms. We use density functional theory (DFT) computations on small-molecule computational models of the [FeFe]H2ase active site to address this problem. Specifically, a series of structural candidates are geometry optimized and their infrared (IR) spectra are simulated using the computed C-O and C-N stretching frequencies and infrared intensities. Structural assignments are made by comparing these spectra to the experimentally determined IR spectra for each form. The H red form is assigned as a mixture of an Fe(I)Fe(I) form with an open site on the distal iron center and either a Fe(I)Fe(I) form in which the distal cyanide has been protonated or a Fe(II)Fe(II) form with a bridging hydride ligand. The Hox form is assigned as a valence-localized Fe(I)Fe(II) redox level with an open site at the distal iron. The Hox(air)(ox) form is assigned as an Fe(II)Fe(II) redox level with OH(-) or OOH(-) bound to the distal iron center that may or may not have an oxygen atom bound to one of the sulfur atoms of the dithiolate linker. Comparisons of the computed IR spectra of the (12)CO and (13)CO inhibited form with the experimental IR spectra show that exogenous CO binds terminally to the distal iron center.

  5. Active chemisorption sites in functionalized ionic liquids for carbon capture.

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

    Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined.

  6. Active chemisorption sites in functionalized ionic liquids for carbon capture.

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

    Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined. PMID:27243042

  7. Studies on the active site of pig plasma amine oxidase.

    PubMed Central

    Collison, D; Knowles, P F; Mabbs, F E; Rius, F X; Singh, I; Dooley, D M; Cote, C E; McGuirl, M

    1989-01-01

    Amine oxidase from pig plasma (PPAO) has two bound Cu2+ ions and at least one pyrroloquinoline quinone (PQQ) moiety as cofactors. It is shown that recovery of activity by copper-depleted PPAO is linear with respect to added Cu2+ ions. Recovery of e.s.r. and optical spectral characteristics of active-site copper parallel the recovery of catalytic activity. These results are consistent with both Cu2+ ions contributing to catalysis. Further e.s.r. studies indicate that the two copper sites in PPAO, unlike those in amine oxidases from other sources, are chemically distinct. These comparative studies establish that non-identity of the Cu2+ ions in PPAO is not a requirement for amine oxidase activity. It is shown through the use of a new assay procedure that there are two molecules of PQQ bound per molecule of protein in PPAO; only the more reactive of these PQQ moieties is required for activity. PMID:2559715

  8. Influence of the proton pump inhibitor lansoprazole on distribution and activity of doxorubicin in solid tumors

    PubMed Central

    Yu, Man; Lee, Carol; Wang, Marina; Tannock, Ian F

    2015-01-01

    Cellular causes of resistance and limited drug distribution within solid tumors limit therapeutic efficacy of anticancer drugs. Acidic endosomes in cancer cells mediate autophagy, which facilitates survival of stressed cells, and may contribute to drug resistance. Basic drugs (e.g. doxorubicin) are sequestered in acidic endosomes, thereby diverting drugs from their target DNA and decreasing penetration to distal cells. Proton pump inhibitors (PPIs) may raise endosomal pH, with potential to improve drug efficacy and distribution in solid tumors. We determined the effects of the PPI lansoprazole to modify the activity of doxorubicin. To gain insight into its mechanisms, we studied the effects of lansoprazole on endosomal pH, and on the spatial distribution of doxorubicin, and of biomarkers reflecting its activity, using in vitro and murine models. Lansoprazole showed concentration-dependent effects to raise endosomal pH and to inhibit endosomal sequestration of doxorubicin in cultured tumor cells. Lansoprazole was not toxic to cancer cells but potentiated the cytotoxicity of doxorubicin and enhanced its penetration through multilayered cell cultures. In solid tumors, lansoprazole improved the distribution of doxorubicin but also increased expression of biomarkers of drug activity throughout the tumor. Combined treatment with lansoprazole and doxorubicin was more effective in delaying tumor growth as compared to either agent alone. Together, lansoprazole enhances the therapeutic effects of doxorubicin both by improving its distribution and increasing its activity in solid tumors. Use of PPIs to improve drug distribution and to inhibit autophagy represents a promising strategy to enhance the effectiveness of anticancer drugs in solid tumors. PMID:26212113

  9. Following [FeFe] Hydrogenase Active Site Intermediates by Time-Resolved Mid-IR Spectroscopy.

    PubMed

    Mirmohades, Mohammad; Adamska-Venkatesh, Agnieszka; Sommer, Constanze; Reijerse, Edward; Lomoth, Reiner; Lubitz, Wolfgang; Hammarström, Leif

    2016-08-18

    Time-resolved nanosecond mid-infrared spectroscopy is for the first time employed to study the [FeFe] hydrogenase from Chlamydomonas reinhardtii and to investigate relevant intermediates of the enzyme active site. An actinic 355 nm, 10 ns laser flash triggered photodissociation of a carbonyl group from the CO-inhibited state Hox-CO to form the state Hox, which is an intermediate of the catalytic proton reduction cycle. Time-resolved infrared spectroscopy allowed us to directly follow the subsequent rebinding of the carbonyl, re-forming Hox-CO, and determine the reaction half-life to be t1/2 ≈ 13 ± 5 ms at room temperature. This gives direct information on the dynamics of CO inhibition of the enzyme. PMID:27494400

  10. Following [FeFe] Hydrogenase Active Site Intermediates by Time-Resolved Mid-IR Spectroscopy.

    PubMed

    Mirmohades, Mohammad; Adamska-Venkatesh, Agnieszka; Sommer, Constanze; Reijerse, Edward; Lomoth, Reiner; Lubitz, Wolfgang; Hammarström, Leif

    2016-08-18

    Time-resolved nanosecond mid-infrared spectroscopy is for the first time employed to study the [FeFe] hydrogenase from Chlamydomonas reinhardtii and to investigate relevant intermediates of the enzyme active site. An actinic 355 nm, 10 ns laser flash triggered photodissociation of a carbonyl group from the CO-inhibited state Hox-CO to form the state Hox, which is an intermediate of the catalytic proton reduction cycle. Time-resolved infrared spectroscopy allowed us to directly follow the subsequent rebinding of the carbonyl, re-forming Hox-CO, and determine the reaction half-life to be t1/2 ≈ 13 ± 5 ms at room temperature. This gives direct information on the dynamics of CO inhibition of the enzyme.

  11. Analysis of Hydrogen Tunneling in an Enzyme Active Site using von Neumann Measurements

    PubMed Central

    Sumner, Isaiah; Iyengar, Srinivasan S.

    2010-01-01

    We build on our earlier quantum wavepacket study of hydrogen transfer in the biological enzyme, soybean lipoxygenase-1, by using von Neumann quantum measurement theory to gain qualitative insights into the transfer event. We treat the enzyme active site as a measurement device which acts on the tunneling hydrogen nucleus via the potential it exerts at each configuration. A series of changing active site geometries during the tunneling process effects a sequential projection of the initial, reactant state onto the final, product state. We study this process using several different kinds of von Neumann measurements and show how a discrete sequence of such measurements not only progressively increases the projection of the hydrogen nuclear wavepacket onto the product side but also favors proton over deuteron transfer. Several qualitative features of the hydrogen tunneling problem found in wavepacket dynamics studies are also recovered here. These include the shift in the “transition state” towards the reactant as a result of nuclear quantization, greater participation of excited states in the case of deuterium, and presence of critical points along the reaction coordinate that facilitate hydrogen and deuterium transfer and coincide with surface crossings. To further “tailor” the dynamics, we construct a perturbation to the sequence of measurements, that is a perturbation to the dynamical sequence of active site geometry evolution, which leads us to insight on the existence of sensitive regions of the reaction profile where subtle changes to the dynamics of the active site can have an effect on the hydrogen and deuterium transfer process. PMID:22933858

  12. A Base-Independent Repair Mechanism for DNA Glycosylase—No Discrimination Within the Active Site

    PubMed Central

    Blank, Iris D.; Sadeghian, Keyarash; Ochsenfeld, Christian

    2015-01-01

    The ubiquitous occurrence of DNA damages renders its repair machinery a crucial requirement for the genomic stability and the survival of living organisms. Deficiencies in DNA repair can lead to carcinogenesis, Alzheimer, or Diabetes II, where increased amounts of oxidized DNA bases have been found in patients. Despite the highest mutation frequency among oxidized DNA bases, the base-excision repair process of oxidized and ring-opened guanine, FapydG (2,6-diamino-4-hydroxy-5-formamidopyrimidine), remained unclear since it is difficult to study experimentally. We use newly-developed linear-scaling quantum-chemical methods (QM) allowing us to include up to 700 QM-atoms and achieving size convergence. Instead of the widely assumed base-protonated pathway we find a ribose-protonated repair mechanism which explains experimental observations and shows strong evidence for a base-independent repair process. Our results also imply that discrimination must occur during recognition, prior to the binding within the active site. PMID:26013033

  13. Linkage between proton binding and amidase activity in human alpha-thrombin: effect of ions and temperature.

    PubMed

    Di Cera, E; De Cristofaro, R; Albright, D J; Fenton, J W

    1991-08-13

    The amidase activity of human alpha-thrombin has been studied at steady state in the pH range 6-10, as a function of NaCl concentration from 1 mM to 1 M and temperature from 10 to 40 degrees C. The Michaelis-Menten constant, Km, shows a bell-shaped dependence over this pH range with a minimum around pH 7.5 in the presence of 0.1 M NaCl at 25 degrees C. The catalytic constant, kcat, also has a bell-shaped pH dependence with multiple inflection points that are more evident at low NaCl concentrations and a maximum around pH 8.2 in the presence of 0.1 M NaCl at 25 degrees C. A detailed analysis of the results in terms of a general linkage scheme has allowed a thorough characterization of the linkage between proton and substrate binding and its dependence on NaCl concentration, as well as the relevant entropic and enthalpic contributions to binding and catalytic events. Formulation of detailed partition functions for each enzyme intermediate involved in the catalytic cycle suggests that (at least) three groups are responsible for the control of thrombin amidase activity as a function of pH. One group is to be identified with the active site His, due to its pK values in the free enzyme and the adduct and its enthalpy of ionization. The effect of NaCl concentration on amidase activity seems to be extremely specific. Comparative steady-state measurements carried out in the presence of NaCl, NaBr, NaI, KCl, and MgCl2 show that human alpha-thrombin is capable of discriminating among different cations and anions. This suggests that small ions participate as allosteric effectors in the regulation of thrombin activity. The linkage with NaCl is strongly pH dependent and increases with decreasing pH. The present results provide information on the basic aspects of human alpha-thrombin activity and regulation and enable a rigorous thermodynamic approach to other important regulatory interactions in human alpha-thrombin and its structurally perturbed derivatives. PMID:1868067

  14. Computer simulation of the active site of human serum cholinesterase

    SciTech Connect

    Kefang Jiao; Song Li; Zhengzheng Lu

    1996-12-31

    The first 3D-structure of acetylchelinesterase from Torpedo California electric organ (T.AChE) was published by JL. Sussman in 1991. We have simulated 3D-structure of human serum cholinesterase (H.BuChE) and the active site of H.BuChE. It is discovered by experiment that the residue of H.BuChE is still active site after a part of H.BuChE is cut. For example, the part of 21KD + 20KD is active site of H.BuChE. The 20KD as it is. Studies on these peptides by Hemelogy indicate that two active peptides have same negative electrostatic potential maps diagram. These negative electrostatic areas attached by acetyl choline with positive electrostatic potency. We predict that 147...236 peptide of AChE could be active site because it was as 20KD as with negative electrostatic potential maps. We look forward to proving from other ones.

  15. SU-E-T-230: Measurement of Proton-Activated Positron Emission with PRESAGE 3-D Dosimeters

    SciTech Connect

    Carroll, M; Mawlawi, O; Ibbott, G; Adamovics, J

    2014-06-01

    Purpose: Measurement of positron emission following proton beam irradiation of a target has been studied as a method of in-vivo dosimetry. Relative dosimetry studies between a phantom and treatment plan are susceptible to range uncertainties from material heterogeneities and setup error. By using the radiochromic polyurethane dosimeter PRESAGE, we can correlate the proton dose distribution to the PET activity measurement within a single detector. The PRESAGE formulation used was developed for high-LET proton radiotherapy, has similar density and RLSP to tissue, and consists of a greater carbon component, which gives it a higher positron signal than many other 3D detectors. Methods: Three cylindrical PRESAGE dosimeters were irradiated semi-uniformly to 500 cGy with 180- MeV protons. The beam was directed along the dosimeter axis and delivered a 2-cm SOBP at the center of the dosimeter. The dosimeters were rushed to a nearby PET/CT where imaging began within 15 minutes, less than a single half-life of 11C. A 3-hr measurement captured the full activation decay. Afterwards, the dose profiles were measured by optical-CT. A direct comparison between the measured dose and the positron emission was performed using CERR software. Results: The correlations between dose distributions and PET activity were consistent with previous studies in that the proximal region of the SOBP displayed the highest activity. The spatial distributions between the dose and activity were similar. Along the central axis of the beam, we found a shift in the distal 80% of 1 cm. The lateral profile showed good agreement between dose and activity. PET imaging times between 30-min and 3-hrs showed <5% discrepancy. Conclusion: PRESAGE dosimeters offer a strong and unique potential to accurately correlate dosimetric and PET activation information. Implementation in an anthropomorphic phantom could be used to study representative treatment plans. NIH grant 5R01CA100835.

  16. Exotic Protonated Species Produced by UV-Induced Photofragmentation of a Protonated Dimer: Metastable Protonated Cinchonidine.

    PubMed

    Alata, Ivan; Scuderi, Debora; Lepere, Valeria; Steinmetz, Vincent; Gobert, Fabrice; Thiao-Layel, Loïc; Le Barbu-Debus, Katia; Zehnacker-Rentien, Anne

    2015-10-01

    A metastable protonated cinchona alkaloid was produced in the gas phase by UV-induced photodissociation (UVPD) of its protonated dimer in a Paul ion trap. The infrared multiple photon dissociation (IRMPD) spectrum of the molecular ion formed by UVPD was obtained and compared to DFT calculations to characterize its structure. The protonation site obtained thereby is not accessible by classical protonation ways. The protonated monomer directly formed in the ESI source or by collision-induced dissociation (CID) of the dimer undergoes protonation at the most basic alkaloid nitrogen. In contrast, protonation occurs at the quinoline aromatic ring nitrogen in the UVPD-formed monomer. PMID:26347997

  17. Exotic Protonated Species Produced by UV-Induced Photofragmentation of a Protonated Dimer: Metastable Protonated Cinchonidine.

    PubMed

    Alata, Ivan; Scuderi, Debora; Lepere, Valeria; Steinmetz, Vincent; Gobert, Fabrice; Thiao-Layel, Loïc; Le Barbu-Debus, Katia; Zehnacker-Rentien, Anne

    2015-10-01

    A metastable protonated cinchona alkaloid was produced in the gas phase by UV-induced photodissociation (UVPD) of its protonated dimer in a Paul ion trap. The infrared multiple photon dissociation (IRMPD) spectrum of the molecular ion formed by UVPD was obtained and compared to DFT calculations to characterize its structure. The protonation site obtained thereby is not accessible by classical protonation ways. The protonated monomer directly formed in the ESI source or by collision-induced dissociation (CID) of the dimer undergoes protonation at the most basic alkaloid nitrogen. In contrast, protonation occurs at the quinoline aromatic ring nitrogen in the UVPD-formed monomer.

  18. Linkage between proton binding and amidase activity in human gamma-thrombin.

    PubMed

    De Cristofaro, R; Fenton, J W; Di Cera, E

    1992-02-01

    The amidase activity of human gamma-thrombin has been studied in the pH range 6-10 as a function of NaCl concentration and temperature. As recently found for human alpha-thrombin [Di Cera, E., De Cristofaro, R., Albright, D.J., & Fenton, J.W., II (1991) Biochemistry 30, 7913-7924], the Michaelis-Menten constant, Km, shows a bell-shaped dependence over this pH range with a minimum around pH 7.9 in the presence of 0.1 M NaCl at 25 degrees C. The catalytic constant, kcat, has a bell-shaped pH dependence with a maximum around pH 8.6. A thermodynamic analysis of these parameters has enabled a characterization of the linkage between proton and substrate binding, its dependence on NaCl concentration, and the relevant entropic and enthalpic contributions to binding and catalytic events. Three groups seem to be responsible for the control of gamma-thrombin amidase activity as a function of pH. One of these groups has pK values that are significantly different from those found for alpha-thrombin, and all groups show slightly perturbed enthalpies of ionization. The dependence of gamma-thrombin amidase activity on NaCl concentration is different from that of alpha-thrombin. Increasing NaCl concentration always decreases the substrate affinity for the enzyme in the case of alpha-thrombin, regardless of pH. In the case of gamma-thrombin, such an effect is observed only in the pH range 7.5-9, and a reversed linkage is observed at pH less than 7 and greater than 9.5.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1310421

  19. Aconitase: its source of catalytic protons.

    PubMed

    Kuo, D J; Rose, I A

    1987-12-01

    An ordinary isotope partition experiment was performed to determine the rate of dissociation of the proton from the donor site for the hydration of cis-aconitate. Aconitase in [3H]water was efficiently diluted into well-mixed solutions of cis-aconitate. Citrate and isocitrate that were formed within 2 s were more heavily labeled than could be explained by consideration of an isotope effect in the processing of one proton per enzyme equivalent. Control experiments indicate that mixing was much more rapid than catalytic turnover, ruling out incompletely diluted [3H]water as a significant isotope source. Therefore, it appears that significantly more than one enzyme-bound tritium atom (protons) must have been used in the course of the multiple turnover of the enzyme after the dilution was complete. Isotope incorporation reached values in excess of four proton equivalents as a limit with simple Michaelis dependence on cis-aconitate. From the half-saturation concentration value for trapping, 0.15 mM, the t 1/2 for exchange of each of these protons with solvent appears to be approximately 0.1 s at 0 degrees C. The large number of protons trapped seems to suggest the existence of a structurally stabilized pool of protons, or water, that communicates between the active site base and the medium in the hydration of cis-aconitate. The proton abstracted in the dehydration of [3H]citrate is transferred directly to undissociated cis-aconitate to form isocitrate without dilution, or cis-aconitate having dissociated, the tritium passes to the medium, presumably through the pool of bound protons indicated above. All of the citrate-derived protons can be found in isocitrate if cis-aconitate is added in sufficient concentration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2827757

  20. Resonant active sites in catalytic ammonia synthesis: A structural model

    NASA Astrophysics Data System (ADS)

    Cholach, Alexander R.; Bryliakova, Anna A.; Matveev, Andrey V.; Bulgakov, Nikolai N.

    2016-03-01

    Adsorption sites Mn consisted of n adjacent atoms M, each bound to the adsorbed species, are considered within a realistic model. The sum of bonds Σ lost by atoms in a site in comparison with the bulk atoms was used for evaluation of the local surface imperfection, while the reaction enthalpy at that site was used as a measure of activity. The comparative study of Mn sites (n = 1-5) at basal planes of Pt, Rh, Ir, Fe, Re and Ru with respect to heat of N2 dissociative adsorption QN and heat of Nad + Had → NHad reaction QNH was performed using semi-empirical calculations. Linear QN(Σ) increase and QNH(Σ) decrease allowed to specify the resonant Σ for each surface in catalytic ammonia synthesis at equilibrium Nad coverage. Optimal Σ are realizable for Ru2, Re2 and Ir4 only, whereas other centers meet steric inhibition or unreal crystal structure. Relative activity of the most active sites in proportion 5.0 × 10- 5: 4.5 × 10- 3: 1: 2.5: 3.0: 1080: 2270 for a sequence of Pt4, Rh4, Fe4(fcc), Ir4, Fe2-5(bcc), Ru2, Re2, respectively, is in agreement with relevant experimental data. Similar approach can be applied to other adsorption or catalytic processes exhibiting structure sensitivity.

  1. Bovine plasma amine oxidase (PAO) oxidizes substrate by a proton activation mechanism

    SciTech Connect

    Hartmann, C.; Klinman, J.P.

    1986-05-01

    PAO catalyzes the oxidative deamination of amines to aldehydes, concomitant with a 2e/sup -/ reduction of O/sub 2/ to H/sub 2/O/sub 2/. Several investigators have proposed recently that the organic cofactor in PAO is pyrroloquinoline quinone (PQQ), hitherto seen exclusively in prokaryotes. The structure and properties of PQQ predict first, that substrate and PAO will form a covalent adduct and second, that substrate will be oxidized via proton abstraction. In earlier studies from this laboratory, steady state isotope effects, in conjunction with an intrinsic isotope effect, have been shown to provide microscopic rate constants from complex mechanisms. In this study, V, D/sub V/, V/K and /sup D/(V/K) have been measured for the oxidation of a series of nine ring-substituted benzylamines and (1-/sup 2/H/sub 2/)-benzylamines with PAO. The series of substrates was chosen to minimize collinearity in the electronic and hydrophobic properties of ring substituents. Computed rate constants for the C-H bond cleavage step indicate a strong correlation with electron withdrawing substituents, rho = 1.3, confirming the formation of a discrete carbanion intermediate upon substrate activation. Additional studies are in progress, with the objective of trapping and characterizing the putative adduct between PAO and substrates.

  2. Proton: The Particle

    SciTech Connect

    Suit, Herman

    2013-11-01

    The purpose of this article is to review briefly the nature of protons: creation at the Big Bang, abundance, physical characteristics, internal components, and life span. Several particle discoveries by proton as the experimental tool are considered. Protons play important roles in science, medicine, and industry. This article was prompted by my experience in the curative treatment of cancer patients by protons and my interest in the nature of protons as particles. The latter has been stimulated by many discussions with particle physicists and reading related books and journals. Protons in our universe number ≈10{sup 80}. Protons were created at 10{sup −6} –1 second after the Big Bang at ≈1.37 × 10{sup 10} years beforethe present. Proton life span has been experimentally determined to be ≥10{sup 34} years; that is, the age of the universe is 10{sup −24}th of the minimum life span of a proton. The abundance of the elements is hydrogen, ≈74%; helium, ≈24%; and heavier atoms, ≈2%. Accordingly, protons are the dominant baryonic subatomic particle in the universe because ≈87% are protons. They are in each atom in our universe and thus involved in virtually every activity of matter in the visible universe, including life on our planet. Protons were discovered in 1919. In 1968, they were determined to be composed of even smaller particles, principally quarks and gluons. Protons have been the experimental tool in the discoveries of quarks (charm, bottom, and top), bosons (W{sup +}, W{sup −}, Z{sup 0}, and Higgs), antiprotons, and antineutrons. Industrial applications of protons are numerous and important. Additionally, protons are well appreciated in medicine for their role in radiation oncology and in magnetic resonance imaging. Protons are the dominant baryonic subatomic particle in the visible universe, comprising ≈87% of the particle mass. They are present in each atom of our universe and thus a participant in every activity involving matter.

  3. Proton: the particle.

    PubMed

    Suit, Herman

    2013-11-01

    The purpose of this article is to review briefly the nature of protons: creation at the Big Bang, abundance, physical characteristics, internal components, and life span. Several particle discoveries by proton as the experimental tool are considered. Protons play important roles in science, medicine, and industry. This article was prompted by my experience in the curative treatment of cancer patients by protons and my interest in the nature of protons as particles. The latter has been stimulated by many discussions with particle physicists and reading related books and journals. Protons in our universe number ≈10(80). Protons were created at 10(-6) -1 second after the Big Bang at ≈1.37 × 10(10) years beforethe present. Proton life span has been experimentally determined to be ≥10(34) years; that is, the age of the universe is 10(-24)th of the minimum life span of a proton. The abundance of the elements is hydrogen, ≈74%; helium, ≈24%; and heavier atoms, ≈2%. Accordingly, protons are the dominant baryonic subatomic particle in the universe because ≈87% are protons. They are in each atom in our universe and thus involved in virtually every activity of matter in the visible universe, including life on our planet. Protons were discovered in 1919. In 1968, they were determined to be composed of even smaller particles, principally quarks and gluons. Protons have been the experimental tool in the discoveries of quarks (charm, bottom, and top), bosons (W(+), W(-), Z(0), and Higgs), antiprotons, and antineutrons. Industrial applications of protons are numerous and important. Additionally, protons are well appreciated in medicine for their role in radiation oncology and in magnetic resonance imaging. Protons are the dominant baryonic subatomic particle in the visible universe, comprising ≈87% of the particle mass. They are present in each atom of our universe and thus a participant in every activity involving matter.

  4. Proton: the particle.

    PubMed

    Suit, Herman

    2013-11-01

    The purpose of this article is to review briefly the nature of protons: creation at the Big Bang, abundance, physical characteristics, internal components, and life span. Several particle discoveries by proton as the experimental tool are considered. Protons play important roles in science, medicine, and industry. This article was prompted by my experience in the curative treatment of cancer patients by protons and my interest in the nature of protons as particles. The latter has been stimulated by many discussions with particle physicists and reading related books and journals. Protons in our universe number ≈10(80). Protons were created at 10(-6) -1 second after the Big Bang at ≈1.37 × 10(10) years beforethe present. Proton life span has been experimentally determined to be ≥10(34) years; that is, the age of the universe is 10(-24)th of the minimum life span of a proton. The abundance of the elements is hydrogen, ≈74%; helium, ≈24%; and heavier atoms, ≈2%. Accordingly, protons are the dominant baryonic subatomic particle in the universe because ≈87% are protons. They are in each atom in our universe and thus involved in virtually every activity of matter in the visible universe, including life on our planet. Protons were discovered in 1919. In 1968, they were determined to be composed of even smaller particles, principally quarks and gluons. Protons have been the experimental tool in the discoveries of quarks (charm, bottom, and top), bosons (W(+), W(-), Z(0), and Higgs), antiprotons, and antineutrons. Industrial applications of protons are numerous and important. Additionally, protons are well appreciated in medicine for their role in radiation oncology and in magnetic resonance imaging. Protons are the dominant baryonic subatomic particle in the visible universe, comprising ≈87% of the particle mass. They are present in each atom of our universe and thus a participant in every activity involving matter. PMID:24074929

  5. Multi-site Phosphorylation Regulates Bim Stability and Apoptotic Activity

    PubMed Central

    Hübner, Anette; Barrett, Tamera; Flavell, Richard A.; Davis, Roger J.

    2008-01-01

    The pro-apoptotic BH3-only protein Bim is established to be an important mediator of signaling pathways that induce cell death. Multi-site phosphorylation of Bim by several members of the MAP kinase group is implicated as a regulatory mechanism that controls the apoptotic activity of Bim. To test the role of Bim phosphorylation in vivo, we constructed mice with a series of mutant alleles that express phosphorylation-defective Bim proteins. We show that mutation of the phosphorylation site Thr-112 causes decreased binding of Bim to the anti-apoptotic protein Bcl2 and can increase cell survival. In contrast, mutation of the phosphorylation sites Ser-55, Ser-65, and Ser-73 can cause increased apoptosis because of reduced proteasomal degradation of Bim. Together, these data indicate that phosphorylation can regulate Bim by multiple mechanisms and that the phosphorylation of Bim on different sites can contribute to the sensitivity of cellular apoptotic responses. PMID:18498746

  6. A peroxide bridge between Fe and Cu ions in the O2 reduction site of fully oxidized cytochrome c oxidase could suppress the proton pump.

    PubMed

    Aoyama, Hiroshi; Muramoto, Kazumasa; Shinzawa-Itoh, Kyoko; Hirata, Kunio; Yamashita, Eiki; Tsukihara, Tomitake; Ogura, Takashi; Yoshikawa, Shinya

    2009-02-17

    The fully oxidized form of cytochrome c oxidase, immediately after complete oxidation of the fully reduced form, pumps protons upon each of the initial 2 single-electron reduction steps, whereas protons are not pumped during single-electron reduction of the fully oxidized "as-isolated" form (the fully oxidized form without any reduction/oxidation treatment) [Bloch D, et al. (2004) The catalytic cycle of cytochrome c oxidase is not the sum of its two halves. Proc Natl Acad Sci USA 101:529-533]. For identification of structural differences causing the remarkable functional difference between these 2 distinct fully oxidized forms, the X-ray structure of the fully oxidized as-isolated bovine heart cytochrome c oxidase was determined at 1.95-A resolution by limiting the X-ray dose for each shot and by using many (approximately 400) single crystals. This minimizes the effects of hydrated electrons induced by the X-ray irradiation. The X-ray structure showed a peroxide group bridging the 2 metal sites in the O(2) reduction site (Fe(3+)-O(-)-O(-)-Cu(2+)), in contrast to a ferric hydroxide (Fe(3+)-OH(-)) in the fully oxidized form immediately after complete oxidation from the fully reduced form, as has been revealed by resonance Raman analyses. The peroxide-bridged structure is consistent with the reductive titration results showing that 6 electron equivalents are required for complete reduction of the fully oxidized as-isolated form. The structural difference between the 2 fully oxidized forms suggests that the bound peroxide in the O(2) reduction site suppresses the proton pumping function. PMID:19164527

  7. Two-dimensional NMR studies of squash family inhibitors. Sequence-specific proton assignments and secondary structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III

    SciTech Connect

    Krisnamoorthi, R.; Yuxi Gong; Chanlan Sun Lin ); VanderVelde, D. )

    1992-01-28

    The solution structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III*) was investigated by two-dimensional proton nuclear magnetic resonance (2D NMR) spectroscopy. CMTI-III*, prepared by reacting CMTI-III with trypsin which cleaved the Arg5-Ile6 peptide bond, had the two fragments held together by a disulfide linkage. Sequence-specific {sup 1}H NMR resonance assignments were made for all the 29 amino acid residues of the protein. The secondary structure of CMTI-III*, as deduced from NOESY cross peaks and identification of slowly exchanging hydrogens, contains two turns, a 3{sub 10}-helix, and a triple-stranded {beta}-sheet. Sequential proton assignments were also made for the virgin inhibitor, CMTI-III, at pH 4.71, 30C. Comparison of backbone hydrogen chemical shifts of CMTI-III and CMTI-III* revealed significant changes for residues located far away from the reactive-site region as well as for those located near it, indicating tertiary structural changes that are transmitted through most of the 29 residues of the inhibitor protein. These chemical shift changes were relatively small compared to changes that occurred upon hydrolysis of the reactive-site peptide bond between Arg 5 and Ile6 in CMTI-III.

  8. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    PubMed

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  9. Chemical Modification of Papain and Subtilisin: An Active Site Comparison

    ERIC Educational Resources Information Center

    St-Vincent, Mireille; Dickman, Michael

    2004-01-01

    An experiment using methyle methanethiosulfonate (MMTS) and phenylmethylsulfonyl flouride (PMSF) to specifically modify the cysteine and serine residues in the active sites of papain and subtilism respectively is demonstrated. The covalent modification of these enzymes and subsequent rescue of papain shows the beginning biochemist that proteins…

  10. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  11. Surface Activation of Electrocatalysis at Oxide Electrodes. Concerted Electron-Proton Transfer

    SciTech Connect

    Gagliardi, Christopher J.; Jurss, Jonah W.; Thorp, H. Holden; Meyer, Thomas J.

    2011-03-21

    Dramatic rate enhancements are observed for the oxidation of phenols, including tyrosine, at indium-tin oxide electrodes modified by the addition of the electron-transfer relays [MII(bpy)2(4,4'-(HO)2P(O)CH2)2bpy)]2+ (M = Ru, Os) with clear evidence for the importance of proton-coupled electron transfer and concerted electron-proton transfer.

  12. The protonation state of catalytic residues in the resting state of KasA revisited: detailed mechanism for the activation of KasA by its own substrate.

    PubMed

    Lee, Wook; Engels, Bernd

    2014-02-11

    Mycobacterium tuberculosis is the causative pathogen of tuberculosis, the second leading cause of death from an infectious disease globally. β-Ketoacyl ACP synthase I (KasA) is essential for the survival of M. tuberculosis, because it is one of the key enzymes in the biosynthetic pathway of mycolic acid, the building block of the cell wall in M. tuberculosis. To distinguish among the various suggested mechanisms of KasA that are based on different protonation states of the active site, we characterize its resting state by various theoretical approaches ranging from first-principle-based quantum mechanical/molecular mechanical molecular dynamics simulations (QM/MM MD) with large QM parts to force field-based MD and free energy perturbation computations. In contrast to a previous study that used less reliable semiempirical approaches in combination with smaller QM parts, our improved computations predict that the most important active site residues, Cys171 and His311, are neutral. Because the neutral catalytic residues are too unreactive to attack the substrate, the question of how their activation is achieved arises. Combining our computed results with structural information about the malonyl binding pocket, we devised a detailed model about the activation mechanism. A conformational change of Phe404 possibly triggered by the substrate is central for the activation because it switches KasA to the sufficiently reactive zwitterionic state.

  13. The protonation state of catalytic residues in the resting state of KasA revisited: detailed mechanism for the activation of KasA by its own substrate.

    PubMed

    Lee, Wook; Engels, Bernd

    2014-02-11

    Mycobacterium tuberculosis is the causative pathogen of tuberculosis, the second leading cause of death from an infectious disease globally. β-Ketoacyl ACP synthase I (KasA) is essential for the survival of M. tuberculosis, because it is one of the key enzymes in the biosynthetic pathway of mycolic acid, the building block of the cell wall in M. tuberculosis. To distinguish among the various suggested mechanisms of KasA that are based on different protonation states of the active site, we characterize its resting state by various theoretical approaches ranging from first-principle-based quantum mechanical/molecular mechanical molecular dynamics simulations (QM/MM MD) with large QM parts to force field-based MD and free energy perturbation computations. In contrast to a previous study that used less reliable semiempirical approaches in combination with smaller QM parts, our improved computations predict that the most important active site residues, Cys171 and His311, are neutral. Because the neutral catalytic residues are too unreactive to attack the substrate, the question of how their activation is achieved arises. Combining our computed results with structural information about the malonyl binding pocket, we devised a detailed model about the activation mechanism. A conformational change of Phe404 possibly triggered by the substrate is central for the activation because it switches KasA to the sufficiently reactive zwitterionic state. PMID:24479625

  14. 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

  15. N-methyl-D-aspartate recognition site ligands modulate activity at the coupled glycine recognition site.

    PubMed

    Hood, W F; Compton, R P; Monahan, J B

    1990-03-01

    In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine-insensitive [3H]glycine binding are altered in the presence of N-methyl-D-aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L-glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 microM vs. 0.129 microM). Similar decreases in affinity are observed for the glycine site agonists, D-serine and 1-aminocyclopropane-1-carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1-hydroxy-3-amino-2-pyrrolidone and 1-aminocyclobutane-1-carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L-glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.

  16. Enumerating Pathways of Proton Abstraction Based on a Spatial and Electrostatic Analysis of Residues in the Catalytic Site

    PubMed Central

    Chakraborty, Sandeep

    2012-01-01

    The pathways of proton abstraction (PA), a key aspect of most catalytic reactions, is often controversial and highly debated. Ultrahigh-resolution diffraction studies, molecular dynamics, quantum mechanics and molecular mechanic simulations are often adopted to gain insights in the PA mechanisms in enzymes. These methods require expertise and effort to setup and can be computationally intensive. We present a push button methodology – Proton abstraction Simulation (PRISM) – to enumerate the possible pathways of PA in a protein with known 3D structure based on the spatial and electrostatic properties of residues in the proximity of a given nucleophilic residue. Proton movements are evaluated in the vicinity of this nucleophilic residue based on distances, potential differences, spatial channels and characteristics of the individual residues (polarity, acidic, basic, etc). Modulating these parameters eliminates their empirical nature and also might reveal pathways that originate from conformational changes. We have validated our method using serine proteases and concurred with the dichotomy in PA in Class A β-lactamases, both of which are hydrolases. The PA mechanism in a transferase has also been corroborated. The source code is made available at www.sanchak.com/prism. PMID:22745790

  17. A Ta/W mixed addenda heteropolyacid with excellent acid catalytic activity and proton-conducting property

    NASA Astrophysics Data System (ADS)

    Li, Shujun; Peng, Qingpo; Chen, Xuenian; Wang, Ruoya; Zhai, Jianxin; Hu, Weihua; Ma, Fengji; Zhang, Jie; Liu, Shuxia

    2016-11-01

    A new HPAs H20[P8W60Ta12(H2O)4(OH)8O236]·125H2O (H-1) which comprises a Ta/W mixed addenda heteropolyanion, 20 protons, and 125 crystalline water molecules has been prepared through ion-exchange method. The structure and properties of H-1 have been explored in detail. AC impedance measurements indicate that H-1 is a good solid state proton conducting material at room temperature with a conductivity value of 7.2×10-3 S cm-1 (25 °C, 30% RH). Cyclic voltammograms of H-1 indicate the electrocatalytic activity towards the reduction of nitrite. Hammett acidity constant H0 of H-1 in CH3CN is -2.91, which is the strongest among the present known HPAs. Relatively, H-1 exhibits excellent catalytic activities toward acetal reaction.

  18. Control of active sites in selective flocculation: I -- Mathematical model

    SciTech Connect

    Behl, S.; Moudgil, B.M.; Prakash, T.S. . Dept. of Materials Science and Engineering)

    1993-12-01

    Heteroflocculation has been determined to be another major reason for loss in selectivity for flocculation process. In a mathematical model developed earlier, conditions for controlling heteroflocculation were discussed. Blocking active sites to control selective adsorption of a flocculant oil a desirable solid surface is discussed. It has been demonstrated that the lower molecular weight fraction of a flocculant which is incapable of flocculating the particles is an efficient site blocking agent. The major application of selective flocculation has been in mineral processing but many potential uses exist in biological and other colloidal systems. These include purification of ceramic powders, separating hazardous solids from chemical waste, and removal of deleterious components from paper pulp.

  19. The site of activation of factor X by cancer procoagulant.

    PubMed

    Gordon, S G; Mourad, A M

    1991-12-01

    Cancer procoagulant (CP) is a cysteine proteinase found in a variety of malignant cells and tissues and in human amnion-chorion tissue. It initiates coagulation by activating factor X. However, the amino acid sequence of the substrate protein that determines the cleavage site of cysteine proteinases is different from that of the serine proteinases that normally activate factor X, such as factor IXa, VIIa and Russell's Viper Venom (RVV). Therefore, it was of interest to determine the site of cleavage of human factor X by CP. Purified CP was incubated with purified factor X and the reaction mixture was electrophoresed on a 10% Tris-tricine SDS-PAGE gel. The proteins were electroeluted on to a polyvinylidene difluoride (PVDF) membrane, and stained with Coomassie blue. The heavy chain of activated factor X was cut out of the PVDF membrane and sequenced with an Applied Biosystems 477A with on-line HPLC. The primary cleavage sequence was Asp-Ala-Ala-Asp-Leu-Asp-Pro-; two other secondary sequences Ser-Ile-Thr-Trp-Lys-Pro- and Glu-Asn-Pro-Phe-Asp-Leu were found. The penultimate amino acid on the carbonyl side of the hydrolysed amide bond plays a critical role for the recognition of the cleavage site of cysteine proteinases. These data indicate that the penultimate amino acid for the primary cleavage site of factor X by CP is proline-20 and for the secondary sites, proline-13 and proline-28. This is in contrast to arginine-52 that determines the specificity of the cleavage by normal serine proteinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. 1H-NMR comparative study of the active site in shark (Galeorhinus japonicus), horse, and sperm whale deoxy myoglobins.

    PubMed

    Yamamoto, Y; Iwafune, K; Chûjô, R; Inoue, Y; Imai, K; Suzuki, T

    1992-09-01

    1H-NMR spectra of deoxy myoglobins (Mbs) from shark (Galeorhinus japonicus), horse, and sperm whale have been studied to gain insights into their active site structure. It has been demonstrated for the first time that nuclear Overhauser effect (NOE) can be observed between heme peripheral side-chain proton resonances of these paramagnetic complexes. Val-E11 methyl and His-F8 C delta H proton resonances of these Mbs were also assigned from the characteristic shift and line width. The hyperfine shift of the former resonance was used to calculate the magnetic anisotropy of the protein. The shift analysis of the latter resonance, together with the previously assigned His-F8 N delta H proton resonance, revealed that the strain on the Fe-N epsilon bond is in the order horse Mb approximately whale Mb < shark Mb and that the hydrogen bond strength of the His-F8 N delta H proton to the main-chain carbonyl oxygen in the preceding turn of the F helix is in the order shark Mb < horse Mb < whale Mb. Weaker Feporphyrin interaction in shark Mb was manifested in a smaller shift of the heme methyl proton resonance and appears to result from distortion of the coordination geometry in this Mb. Larger strain on the Fe-N epsilon bond in shark Mb should be to some extent attributed to its lowered O2 affinity (P50 = 1.1 mmHg at 20 degrees C), compared to whale and horse Mbs.

  1. Active Site, Catalytic Cycle, and Iodination Reactions of Vanadium Iodoperoxidase: A Computational Study.

    PubMed

    Pacios, Luis F; Gálvez, Oscar

    2010-05-11

    A combined computational study using molecular surfaces and Poisson-Boltzmann electrostatic potentials for proteins and quantum calculations on complexes representing the vanadate cofactor throughout the catalytic cycle is employed to study the activity of vanadium iodoperoxidase (VIPO) from alga Laminaria digitata . A model structure of VIPO is compared with available crystal structures of chloroperoxidases (VClPOs) and bromoperoxidases (VBrPOs) focusing on properties of the active site that concern halogen specificity. It is found that VIPO displays distinctive features regarding electrostatic potentials at the site cavity and the local topography of the cavity entrance. Quantum calculations on cofactor stages throughout the catalytic cycle reveal that, while steps involving binding of hydrogen peroxide and halide oxidization agree with available data on VBrPO, final formation and subsequent release of hypohalous acid could follow a different pathway consisting of His476-assisted protonation of bonded hypoiodite and further displacement by a water molecule. Ab initio free energies of reaction computed to explore iodination of organic substrates predict strongly exoergonic reactions with HOI, whereas other possible iodination reagents give thermodynamically disfavored reactions.

  2. Active-Site-Accessible, Porphyrinic Metal;#8722;Organic Framework Materials

    SciTech Connect

    Farha, Omar K.; Shultz, Abraham M.; Sarjeant, Amy A.; Nguyen, SonBinh T.; Hupp, Joseph T.

    2012-02-06

    On account of their structural similarity to cofactors found in many metallo-enzymes, metalloporphyrins are obvious potential building blocks for catalytically active, metal-organic framework (MOF) materials. While numerous porphyrin-based MOFs have already been described, versions featuring highly accessible active sites and permanent microporosity are remarkably scarce. Indeed, of the more than 70 previously reported porphyrinic MOFs, only one has been shown to be both permanently microporous and contain internally accessible active sites for chemical catalysis. Attempts to generalize the design approach used in this single successful case have failed. Reported here, however, is the synthesis of an extended family of MOFs that directly incorporate a variety of metalloporphyrins (specifically Al{sup 3+}, Zn{sup 2+}, Pd{sup 2+}, Mn{sup 3+}, and Fe{sup 3+} complexes). These robust porphyrinic materials (RPMs) feature large channels and readily accessible active sites. As an illustrative example, one of the manganese-containing RPMs is shown to be catalytically competent for the oxidation of alkenes and alkanes.

  3. Proton-Coupled Electron Transfer

    SciTech Connect

    Weinberg, Dave; Gagliardi, Christopher J.; Hull, Jonathan F; Murphy, Christine Fecenko; Kent, Caleb A.; Westlake, Brittany C.; Paul, Amit; Ess, Daniel H; McCafferty, Dewey Granville; Meyer, Thomas J

    2012-07-11

    Proton-Coupled Electron Transfer (PCET) describes reactions in which there is a change in both electron and proton content between reactants and products. It originates from the influence of changes in electron content on acid-base properties and provides a molecular-level basis for energy transduction between proton transfer and electron transfer. Coupled electron-proton transfer or EPT is defined as an elementary step in which electrons and protons transfer from different orbitals on the donor to different orbitals on the acceptor. There is (usually) a clear distinction between EPT and H-atom transfer (HAT) or hydride transfer, in which the transferring electrons and proton come from the same bond. Hybrid mechanisms exist in which the elementary steps are different for the reaction partners. EPT pathways such as PhO•/PhOH exchange have much in common with HAT pathways in that electronic coupling is significant, comparable to the reorganization energy with H{sub DA} ~ λ. Multiple-Site Electron-Proton Transfer (MS-EPT) is an elementary step in which an electron-proton donor transfers electrons and protons to different acceptors, or an electron-proton acceptor accepts electrons and protons from different donors. It exploits the long-range nature of electron transfer while providing for the short-range nature of proton transfer. A variety of EPT pathways exist, creating a taxonomy based on what is transferred, e.g., 1e-/2H+ MS-EPT. PCET achieves “redox potential leveling” between sequential couples and the buildup of multiple redox equivalents, which is of importance in multielectron catalysis. There are many examples of PCET and pH-dependent redox behavior in metal complexes, in organic and biological molecules, in excited states, and on surfaces. Changes in pH can be used to induce electron transfer through films and over long distances in molecules. Changes in pH, induced by local electron transfer, create pH gradients and a driving

  4. Combining density functional theory (DFT) and collision cross-section (CCS) calculations to analyze the gas-phase behaviour of small molecules and their protonation site isomers.

    PubMed

    Boschmans, Jasper; Jacobs, Sam; Williams, Jonathan P; Palmer, Martin; Richardson, Keith; Giles, Kevin; Lapthorn, Cris; Herrebout, Wouter A; Lemière, Filip; Sobott, Frank

    2016-06-20

    Electrospray ion mobility-mass spectrometry (IM-MS) data show that for some small molecules, two (or even more) ions with identical sum formula and mass, but distinct drift times are observed. In spite of showing their own unique and characteristic fragmentation spectra in MS/MS, no configurational or constitutional isomers are found to be present in solution. Instead the observation and separation of such ions appears to be inherent to their gas-phase behaviour during ion mobility experiments. The origin of multiple drift times is thought to be the result of protonation site isomers ('protomers'). Although some important properties of protomers have been highlighted by other studies, correlating the experimental collision cross-sections (CCSs) with calculated values has proven to be a major difficulty. As a model, this study uses the pharmaceutical compound melphalan and a number of related molecules with alternative (gas-phase) protonation sites. Our study combines density functional theory (DFT) calculations with modified MobCal methods (e.g. nitrogen-based Trajectory Method algorithm) for the calculation of theoretical CCS values. Calculated structures can be linked to experimentally observed signals, and a strong correlation is found between the difference of the calculated dipole moments of the protomer pairs and their experimental CCS separation.

  5. Combining density functional theory (DFT) and collision cross-section (CCS) calculations to analyze the gas-phase behaviour of small molecules and their protonation site isomers.

    PubMed

    Boschmans, Jasper; Jacobs, Sam; Williams, Jonathan P; Palmer, Martin; Richardson, Keith; Giles, Kevin; Lapthorn, Cris; Herrebout, Wouter A; Lemière, Filip; Sobott, Frank

    2016-06-20

    Electrospray ion mobility-mass spectrometry (IM-MS) data show that for some small molecules, two (or even more) ions with identical sum formula and mass, but distinct drift times are observed. In spite of showing their own unique and characteristic fragmentation spectra in MS/MS, no configurational or constitutional isomers are found to be present in solution. Instead the observation and separation of such ions appears to be inherent to their gas-phase behaviour during ion mobility experiments. The origin of multiple drift times is thought to be the result of protonation site isomers ('protomers'). Although some important properties of protomers have been highlighted by other studies, correlating the experimental collision cross-sections (CCSs) with calculated values has proven to be a major difficulty. As a model, this study uses the pharmaceutical compound melphalan and a number of related molecules with alternative (gas-phase) protonation sites. Our study combines density functional theory (DFT) calculations with modified MobCal methods (e.g. nitrogen-based Trajectory Method algorithm) for the calculation of theoretical CCS values. Calculated structures can be linked to experimentally observed signals, and a strong correlation is found between the difference of the calculated dipole moments of the protomer pairs and their experimental CCS separation. PMID:27264846

  6. Functional constituents of the active site of human neutrophil collagenase.

    PubMed

    Mookhtiar, K A; Wang, F; Van Wart, H E

    1986-05-01

    A series of chemical modification reactions has been carried out to identify functional constituents of the active site of human neutrophil collagenase. The enzyme is reversibly inhibited by the transition metal chelating agent 1,10-phenanthroline, and inhibition is fully reversed by zinc. Removal of weakly bound metal ions by gel filtration inactivates collagenase, and activity is fully restored on immediate readdition of calcium. The enzyme is unaffected by reagents that modify serine, cysteine, and arginine residues. However, reaction with the carboxyl reagents cyclohexylmorpholinocarbodiimide and Woodward's Reagent K lowers the activity of the enzyme substantially. Acetylimidazole inactivates the enzyme, but activity is completely restored on addition of hydroxylamine. The enzyme is also inactivated by tetranitromethane, indicating that it contains an essential tyrosine residue. Acylation of collagenase with diethyl pyrocarbonate, diketene, acetic anhydride, or trinitrobenzenesulfonate inactivates the enzyme, and activity is not restored on addition of hydroxylamine, indicating the presence of an essential lysine residue.

  7. HYSCORE Analysis of the Effects of Substrates on Coordination of Water to the Active Site Iron in Tyrosine Hydroxylase.

    PubMed

    McCracken, John; Eser, Bekir E; Mannikko, Donald; Krzyaniak, Matthew D; Fitzpatrick, Paul F

    2015-06-23

    Tyrosine hydroxylase is a mononuclear non-heme iron monooxygenase found in the central nervous system that catalyzes the hydroxylation of tyrosine to yield L-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. Catalysis requires the binding of tyrosine, a tetrahydropterin, and O₂ at an active site that consists of a ferrous ion coordinated facially by the side chains of two histidines and a glutamate. We used nitric oxide as a surrogate for O₂ to poise the active site iron in an S = ³/₂ {FeNO}⁷ form that is amenable to electron paramagnetic resonance (EPR) spectroscopy. The pulsed EPR method of hyperfine sublevel correlation (HYSCORE) spectroscopy was then used to probe the ligands at the remaining labile coordination sites on iron. For the complex formed by the addition of tyrosine and nitric oxide, TyrH/NO/Tyr, orientation-selective HYSCORE studies provided evidence of the coordination of one H₂O molecule characterized by proton isotropic hyperfine couplings (A(iso) = 0.0 ± 0.3 MHz) and dipolar couplings (T = 4.4 and 4.5 ± 0.2 MHz). These data show complex HYSCORE cross peak contours that required the addition of a third coupled proton, characterized by an A(iso) of 2.0 MHz and a T of 3.8 MHz, to the analysis. This proton hyperfine coupling differed from those measured previously for H₂O bound to {FeNO}⁷ model complexes and was assigned to a hydroxide ligand. For the complex formed by the addition of tyrosine, 6-methyltetrahydropterin, and NO, TyrH/NO/Tyr/6-MPH₄, the HYSCORE cross peaks attributed to H₂O and OH⁻ for the TyrH/NO/Tyr complex were replaced by a cross peak due to a single proton characterized by an A(iso) of 0.0 MHz and a dipolar coupling (T = 3.8 MHz). This interaction was assigned to the N₅ proton of the reduced pterin.

  8. Nest predation increases with parental activity: Separating nest site and parental activity effects

    USGS Publications Warehouse

    Martin, T.E.; Scott, J.; Menge, C.

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection.

  9. Sodium and proton effects on inward proton transport through Na/K pumps.

    PubMed

    Mitchell, Travis J; Zugarramurdi, Camila; Olivera, J Fernando; Gatto, Craig; Artigas, Pablo

    2014-06-17

    The Na/K pump hydrolyzes ATP to export three intracellular Na (Nai) as it imports two extracellular K (Ko) across animal plasma membranes. Within the protein, two ion-binding sites (sites I and II) can reciprocally bind Na or K, but a third site (site III) exclusively binds Na in a voltage-dependent fashion. In the absence of Nao and Ko, the pump passively imports protons, generating an inward current (IH). To elucidate the mechanisms of IH, we used voltage-clamp techniques to investigate the [H]o, [Na]o, and voltage dependence of IH in Na/K pumps from ventricular myocytes and in ouabain-resistant pumps expressed in Xenopus oocytes. Lowering pHo revealed that Ho both activates IH (in a voltage-dependent manner) and inhibits it (in a voltage-independent manner) by binding to different sites. Nao effects depend on pHo; at pHo where no Ho inhibition is observed, Nao inhibits IH at all concentrations, but when applied at pHo that inhibits pump-mediated current, low [Na]o activates IH and high [Na]o inhibits it. Our results demonstrate that IH is a property inherent to Na/K pumps, not linked to the oocyte expression environment, explains differences in the characteristics of IH previously reported in the literature, and supports a model in which 1), protons leak through site III; 2), binding of two Na or two protons to sites I and II inhibits proton transport; and 3), pumps with mixed Na/proton occupancy of sites I and II remain permeable to protons.

  10. Sodium and proton effects on inward proton transport through Na/K pumps.

    PubMed

    Mitchell, Travis J; Zugarramurdi, Camila; Olivera, J Fernando; Gatto, Craig; Artigas, Pablo

    2014-06-17

    The Na/K pump hydrolyzes ATP to export three intracellular Na (Nai) as it imports two extracellular K (Ko) across animal plasma membranes. Within the protein, two ion-binding sites (sites I and II) can reciprocally bind Na or K, but a third site (site III) exclusively binds Na in a voltage-dependent fashion. In the absence of Nao and Ko, the pump passively imports protons, generating an inward current (IH). To elucidate the mechanisms of IH, we used voltage-clamp techniques to investigate the [H]o, [Na]o, and voltage dependence of IH in Na/K pumps from ventricular myocytes and in ouabain-resistant pumps expressed in Xenopus oocytes. Lowering pHo revealed that Ho both activates IH (in a voltage-dependent manner) and inhibits it (in a voltage-independent manner) by binding to different sites. Nao effects depend on pHo; at pHo where no Ho inhibition is observed, Nao inhibits IH at all concentrations, but when applied at pHo that inhibits pump-mediated current, low [Na]o activates IH and high [Na]o inhibits it. Our results demonstrate that IH is a property inherent to Na/K pumps, not linked to the oocyte expression environment, explains differences in the characteristics of IH previously reported in the literature, and supports a model in which 1), protons leak through site III; 2), binding of two Na or two protons to sites I and II inhibits proton transport; and 3), pumps with mixed Na/proton occupancy of sites I and II remain permeable to protons. PMID:24940773

  11. Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation*

    PubMed Central

    Zhu, Shaotong; Vik, Steven B.

    2015-01-01

    Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441–445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu2+ ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50–90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10–20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus. PMID:26134569

  12. Active sites in char gasification: Final technical report

    SciTech Connect

    Wojtowicz, M.; Lilly, W.D.; Perkins, M.T.; Hradil, G.; Calo, J.M.; Suuberg, E.M.

    1987-09-01

    Among the key variables in the design of gasifiers and combustors is the reactivity of the chars which must be gasified or combusted. Significant loss of unburned char is unacceptable in virtually any process; the provision of sufficient residence time for complete conversion is essential. A very wide range of reactivities are observed, depending upon the nature of the char in a process. The current work focuses on furthering the understanding of gasification reactivities of chars. It has been well established that the reactivity of char to gasification generally depends upon three principal factors: (1) the concentration of ''active sites'' in the char; (2) mass transfer within the char; and (3) the type and concentration of catalytic impurities in the char. The present study primarily addresses the first factor. The subject of this research is the origin, nature, and fate of active sites in chars derived from parent hydrocarbons with coal-like structure. The nature and number of the active sites and their reactivity towards oxygen are examined in ''model'' chars derived from phenol-formaldehyde type resins. How the active sites are lost by the process of thermal annealing during heat treatment of chars are studied, and actual rate for the annealing process is derived. Since intrinsic char reactivities are of primary interest in the present study, a fair amount of attention was given to the model char synthesis and handling so that the effect of catalytic impurities and oxygen-containing functional groups in the chemical structure of the material were minimized, if not completely eliminated. The project would not be considered complete without comparing characteristic features of synthetic chars with kinetic behavior exhibited by natural chars, including coal chars.

  13. Range degradation and distal edge behavior of proton radiotherapy beams using 11C activation and Monte Carlo simulation

    NASA Astrophysics Data System (ADS)

    Elmekawy, Ahmed Farouk

    The distal edge of therapeutic proton radiation beams was investigated by different methods. Proton beams produced at the Hampton University Proton Therapy Institute (HUPTI) were used to irradiate a Polymethylmethacrylate (PMMA) phantom for three different ranges (13.5, 17.0 and 21.0 cm) to investigate the distal slope dependence of the Bragg peak. The activation of 11 C was studied by scanning the phantom less than 10 minutes post-irradiation with a Philips Big Bore Gemini(c) PET/CT. The DICOM images were imported into the Varian Eclipse(c) Treatment Planning System (TPS) for analysis and then analyzed by ImageJ(c) . The distal slope ranged from ?0.1671 +/- 0.0036 to -0.1986 +/- 0.0052 (pixel intensity/slice number) for ranges 13.5 to 21.0 cm respectively. A realistic description of the setup was modeled using the GATE 7.0 Monte Carlo simulation tool and compared to the experiment data. The results show the distal slope ranged from -0.1158+/-0.0133 to -0.0787+/-0.002 (Gy/mm). Additionally, low activity, 11C were simulated to study the 11C reconstructed half-life dependence versus the initial activity for six ranges chosen around the previous activation study. The results of the expected/nominal half-life vs. activity ranged from -5 x 10-4 +/- 2.8104 x 10-4 to 1.6 x 10-3 +/- 9.44 x 10-4 (%diff./Bq). The comparison between two experiments with proton beams on a PMMA phantom and multi-layer ion chamber, and two GATE simulations of a proton beam incident on a water phantom and 11C PET study show that: (i) the distal fall-off variation of the steepness of the slopes are found to be similar thus validating the sensitivity of the PET technique to the range degradation and (ii) the average of the super-ratios difference between all studies observed is primarily due to the difference in the dose deposited in the media.

  14. Brownian aggregation rate of colloid particles with several active sites

    SciTech Connect

    Nekrasov, Vyacheslav M.; Yurkin, Maxim A.; Chernyshev, Andrei V.; Polshchitsin, Alexey A.; Yakovleva, Galina E.; Maltsev, Valeri P.

    2014-08-14

    We theoretically analyze the aggregation kinetics of colloid particles with several active sites. Such particles (so-called “patchy particles”) are well known as chemically anisotropic reactants, but the corresponding rate constant of their aggregation has not yet been established in a convenient analytical form. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shown to produce accurate results in comparison with more sophisticated approaches. Also, to account for the case of a low number of active sites per particle we used Monte Carlo stochastic algorithm based on Gillespie method. Simulations showed that such discrete model is required when this number is less than 10. Finally, we applied the developed approach to the simulation of immunoagglutination, assuming that the formed clusters have fractal structure.

  15. Active Sites Environmental Monitoring Program: FY 1991 report

    SciTech Connect

    Ashwood, T.L.; Hicks, D.S.; Morrissey, C.M.

    1992-11-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from April 1991 through September 1991. The ASEMP was established in 1989 by Solid Waste Operations (SWO) and the Environmental Sciences Division, both of Oak Ridge National Laboratory, to provide early detection and performance monitoring at active low-level (radioactive) waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. A new set of action levels was developed on the basis of a statistical analysis of background contamination. These new action levels have been used to evaluate results in this report. Results of ASEMP monitoring continue to demonstrate that no LLW (except [sup 3]H) is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II, which began in early FY 1991, was >90% complete at the end of September 1991. Results of sampling of groundwater and surface waters is presented.

  16. Inhibition and active-site modelling of prolidase.

    PubMed

    King, G F; Crossley, M J; Kuchel, P W

    1989-03-15

    Consideration of the active-site model of prolidase led us to examine azetidine, pyrrolidine and piperidine substrate analogs as potential in vivo inhibitors of the enzyme. One of these, N-benzyloxycarbonyl-L-proline, was shown to be a potent competitive inhibitor of porcine kidney prolidase (Ki = 90 microM); its rapid protein-mediated permeation of human and sheep erythrocytes suggests that it may be effective in vivo. The higher homolog, N-benzyloxycarbonyl-L-pipecolic acid, was also a potent inhibitor of the enzyme while the antihypertensive drugs, captopril and enalaprilat, were shown to have mild and no inhibitory effects, respectively. Analysis of inhibitor action and consideration of X-ray crystallographic data of relevant Mn2+ complexes allowed the active-site model of prolidase to be further refined; a new model is presented in which the substrate acts as a bidentate ligand towards the active-site manganous ion. Various aspects of the new model help to explain why Mn2+ has been 'chosen' by the enzyme in preference to other biologically available metal ions. PMID:2924773

  17. Energy spectra of protons, deuterium, and helium nuclei during quiet solar activity periods in 1996-97

    NASA Astrophysics Data System (ADS)

    Kecskemety, Karoly

    A background reduction method based on pulse-height analysis (see accompanying paper by Valtonen et al.) is applied for the data of the ERNE and EPHIN energetic particle telescopes aboard SOHO. Energy spectra of protons, deuterons, 3He and 4He nuclei have been obtained during very low activity periods in 1996-97 in the energy range of 1.3 to 22 MeV/n. The proton spectra are comparable to the lowest spectra of the Ulysses (COSPIN/LET, 1996) and near-Earth IMP-8 (1985-87) measurements. Deuterons and 3He are only significantly seen above about 10 MeV/n, their spectra are consistent with an increase proportional to kinetic energy, expected for purely galactic origin.

  18. Enhanced Proton Translocating Pyrophosphatase Activity Improves Nitrogen Use Efficiency in Romaine Lettuce1[C][W][OA

    PubMed Central

    Paez-Valencia, Julio; Sanchez-Lares, Jonathan; Marsh, Ellen; Dorneles, Liane T.; Santos, Mirella P.; Sanchez, Diego; Winter, Alexander; Murphy, Sean; Cox, Jennifer; Trzaska, Marcin; Metler, Jason; Kozic, Alex; Facanha, Arnoldo R.; Schachtman, Daniel; Sanchez, Charles A.; Gaxiola, Roberto A.

    2013-01-01

    Plant nitrate (NO3−) acquisition depends on the combined activities of root high- and low-affinity NO3− transporters and the proton gradient generated by the plasma membrane H+-ATPase. These processes are coordinated with photosynthesis and the carbon status of the plant. Here, we present the characterization of romaine lettuce (Lactuca sativa ‘Conquistador’) plants engineered to overexpress an intragenic gain-of-function allele of the type I proton translocating pyrophosphatase (H+-PPase) of Arabidopsis (Arabidopsis thaliana). The proton-pumping and inorganic pyrophosphate hydrolytic activities of these plants are augmented compared with control plants. Immunohistochemical data show a conspicuous increase in H+-PPase protein abundance at the vasculature of the transgenic plants. Transgenic plants displayed an enhanced rhizosphere acidification capacity consistent with the augmented plasma membrane H+-ATPase proton transport values, and ATP hydrolytic capacities evaluated in vitro. These transgenic lines outperform control plants when challenged with NO3− limitations in laboratory, greenhouse, and field scenarios. Furthermore, we report the characterization of a lettuce LsNRT2.1 gene that is constitutive up-regulated in the transgenic plants. Of note, the expression of the LsNRT2.1 gene in control plants is regulated by NO3− and sugars. Enhanced accumulation of 15N-labeled fertilizer by transgenic lettuce compared with control plants was observed in greenhouse experiments. A negative correlation between the level of root soluble sugars and biomass is consistent with the strong root growth that characterizes these transgenic plants. PMID:23307651

  19. The depletion and regeneration of dissolution-active sites at the mineral-water interface: II. regeneration of active sites on α-Fe 2O 3 at pH 3 and pH 6

    NASA Astrophysics Data System (ADS)

    Samson, Sherry D.; Eggleston, Carrick M.

    2000-11-01

    Periods of transient nonsteady state dissolution can contain much information about dissolution mechanisms. Here, pH-jump-induced dissolution transients are used to explore the kinetics of production, at pH 3 and pH 6, of α-Fe 2O 3 surface sites active for dissolution at pH 1. We find that such sites are generated in a matter of minutes or less at higher pH. The steady state dissolution rate of hematite at pH 1 is ≤10.7 pmol m -2 s -1, whereas the rate of active site production at pH 6 in the first 30 min. of aging is at least 119 pmol m -2 s -1. Apparently, active sites are produced relatively slowly at low pH and relatively rapidly at circumneutral pH, despite the fact that dissolution rates are near a minimum at circumneutral pH. Using aqueous water exchange rates as a proxy for surface ligand exchange rates, this is qualitatively consistent with relatively slow water exchange by aqueous Fe 3+ ions at low pH and relatively rapid water exchange by Fe 3+ hydrolysis products (e.g., Fe(OH) 2+) at circumneutral pH. Consequently, the highest overall dissolution rates are achieved not at steady state at low pH, but by cycling between neutral and low pH. Our results call into question the assumption that oxide mineral surfaces, particularly those of iron and aluminum oxides, are inert on the time scale of proton or ligand adsorption (e.g., during the acid-base titrations typically used to measure oxide surface charge due to proton adsorption).

  20. In situ probing of the active site geometry of ultrathin nanowires for the oxygen reduction reaction

    DOE PAGES

    Liu, Haiqing; Wong, Stanislaus S.; An, Wei; Li, Yuanyuan; Frenkel, Anatoly I.; Sasaki, Kotaro; Koenigsmann, Christopher; Su, Dong; Anderson, Rachel M.; Crooks, Richard M.; et al

    2015-09-24

    To create truly effective electrocatalysts for the cathodic reaction governing proton exchange membrane fuel cells (PEMFC), namely the oxygen reduction reaction (ORR), necessitates an accurate and detailed structural understanding of these electrocatalysts, especially at the nanoscale, and to precisely correlate that structure with demonstrable performance enhancement. To address this key issue, we have combined and interwoven theoretical calculations with experimental, spectroscopic observations in order to acquire useful structural insights into the active site geometry with implications for designing optimized nanoscale electrocatalysts with rationally predicted properties. Specifically, we have probed ultrathin (~2 nm) core–shell Pt~Pd9Au nanowires, which have been previously shownmore » to be excellent candidates for ORR in terms of both activity and long-term stability, from the complementary perspectives of both DFT calculations and X-ray absorption spectroscopy (XAS). The combination and correlation of data from both experimental and theoretical studies has revealed for the first time that the catalytically active structure of our ternary nanowires can actually be ascribed to a PtAu~Pd configuration, comprising a PtAu binary shell and a pure inner Pd core. Moreover, we have plausibly attributed the resulting structure to a specific synthesis step, namely the Cu underpotential deposition (UPD) followed by galvanic replacement with Pt. Thus, the fundamental insights gained into the performance of our ultrathin nanowires from our demonstrated approach will likely guide future directed efforts aimed at broadly improving upon the durability and stability of nanoscale electrocatalysts in general.« less

  1. In situ probing of the active site geometry of ultrathin nanowires for the oxygen reduction reaction

    SciTech Connect

    Liu, Haiqing; Wong, Stanislaus S.; An, Wei; Li, Yuanyuan; Frenkel, Anatoly I.; Sasaki, Kotaro; Koenigsmann, Christopher; Su, Dong; Anderson, Rachel M.; Crooks, Richard M.; Adzic, Radoslav R.; Liu, Ping

    2015-09-24

    To create truly effective electrocatalysts for the cathodic reaction governing proton exchange membrane fuel cells (PEMFC), namely the oxygen reduction reaction (ORR), necessitates an accurate and detailed structural understanding of these electrocatalysts, especially at the nanoscale, and to precisely correlate that structure with demonstrable performance enhancement. To address this key issue, we have combined and interwoven theoretical calculations with experimental, spectroscopic observations in order to acquire useful structural insights into the active site geometry with implications for designing optimized nanoscale electrocatalysts with rationally predicted properties. Specifically, we have probed ultrathin (~2 nm) core–shell Pt~Pd9Au nanowires, which have been previously shown to be excellent candidates for ORR in terms of both activity and long-term stability, from the complementary perspectives of both DFT calculations and X-ray absorption spectroscopy (XAS). The combination and correlation of data from both experimental and theoretical studies has revealed for the first time that the catalytically active structure of our ternary nanowires can actually be ascribed to a PtAu~Pd configuration, comprising a PtAu binary shell and a pure inner Pd core. Moreover, we have plausibly attributed the resulting structure to a specific synthesis step, namely the Cu underpotential deposition (UPD) followed by galvanic replacement with Pt. Thus, the fundamental insights gained into the performance of our ultrathin nanowires from our demonstrated approach will likely guide future directed efforts aimed at broadly improving upon the durability and stability of nanoscale electrocatalysts in general.

  2. Proton beam simulation with MCNPX/CINDER'90: Germanium metal activation estimates below 30MeV relevant to the bulk production of arsenic radioisotopes.

    PubMed

    Fassbender, M; Taylor, W; Vieira, D; Nortier, M; Bach, H; John, K

    2012-01-01

    Germanium metal targets encapsulated in Nb shells were irradiated in a proton beam. Proton and secondary neutron beam fluences as well as radionuclide activity formation were modeled using MCNPX in combination with CINDER90. Targets were chemically processed using distillation and anion exchange. Good agreement between the measured radiochemical yields and MCNPX/CINDER90 estimates was observed. A target of pentavalent (73,74)As radioarsenic for neutron activation studies was prepared.

  3. Proton beam simulation with MCNPX/CINDER'90: Germanium metal activation estimates below 30MeV relevant to the bulk production of arsenic radioisotopes.

    PubMed

    Fassbender, M; Taylor, W; Vieira, D; Nortier, M; Bach, H; John, K

    2012-01-01

    Germanium metal targets encapsulated in Nb shells were irradiated in a proton beam. Proton and secondary neutron beam fluences as well as radionuclide activity formation were modeled using MCNPX in combination with CINDER90. Targets were chemically processed using distillation and anion exchange. Good agreement between the measured radiochemical yields and MCNPX/CINDER90 estimates was observed. A target of pentavalent (73,74)As radioarsenic for neutron activation studies was prepared. PMID:21890369

  4. Druggability analysis and classification of protein tyrosine phosphatase active sites

    PubMed Central

    Ghattas, Mohammad A; Raslan, Noor; Sadeq, Asil; Al Sorkhy, Mohammad; Atatreh, Noor

    2016-01-01

    Protein tyrosine phosphatases (PTP) play important roles in the pathogenesis of many diseases. The fact that no PTP inhibitors have reached the market so far has raised many questions about their druggability. In this study, the active sites of 17 PTPs were characterized and assessed for its ability to bind drug-like molecules. Consequently, PTPs were classified according to their druggability scores into four main categories. Only four members showed intermediate to very druggable pocket; interestingly, the rest of them exhibited poor druggability. Particularly focusing on PTP1B, we also demonstrated the influence of several factors on the druggability of PTP active site. For instance, the open conformation showed better druggability than the closed conformation, while the tight-bound water molecules appeared to have minimal effect on the PTP1B druggability. Finally, the allosteric site of PTP1B was found to exhibit superior druggability compared to the catalytic pocket. This analysis can prove useful in the discovery of new PTP inhibitors by assisting researchers in predicting hit rates from high throughput or virtual screening and saving unnecessary cost, time, and efforts via prioritizing PTP targets according to their predicted druggability. PMID:27757011

  5. The role of active site tyrosine 58 in Citrobacter freundii methionine γ-lyase.

    PubMed

    Anufrieva, Natalya V; Faleev, Nicolai G; Morozova, Elena A; Bazhulina, Natalia P; Revtovich, Svetlana V; Timofeev, Vladimir P; Tkachev, Yaroslav V; Nikulin, Alexei D; Demidkina, Tatyana V

    2015-09-01

    In the spatial structure of methionine γ-lyase (MGL, EC 4.4.1.11) from Citrobacter freundii, Tyr58 is located at H-bonding distance to the oxygen atom of the phosphate "handle" of pyridoxal 5'-phosphate (PLP). It was replaced for phenylalanine by site-directed mutagenesis. The X-ray structure of the mutant enzyme was determined at 1.96Å resolution. Comparison of spatial structures and absorption spectra of wild-type and mutant holoenzymes demonstrated that the replacement did not result in essential changes of the conformation of the active site Tyr58Phe MGL. The Kd value of PLP for Tyr58Phe MGL proved to be comparable to the Kd value for the wild-type enzyme. The replacement led to a decrease of catalytic efficiencies in both γ- and β-elimination reactions of about two orders of magnitude as compared to those for the wild-type enzyme. The rates of exchange of C-α- and C-β- protons of inhibitors in D2O catalyzed by the mutant form are comparable with those for the wild-type enzyme. Spectral data on the complexes of the mutant form with the substrates and inhibitors showed that the replacement led to a change of rate the limiting step of the physiological reaction. The results allowed us to conclude that Tyr58 is involved in an optimal positioning of the active site Lys210 at some stages of γ- and β-elimination reactions. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

  6. Mechanistic insights into ferredoxin-NADP(H) reductase catalysis involving the conserved glutamate in the active site.

    PubMed

    Dumit, Verónica I; Essigke, Timm; Cortez, Néstor; Ullmann, G Matthias

    2010-04-01

    Plant-type ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes harboring one molecule of noncovalently bound flavin adenine dinucleotide that catalyze reversible reactions between obligatory one-electron carriers and obligatory two-electron carriers. A glutamate next to the C-terminus is strictly conserved in FNR and has been proposed to function as proton donor/acceptor during catalysis. However, experimental studies of this proposed function led to contradicting conclusions about the role of this glutamate in the catalytic mechanism. In the present work, we study the titration behavior of the glutamate in the active site of FNR using theoretical methods. Protonation probabilities for maize FNR were computed for the reaction intermediates of the catalytic cycle by Poisson-Boltzmann electrostatic calculations and Metropolis Monte Carlo titration. The titration behavior of the highly conserved glutamate was found to vary depending on the bound substrates NADP(H) and ferredoxin and also on the redox states of these substrates and the flavin adenine dinucleotide. Our results support the involvement of the glutamate in the FNR catalytic mechanism not only as a proton donor but also as a key residue for stabilizing and destabilizing reaction intermediates. On the basis of our findings, we propose a model rationalizing the function of the glutamate in the reaction cycle, which allows reinterpretation of previous experimental results.

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

    PubMed

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

    2013-02-01

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

  8. Current activities handbook: formerly utilized sites remedial action program

    SciTech Connect

    1981-02-27

    This volume is one of a series produced under contract with the DOE, by Politech Corporation to develop a legislative and regulatory data base to assist the FUSRAP management in addressing the institutional and socioeconomic issues involved in carrying out the Formerly Utilized Sites Remedial Action Program. This Information Handbook series contains information about all relevant government agencies at the Federal and state levels, the pertinent programs they administer, each affected state legislature, and current Federal and state legislative and regulatory initiatives. This volume is a compilation of information about the activities each of the thirteen state legislatures potentially affected by the Formerly Utilized Sites Remedial Action Program. It contains a description of the state legislative procedural rules and a schedule of each legislative session; a summary of pending relevant legislation; the name and telephone number of legislative and state agency contacts; and the full text of all bills identified.

  9. Enantioselective Collision-Activated Dissociation of Gas-Phase Tryptophan Induced by Chiral Recognition of Protonated uc(l)-Alanine Peptides

    NASA Astrophysics Data System (ADS)

    Fujihara, Akimasa; Matsuyama, Hiroki; Tajiri, Michiko; Wada, Yoshinao; Hayakawa, Shigeo

    2016-06-01

    Enantioselective dissociation in the gas phase is important for enantiomeric enrichment and chiral transmission processes in molecular clouds regarding the origin of homochirality in biomolecules. Enantioselective collision-activated dissociation (CAD) of tryptophan (Trp) and the chiral recognition ability of uc(l)-alanine peptides (uc(l)-Ala n ; n = 2-4) were examined using a linear ion trap mass spectrometer. CAD spectra of gas-phase heterochiral H+(uc(d)-Trp)(uc(l)-Ala n ) and homochiral H+(uc(l)-Trp)(uc(l)-Ala n ) noncovalent complexes were obtained as a function of the peptide size n. The H2O-elimination product was observed in CAD spectra of both heterochiral and homochiral complexes for n = 2 and 4, and in homochiral H+(uc(l)-Trp)(uc(l)-Ala3), indicating that the proton is attached to the uc(l)-alanine peptide, and H2O loss occurs from H+(uc(l)-Ala n ) in the noncovalent complexes. H2O loss did not occur in heterochiral H+(uc(d)-Trp)(uc(l)-Ala3), where NH3 loss and (H2O + CO) loss were the primary dissociation pathways. In heterochiral H+(uc(d)-Trp)(uc(l)-Ala3), the protonation site is the amino group of uc(d)-Trp, and NH3 loss and (H2O + CO) loss occur from H+(uc(d)-Trp). uc(l)-Ala peptides recognize uc(d)-Trp through protonation of the amino group for peptide size n = 3. NH3 loss and (H2O + CO) loss from H+(uc(d)-Trp) proceeds via enantioselective CAD in gas-phase heterochiral H+(uc(d)-Trp)(uc(l)-Ala3) at room temperature, whereas uc(l)-Trp dissociation was not observed in homochiral H+(uc(l)-Trp)(uc(l)-Ala3). These results suggest that enantioselective dissociation induced by chiral recognition of uc(l)-Ala peptides through protonation could play an important role in enantiomeric enrichment and chiral transmission processes of amino acids.

  10. Hydrogen Production Catalyzed by Bidirectional, Biomimetic Models of the [FeFe]-Hydrogenase Active Site.

    PubMed

    Lansing, James C; Camara, James M; Gray, Danielle E; Rauchfuss, Thomas B

    2014-10-27

    Active site mimics of [FeFe]-hydrogenase are shown to be bidirectional catalysts, producing H2 upon treatment with protons and reducing equivalents. This reactivity complements the previously reported oxidation of H2 by these same catalysts in the presence of oxidants. The complex Fe2(adt(Bn))(CO)3(dppv)(PFc*(Et2) ) ([1](0); adt(Bn) = (SCH2)2NBn, dppv = cis-1,2-bis(diphenylphosphino)ethylene, PFc*(Et2) = Et2PCH2C5Me4FeCp*) reacts with excess [H(OEt2)2]BAr(F) 4 (BAr(F) 4 (-) = B(C6H3-3,5-(CF3)2)4 (-)) to give ∼0.5 equiv of H2 and [Fe2(adt(Bn)H)(CO)3(dppv)(PFc*(Et2) )](2+) ([1H](2+)). The species [1H](2+) consists of a ferrocenium ligand, an N-protonated amine, and an Fe(I)Fe(I) core. In the presence of additional reducing equivalents in the form of decamethylferrocene (Fc*), hydrogen evolution is catalytic, albeit slow. The related catalyst Fe2(adt(Bn))(CO)3(dppv)(PMe3) (3) behaves similarly in the presence of Fc*, except that in the absence of excess reducing agent it converts to the catalytically inactive μ-hydride derivative [μ-H3](+). Replacement of the adt in [1](0) with propanedithiolate (pdt) results in a catalytically inactive complex. In the course of synthesizing [FeFe]-hydrogenase mimics, new routes to ferrocenylphosphine ligands and nonamethylferrocene were developed. PMID:25364093

  11. Electrostatic fields in the active sites of lysozymes.

    PubMed

    Sun, D P; Liao, D I; Remington, S J

    1989-07-01

    Considerable experimental evidence is in support of several aspects of the mechanism that has been proposed for the catalytic activity of lysozyme. However, the enzymatically catalyzed hydrolysis of polysaccharides proceeds over 5 orders of magnitude faster than that of model compounds that mimic the configuration of the substrate in the active site of the enzyme. Although several possible explanations for this rate enhancement have been discussed elsewhere, a definitive mechanism has not emerged. Here we report striking results obtained by classical electrodynamics, which suggest that bond breakage and the consequent separation of charge in lysozyme is promoted by a large electrostatic field across the active site cleft, produced in part by a very asymmetric distribution of charged residues on the enzyme surface. Lysozymes unrelated in amino acid sequence have similar distributions of charged residues and electric fields. The results reported here suggest that the electrostatic component of the rate enhancement is greater than 9 kcal.mol-1. Thus, electrostatic interactions may play a more important role in the enzymatic mechanism than has generally been appreciated.

  12. Histidine at the active site of Neurospora tyrosinase.

    PubMed

    Pfiffner, E; Lerch, K

    1981-10-13

    The involvement of histidyl residues as potential ligands to the binuclear active-site copper of Neurospora tyrosinase was explored by dye-sensitized photooxidation. The enzymatic activity of the holoenzyme was shown to be unaffected by exposure to light in the presence of methylene blue; however, irradiation of the apoenzyme under the same conditions led to a progressive loss of its ability to be reactivated with Cu2+. This photoinactivation was paralleled by a decrease in the histidine content whereas the number of histidyl residues in the holoenzyme remained constant. Copper measurements of photooxidized, reconstituted apoenzyme demonstrated the loss of binding of one copper atom per mole of enzyme as a consequence of photosensitized oxidation of three out of nine histidine residues. Their sequence positions were determined by a comparison of the relative yields of the histidine containing peptides of photooxidized holo- and apotyrosinases. The data obtained show the preferential modification of histidyl residues 188, 193, and 289 and suggest that they constitute metal ligands to one of the two active-site copper atoms. Substitution of copper by cobalt was found to afford complete protection of the histidyl residues from being modified by dye-sensitized photooxidation. PMID:6458322

  13. Affecting proton mobility in activated peptide and whole protein ions via lysine guanidination.

    PubMed

    Pitteri, Sharon J; Reid, Gavin E; McLuckey, Scott A

    2004-01-01

    We have evaluated the effect of lysine guanidination in peptides and proteins on the dissociation of protonated ions in the gas phase. The dissociation of guanidinated model peptide ions compared to their unmodified forms showed behavior consistent with concepts of proton mobility as a major factor in determining favored fragmentation channels. Reduction of proton mobility associated with lysine guanidination was reflected by a relative increase in cleavages occurring C-terminal to aspartic acid residues as well as increases in small molecule losses. To evaluate the effect of guanidination on the dissociation behavior of whole protein ions, bovine ubiquitin was selected as a model. Essentially, all of the amide bond cleavages associated with the +10 charge state of fully guanidinated ubiquitin were observed to occur C-terminal to aspartic acid residues, unlike the dissociation behavior of the +10 ion of the unmodified protein, where competing cleavage N-terminal to proline and nonspecific amide bond cleavages were also observed. The +8 and lower charge states of the guanidinated protein showed prominent losses of small neutral molecules. This overall fragmentation behavior is consistent with current hypotheses regarding whole protein dissociation that consider proton mobility and intramolecular charge solvation as important factors in determining favored dissociation channels, and are also consistent with the fragmentation behaviors observed for the guanidinated model peptide ions. Further evaluation of the utility of condensed phase guanidination of whole proteins is necessary but the results described here confirm that guanidination can be an effective strategy for enhancing C-terminal aspartic acid cleavages. Gas phase dissociation exclusively at aspartic acid residues, especially for whole protein ions, could be useful in identifying and characterizing proteins via tandem mass spectrometry of whole protein ions.

  14. 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

  15. Coordinated Activation of Programmed Cell Death and Defense Mechanisms in Transgenic Tobacco Plants Expressing a Bacterial Proton Pump.

    PubMed Central

    Mittler, R.; Shulaev, V.; Lam, E.

    1995-01-01

    In plants, programmed cell death is thought to be activated during the hypersensitive response to certain avirulent pathogens and in the course of several differentiation processes. We describe a transgenic model system that mimics the activation of programmed cell death in higher plants. In this system, expression of a bacterial proton pump in transgenic tobacco plants activates a cell death pathway that may be similar to that triggered by recognition of an incompatible pathogen. Thus, spontaneous lesions that resemble hypersensitive response lesions are formed, multiple defense mechanisms are apparently activated, and systemic resistance is induced in the absence of a pathogen. Interestingly, mutation of a single amino acid in the putative channel of this proton pump renders it inactive with respect to lesion formation and induction of resistance to pathogen challenge. This transgenic model system may provide insights into the mechanisms involved in mediating cell death in higher plants. In addition, it may also be used as a general agronomic tool to enhance disease protection. PMID:12242350

  16. [Effects of exogenous spermidine on lipid peroxidation and membrane proton pump activity of cucumber seedling leaves under high temperature stress].

    PubMed

    Tian, Jing; Guo, Shi-Rong; Sun, Jin; Wang, Li-Ping; Yang, Yan-Juan; Li, Bin

    2011-12-01

    Taking a relatively heat-resistant cucumber (Cucumis sativus) cultivar 'Jinchun No. 4' as test material, a sand culture experiment was conducted in growth chamber to investigate the effects of foliar spraying spermidine (Spd) on the lipid peroxidation, membrane proton pump activity, and corresponding gene expression of cucumber seedling leaves under high temperature stress. Compared with the control, foliar spraying Spd increased the plant height, stem diameter, dry and fresh mass, and leaf area significantly, and inhibited the increase of leaf relative conductivity, malondialdehyde (MDA) content, and lipoxygenase (LOX) activity effectively. Foliar spraying Spd also helped to the increase of leaf plasma membrane- and tonoplast H(+)-ATPase activity, but no significant difference was observed in the gene expression levels. These results suggested that exogenous Spd could significantly decrease the leaf lipid peroxidation and increase the proton pump activity, and thus, stabilize the leaf membrane structure and function, alleviate the damage induced by high temperature stress, and enhance the heat tolerance of cucumber seedlings.

  17. Modifications on the hydrogen bond network by mutations of Escherichia coli copper efflux oxidase affect the process of proton transfer to dioxygen leading to alterations of enzymatic activities

    SciTech Connect

    Kajikawa, Takao; Kataoka, Kunishige; Sakurai, Takeshi

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Proton transfer pathway to dioxygen in CueO was identified. Black-Right-Pointing-Pointer Glu506 is the key amino acid to transport proton. Black-Right-Pointing-Pointer The Ala mutation at Glu506 formed a compensatory proton transfer pathway. Black-Right-Pointing-Pointer The Ile mutation at Glu506 shut down the hydrogen bond network. -- Abstract: CueO has a branched hydrogen bond network leading from the exterior of the protein molecule to the trinuclear copper center. This network transports protons in the four-electron reduction of dioxygen. We replaced the acidic Glu506 and Asp507 residues with the charged and uncharged amino acid residues. Peculiar changes in the enzyme activity of the mutants relative to the native enzyme indicate that an acidic amino acid residue at position 506 is essential for effective proton transport. The Ala mutation resulted in the formation of a compensatory hydrogen bond network with one or two extra water molecules. On the other hand, the Ile mutation resulted in the complete shutdown of the hydrogen bond network leading to loss of enzymatic activities of CueO. In contrast, the hydrogen bond network without the proton transport function was constructed by the Gln mutation. These results exerted on the hydrogen bond network in CueO are discussed in comparison with proton transfers in cytochrome oxidase.

  18. Bacteriorhodopsin mutants containing single tyrosine to phenylalanine substitutions are all active in proton translocation.

    PubMed Central

    Mogi, T; Stern, L J; Hackett, N R; Khorana, H G

    1987-01-01

    To study the possible role of the tyrosine residues in proton translocation by bacteriorhodopsin, we have replaced these residues individually by phenylalanine. The required codon changes were introduced in the bacterioopsin gene by replacement of appropriate restriction fragments by synthetic counterparts containing the desired nucleotide changes. The denatured opsin polypeptides obtained by expression of the mutant genes in Escherichia coli were purified and treated with a mixture of detergents, phospholipids, and retinal in a previously established renaturation procedure. All of the mutant proteins folded to regenerate bacteriorhodopsin-like chromophores. Three mutants with tyrosine to phenylalanine substitutions at positions 57, 83, and 185 regenerated the chromophore more slowly than the wild-type protein, and two of these mutants, Phe-57 and -83, showed slightly blue-shifted chromophores. When reconstituted into liposomes all of the mutant proteins with single Tyr----Phe substitutions pumped protons at rates and levels comparable to those of the wild-type bacteriorhodopsin. We conclude that single substitutions of tyrosine by phenylalanine do not affect folding, retinal binding, or light-dependent proton pumping in bacteriorhodopsin. PMID:3039495

  19. Proton and Electron Threshold Energy Measurements for Extravehicular Activity Space Suits. Chapter 2

    NASA Technical Reports Server (NTRS)

    Moyers, M. F.; Nelson, G. D.; Saganti, P. B.

    2003-01-01

    Construction of ISS will require more than 1000 hours of EVA. Outside of ISS during EVA, astronauts and cosmonauts are likely to be exposed to a large fluence of electrons and protons. Development of radiation protection guidelines requires the determination of the minimum energy of electrons and protons that penetrate the suits at various locations. Measurements of the water-equivalent thickness of both US. and Russian EVA suits were obtained by performing CT scans. Specific regions of interest of the suits were further evaluated using a differential range shift technique. This technique involved measuring thickness ionization curves for 6-MeV electron and 155-MeV proton beams with ionization chambers using a constant source-to-detector distance. The thicknesses were obtained by stacking polystyrene slabs immediately upstream of the detector. The thicknesses of the 50% ionizations relative to the maximum ionizations were determined. The detectors were then placed within the suit and the stack thickness adjusted until the 50% ionization was reestablished. The difference in thickness between the 50% thicknesses was then used with standard range-energy tables to determine the threshold energy for penetration. This report provides a detailed description of the experimental arrangement and results.

  20. Trichodiene synthase. Identification of active site residues by site-directed mutagenesis.

    PubMed

    Cane, D E; Shim, J H; Xue, Q; Fitzsimons, B C; Hohn, T M

    1995-02-28

    Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-14C]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat. In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca. 7-8.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7873527

  1. Mechanistic pathways of mercury removal from the organomercurial lyase active site.

    PubMed

    Silva, Pedro J; Rodrigues, Viviana

    2015-01-01

    Bacterial populations present in Hg-rich environments have evolved biological mechanisms to detoxify methylmercury and other organometallic mercury compounds. The most common resistance mechanism relies on the H(+)-assisted cleavage of the Hg-C bond of methylmercury by the organomercurial lyase MerB. Although the initial reaction steps which lead to the loss of methane from methylmercury have already been studied experimentally and computationally, the reaction steps leading to the removal of Hg(2+) from MerB and regeneration of the active site for a new round of catalysis have not yet been elucidated. In this paper, we have studied the final steps of the reaction catalyzed by MerB through quantum chemical computations at the combined MP2/CBS//B3PW91/6-31G(d) level of theory. While conceptually simple, these reaction steps occur in a complex potential energy surface where several distinct pathways are accessible and may operate concurrently. The only pathway which clearly emerges as forbidden in our analysis is the one arising from the sequential addition of two thiolates to the metal atom, due to the accumulation of negative charges in the active site. The addition of two thiols, in contrast, leads to two feasible mechanistic possibilities. The most straightforward pathway proceeds through proton transfer from the attacking thiol to Cys159 , leading to its removal from the mercury coordination sphere, followed by a slower attack of a second thiol, which removes Cys96. The other pathway involves Asp99 in an accessory role similar to the one observed earlier for the initial stages of the reaction and affords a lower activation enthalpy, around 14 kcal mol(-1), determined solely by the cysteine removal step rather than by the thiol ligation step. Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after

  2. Mechanistic pathways of mercury removal from the organomercurial lyase active site

    PubMed Central

    Rodrigues, Viviana

    2015-01-01

    Bacterial populations present in Hg-rich environments have evolved biological mechanisms to detoxify methylmercury and other organometallic mercury compounds. The most common resistance mechanism relies on the H+-assisted cleavage of the Hg–C bond of methylmercury by the organomercurial lyase MerB. Although the initial reaction steps which lead to the loss of methane from methylmercury have already been studied experimentally and computationally, the reaction steps leading to the removal of Hg2+ from MerB and regeneration of the active site for a new round of catalysis have not yet been elucidated. In this paper, we have studied the final steps of the reaction catalyzed by MerB through quantum chemical computations at the combined MP2/CBS//B3PW91/6-31G(d) level of theory. While conceptually simple, these reaction steps occur in a complex potential energy surface where several distinct pathways are accessible and may operate concurrently. The only pathway which clearly emerges as forbidden in our analysis is the one arising from the sequential addition of two thiolates to the metal atom, due to the accumulation of negative charges in the active site. The addition of two thiols, in contrast, leads to two feasible mechanistic possibilities. The most straightforward pathway proceeds through proton transfer from the attacking thiol to Cys159 , leading to its removal from the mercury coordination sphere, followed by a slower attack of a second thiol, which removes Cys96. The other pathway involves Asp99 in an accessory role similar to the one observed earlier for the initial stages of the reaction and affords a lower activation enthalpy, around 14 kcal mol−1, determined solely by the cysteine removal step rather than by the thiol ligation step. Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after

  3. Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

    PubMed Central

    Fago, Angela; Malte, Hans; Storz, Jay F.; Gorr, Thomas A.

    2013-01-01

    In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3−), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3− levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3− binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. PMID:23720132

  4. The copper active site of CBM33 polysaccharide oxygenases.

    PubMed

    Hemsworth, Glyn R; Taylor, Edward J; Kim, Robbert Q; Gregory, Rebecca C; Lewis, Sally J; Turkenburg, Johan P; Parkin, Alison; Davies, Gideon J; Walton, Paul H

    2013-04-24

    The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme's three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

  5. Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

    PubMed Central

    Jakubík, J; Bacáková, L; Lisá, V; el-Fakahany, E E; Tucek, S

    1996-01-01

    Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation. PMID:8710935

  6. Radiation inactivation study of aminopeptidase: probing the active site

    NASA Astrophysics Data System (ADS)

    Jamadar, V. K.; Jamdar, S. N.; Mohan, Hari; Dandekar, S. P.; Harikumar, P.

    2004-04-01

    Ionizing radiation inactivated purified chicken intestinal aminopeptidase in media saturated with gases in the order N 2O>N 2>air. The D 37 values in the above conditions were 281, 210 and 198 Gy, respectively. OH radical scavengers such as t-butanol and isopropanol effectively nullified the radiation-induced damage in N 2O. The radicals (SCN) 2•-, Br 2•- and I 2•- inactivated the enzyme, pointing to the involvement of aromatic amino acids and cysteine in its catalytic activity. The enzyme exhibited fluorescence emission at 340 nm which is characteristic of tryptophan. The radiation-induced loss of activity was accompanied by a decrease in the fluorescence of the enzyme suggesting a predominant influence on tryptophan residues. The enzyme inhibition was associated with a marked increase in the Km and a decrease in the Vmax and kcat values, suggesting an irreversible alteration in the catalytic site. The above observations were confirmed by pulse radiolysis studies.

  7. Mimicking enzymatic active sites on surfaces for energy conversion chemistry.

    PubMed

    Gutzler, Rico; Stepanow, Sebastian; Grumelli, Doris; Lingenfelder, Magalí; Kern, Klaus

    2015-07-21

    Metal-organic supramolecular chemistry on surfaces has matured to a point where its underlying growth mechanisms are well understood and structures of defined coordination environments of metal atoms can be synthesized in a controlled and reproducible procedure. With surface-confined molecular self-assembly, scientists have a tool box at hand which can be used to prepare structures with desired properties, as for example a defined oxidation number and spin state of the transition metal atoms within the organic matrix. From a structural point of view, these coordination sites in the supramolecular structure resemble the catalytically active sites of metallo-enzymes, both characterized by metal centers coordinated to organic ligands. Several chemical reactions take place at these embedded metal ions in enzymes and the question arises whether these reactions also take place using metal-organic networks as catalysts. Mimicking the active site of metal atoms and organic ligands of enzymes in artificial systems is the key to understanding the selectivity and efficiency of enzymatic reactions. Their catalytic activity depends on various parameters including the charge and spin configuration in the metal ion, but also on the organic environment, which can stabilize intermediate reaction products, inhibits catalytic deactivation, and serves mostly as a transport channel for the reactants and products and therefore ensures the selectivity of the enzyme. Charge and spin on the transition metal in enzymes depend on the one hand on the specific metal element, and on the other hand on its organic coordination environment. These two parameters can carefully be adjusted in surface confined metal-organic networks, which can be synthesized by virtue of combinatorial mixing of building synthons. Different organic ligands with varying functional groups can be combined with several transition metals and spontaneously assemble into ordered networks. The catalytically active metal

  8. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    NASA Astrophysics Data System (ADS)

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-06-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work.

  9. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    PubMed Central

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-01-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work. PMID:27306204

  10. Spectroscopic Definition of the Ferroxidase Site in M Ferritin: Comparison of Binuclear Substrate vs. Cofactor Active Sites

    PubMed Central

    Schwartz, Jennifer K.; Liu, Xiaofeng S.; Tosha, Takehiko; Theil, Elizabeth C.; Solomon, Edward I.

    2008-01-01

    Maxi ferritins, 24 subunit protein nanocages, are essential in humans, plants, bacteria, and other animals for the concentration and storage of iron as hydrated ferric oxide, while minimizing free radical generation or use by pathogens. Formation of the precursors to these ferric oxides is catalyzed at a non-heme biferrous substrate site, which has some parallels with the cofactor sites in other biferrous enzymes. A combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) has been used to probe Fe(II) binding to the substrate active site in frog M ferritin. These data determined that the active site within each subunit consists of two inequivalent five-coordinate (5C) ferrous centers that are weakly anti-ferromagnetically coupled, consistent with a μ-1,3 carboxylate bridge. The active site ligand set is unusual and likely includes a terminal water bound to each Fe(II) center. The Fe(II) ions bind to the active sites in a concerted manner, and cooperativity among the sites in each subunit is observed, potentially providing a mechanism for the control of ferritin iron loading. Differences in geometric and electronic structure – including a weak ligand field, availability of two water ligands at the biferrous substrate site, and the single carboxylate bridge in ferritin – coincide with the divergent reaction pathways observed between this substrate site and the previously studied cofactor active sites. PMID:18576633

  11. The receptor site of the spider toxin PcTx1 on the proton-gated cation channel ASIC1a

    PubMed Central

    Salinas, Miguel; Rash, Lachlan D; Baron, Anne; Lambeau, Gérard; Escoubas, Pierre; Lazdunski, Michel

    2006-01-01

    Acid-sensing ion channels (ASICs) are excitatory neuronal cation channels, involved in physiopathological processes related to extracellular pH fluctuation such as nociception, ischaemia, perception of sour taste and synaptic transmission. The spider peptide toxin psalmotoxin 1 (PcTx1) has previously been shown to inhibit specifically the proton-gated cation channel ASIC1a. To identify the binding site of PcTx1, we produced an iodinated form of the toxin (125I-PcTx1YN) and developed a set of binding and electrophysiological experiments on several chimeras of ASIC1a and the PcTx1-insensitive channels ASIC1b and ASIC2a. We show that 125I-PcTx1YN binds specifically to ASIC1a at a single site, with an IC50 of 128 pm, distinct from the amiloride blocking site. Results obtained from chimeras indicate that PcTx1 does not bind to ASIC1a transmembrane domains (M1 and M2), involved in formation of the ion pore, but binds principally on both cysteine-rich domains I and II (CRDI and CRDII) of the extracellular loop. The post-M1 and pre-M2 regions, although not involved in the binding site, are crucial for the ability of PcTx1 to inhibit ASIC1a current. The linker domain between CRDI and CRDII is important for their correct spatial positioning to form the PcTx1 binding site. These results will be useful for the future identification or design of new molecules acting on ASICs. PMID:16284080

  12. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    PubMed

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.

  13. Eel calcitonin binding site distribution and antinociceptive activity in rats

    SciTech Connect

    Guidobono, F.; Netti, C.; Sibilia, V.; Villa, I.; Zamboni, A.; Pecile, A.

    1986-03-01

    The distribution of binding site for (/sup 125/I)-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing (/sup 125/I)-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain.

  14. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    PubMed

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth. PMID:26940877

  15. Hydride binding to the active site of [FeFe]-hydrogenase.

    PubMed

    Chernev, Petko; Lambertz, Camilla; Brünje, Annika; Leidel, Nils; Sigfridsson, Kajsa G V; Kositzki, Ramona; Hsieh, Chung-Hung; Yao, Shenglai; Schiwon, Rafael; Driess, Matthias; Limberg, Christian; Happe, Thomas; Haumann, Michael

    2014-11-17

    [FeFe]-hydrogenase from green algae (HydA1) is the most efficient hydrogen (H2) producing enzyme in nature and of prime interest for (bio)technology. Its active site is a unique six-iron center (H-cluster) composed of a cubane cluster, [4Fe4S]H, cysteine-linked to a diiron unit, [2Fe]H, which carries unusual carbon monoxide (CO) and cyanide ligands and a bridging azadithiolate group. We have probed the molecular and electronic configurations of the H-cluster in functional oxidized, reduced, and super-reduced or CO-inhibited HydA1 protein, in particular searching for intermediates with iron-hydride bonds. Site-selective X-ray absorption and emission spectroscopy were used to distinguish between low- and high-spin iron sites in the two subcomplexes of the H-cluster. The experimental methods and spectral simulations were calibrated using synthetic model complexes with ligand variations and bound hydride species. Distinct X-ray spectroscopic signatures of electronic excitation or decay transitions in [4Fe4S]H and [2Fe]H were obtained, which were quantitatively reproduced by density functional theory calculations, thereby leading to specific H-cluster model structures. We show that iron-hydride bonds are absent in the reduced state, whereas only in the super-reduced state, ligand rotation facilitates hydride binding presumably to the Fe-Fe bridging position at [2Fe]H. These results are in agreement with a catalytic cycle involving three main intermediates and at least two protonation and electron transfer steps prior to the H2 formation chemistry in [FeFe]-hydrogenases. PMID:25369169

  16. Structural mutations that probe the interactions between the catalytic and dianion activation sites of triosephosphate isomerase.

    PubMed

    Zhai, Xiang; Amyes, Tina L; Wierenga, Rik K; Loria, J Patrick; Richard, John P

    2013-08-27

    Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form d-glyceraldehyde 3-phosphate. The effects of two structural mutations in TIM on the kinetic parameters for catalysis of the reaction of the truncated substrate glycolaldehyde (GA) and the activation of this reaction by phosphite dianion are reported. The P168A mutation results in similar 50- and 80-fold decreases in (kcat/Km)E and (kcat/Km)E·HPi, respectively, for deprotonation of GA catalyzed by free TIM and by the TIM·HPO(3)(2-) complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy or the magnitude of phosphite dianion activation of TIM for catalysis of deprotonation of GA. A loop 7 replacement mutant (L7RM) of TIM from chicken muscle was prepared by substitution of the archaeal sequence 208-TGAG with 208-YGGS. L7RM exhibits a 25-fold decrease in (kcat/Km)E and a larger 170-fold decrease in (kcat/Km)E·HPi for reactions of GA. The mutation has little effect on the observed and intrinsic phosphodianion binding energy and only a modest effect on phosphite dianion activation of TIM. The observation that both the P168A and loop 7 replacement mutations affect mainly the kinetic parameters for TIM-catalyzed deprotonation but result in much smaller changes in the parameters for enzyme activation by phosphite dianion provides support for the conclusion that catalysis of proton transfer and dianion activation of TIM take place at separate, weakly interacting, sites in the protein catalyst.

  17. A Functional EXXEK Motif is Essential for Proton Coupling and Active Glucosinolate Transport by NPF2.11.

    PubMed

    Jørgensen, Morten Egevang; Olsen, Carl Erik; Geiger, Dietmar; Mirza, Osman; Halkier, Barbara Ann; Nour-Eldin, Hussam Hassan

    2015-12-01

    The proton-dependent oligopeptide transporter (POT/PTR) family shares a highly conserved E1X1X2E2RFXYY (E1X1X2E2R) motif across all kingdoms of life. This motif is suggested to have a role in proton coupling and active transport in bacterial homologs. For the plant POT/PTR family, also known as the NRT1/PTR family (NPF), little is known about the role of the E1X1X2E2R motif. Moreover, nothing is known about the role of the X1 and X2 residues within the E1X1X2E2R motif. We used NPF2.11-a proton-coupled glucosinolate (GLS) symporter from Arabidopsis thaliana-to investigate the role of the E1X1X2E2K motif variant in a plant NPF transporter. Using liquid chromatography-mass spectrometry (LC-MS)-based uptake assays and two-electrode voltage clamp (TEVC) electrophysiology, we demonstrate an essential role for the E1X1X2E2K motif for accumulation of substrate by NPF2.11. Our data suggest that the highly conserved E1, E2 and K residues are involved in translocation of protons, as has been proposed for the E1X1X2E2R motif in bacteria. Furthermore, we show that the two residues X1 and X2 in the E1X1X2E2[K/R] motif are conserved as uncharged amino acids in POT/PTRs from bacteria to mammals and that introducing a positive or negative charge in either position hampers the ability to overaccumulate substrate relative to the assay medium. We hypothesize that introducing a charge at X1 and X2 interferes with the function of the conserved glutamate and lysine residues of the E1X1X2E2K motif and affects the mechanism behind proton coupling. PMID:26443378

  18. A Functional EXXEK Motif is Essential for Proton Coupling and Active Glucosinolate Transport by NPF2.11.

    PubMed

    Jørgensen, Morten Egevang; Olsen, Carl Erik; Geiger, Dietmar; Mirza, Osman; Halkier, Barbara Ann; Nour-Eldin, Hussam Hassan

    2015-12-01

    The proton-dependent oligopeptide transporter (POT/PTR) family shares a highly conserved E1X1X2E2RFXYY (E1X1X2E2R) motif across all kingdoms of life. This motif is suggested to have a role in proton coupling and active transport in bacterial homologs. For the plant POT/PTR family, also known as the NRT1/PTR family (NPF), little is known about the role of the E1X1X2E2R motif. Moreover, nothing is known about the role of the X1 and X2 residues within the E1X1X2E2R motif. We used NPF2.11-a proton-coupled glucosinolate (GLS) symporter from Arabidopsis thaliana-to investigate the role of the E1X1X2E2K motif variant in a plant NPF transporter. Using liquid chromatography-mass spectrometry (LC-MS)-based uptake assays and two-electrode voltage clamp (TEVC) electrophysiology, we demonstrate an essential role for the E1X1X2E2K motif for accumulation of substrate by NPF2.11. Our data suggest that the highly conserved E1, E2 and K residues are involved in translocation of protons, as has been proposed for the E1X1X2E2R motif in bacteria. Furthermore, we show that the two residues X1 and X2 in the E1X1X2E2[K/R] motif are conserved as uncharged amino acids in POT/PTRs from bacteria to mammals and that introducing a positive or negative charge in either position hampers the ability to overaccumulate substrate relative to the assay medium. We hypothesize that introducing a charge at X1 and X2 interferes with the function of the conserved glutamate and lysine residues of the E1X1X2E2K motif and affects the mechanism behind proton coupling.

  19. An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

    SciTech Connect

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert

    2010-08-13

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  20. Toxic effects of mercury on PSI and PSII activities, membrane potential and transthylakoid proton gradient in Microsorium pteropus.

    PubMed

    Deng, Chunnuan; Zhang, Daoyong; Pan, Xiangliang; Chang, Fengqin; Wang, Shuzhi

    2013-10-01

    Mercury (Hg) is one of the top toxic metals in environment and it poses a great risk to organisms. This study aimed to elucidate the toxic effects of Hg(2+) on energy conversion of photosystem I (PSI) and photosystem II (PSII), membrane potential and proton gradient of Microsorium pteropus (an aquatic plant species). Contents of chlorophyll a, chlorophyll b and carotenoids, quantum yield and electron transfer of PSI and PSII of M. pteropus exposed to various concentrations of Hg(2+) were measured. With increasing Hg(2+) concentration, quantum yield and electron transport of PSI [Y(I) and ETR(I)] and PSII [Y(II) and ETR(II)] decreased whereas limitation of donor side of PSI [Y(ND)] increased. At ⩾165μgL(-1) Hg(2+), quantum yield of non-light-induced non-photochemical fluorescence quenching in PSII [Y(NO)] significantly increased but quantum yield of light-induced non-photochemical fluorescence quenching [Y(NPQ)] decreased. Membrane potential (Δψ) and proton gradient (ΔpH) of M. pteropus were reduced significantly at 330μg L(-1) Hg(2+) compared to control. Mercury exposure affected multiple sites in PSII and PSI of M. pteropus.

  1. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    SciTech Connect

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L.

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

  2. Active Sites Environmental Monitoring Program: Program plan. Revision 1

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  3. Syntheses of cofacial dimetallodiporphyrins and their applications to dihydrogen activation or proton reduction catalysis: Models for hydrogen electrode catalysts

    SciTech Connect

    Ha, Yunkyoung.

    1992-01-01

    Dihydrogen may be a future fuel source which could help relieve our present, heavy dependence on coal and oil. Electrochemical methods are the most efficient ways of producing H[sub 2]. It is necessary to catalyze this process. In the present work, activation of dihydrogen and proton reduction to dihydrogen have been studied with new cofacial dimetalloporphyrin complexes. A cofacial dirhodium diporphyrin, Rh[sub 2]DPB, was prepared and used to activate dihydrogen in the presence of ancillary ligands. The reaction of Rh[sub 2]DPB with H[sub 2] and CO resulted in the dirhodium diporphyrin dihydride, Rh(H)Rh(H)DPB. When phosphines or phosphites were employed in the reaction between Rh[sub 2]DPB and H[sub 2], a different type of hydride complex was produced. Three-center two-electron bonded hydride structures have been suggested, based on the upfield M-H chemical shifts, large T[sub 1] values, the absence of [sup 1]J[sub HD], and small [sup 2]J[sub P-H] coupling constants. Catalytic dihydrogen evolution via proton reduction has been achieved using a series of cofacial bisorganometallic diporphyrins. These compounds were synthesized by addition of the corresponding Grignard reagent to the dication of the dimetallodiporphyrins. Chemical two-electron reduction of M[sub 2][sup III/III]R[sub 2]DPB, protonation with a suitable acid, and subsequent H[sub 2] elimination with concurrent re-oxidation of the complexes were investigated. Reduction potentials of M[sub 2]R[sub 2]DPB were measured, and the pK[sub a] values of the catalysts estimated. By changing the central metal and modifying axial ligation, more efficient proton reduction and/or dihydrogen oxidation can be realized. Dinuclear centers in close proximity provide methods for activating molecules and/or performing redox reactions with substrates. Complexes derived from these dinuclear systems constitute additional models for understanding reactivity observed at metal surfaces, in homogeneous catalysis, and in clusters.

  4. Proton-sensing GPCR-YAP Signalling Promotes Cancer-associated Fibroblast Activation of Mesenchymal Stem Cells

    PubMed Central

    Zhu, Hongyi; Guo, Shangchun; Zhang, Yuelei; Yin, Junhui; Yin, Wenjing; Tao, Shicong; Wang, Yang; Zhang, Changqing

    2016-01-01

    The pHs of extracellular fluids (ECFs) in normal tissues are commonly maintained at 7.35 to 7.45. The acidification of the ECF is one of the major characteristics of tumour microenvironment. In this study, we report that decreased extracellular pH promotes the transformation of mesenchymal stem cells (MSCs) into cancer-associated fibroblasts (CAFs), termed CAF activation. Furthermore, we demonstrate that GPR68, a proton-sensing G-protein-coupled receptor (GPCR), is required for the pH-dependent regulation of the differentiation of MSCs into CAFs. We then identify Yes-associated protein 1 (YAP) as a downstream effector of GPR68 for CAF activation. Finally, we show that knockdown of GPR68 in MSCs can prevent the CAF activation under cancer microenvironment. Systemic transplantation of GPR68-silenced MSCs suppresses in-situ tumour growth and prolong life span after cancer graft. PMID:27019624

  5. 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.

  6. 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

  7. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site.

    PubMed

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. PMID:25902402

  8. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site.

    PubMed

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.

  9. A-site deficient La0.2Sr0.7TiO3-δ anode material for proton conducting ethane fuel cell to cogenerate ethylene and electricity

    NASA Astrophysics Data System (ADS)

    Liu, Subiao; Behnamian, Yashar; Chuang, Karl T.; Liu, Qingxia; Luo, Jing-Li

    2015-12-01

    A site deficient La0.2Sr0.7TiO3-δ (LSTA) and a highly proton conductive electrolyte BaCe0.7Zr0.1Y0.2O3-δ (BCZY) are synthesized by using solid state reaction method. The performance of the electrolyte-supported single cell, comprised of LSTA + Cr2O3 + Cu//BCZY//(La0.60Sr0.40)0.95Co0.20Fe0.80O3-δ (LSCF)+BCZY, is fabricated and investigated. LSTA shows remarkably high electrical performance, with a conductivity as high as 27.78 Scm-1 at 1150 °C in a 10% H2/N2 reducing atmosphere. As a main anode component, it shows good catalytic activity towards the oxidation of ethane, causing the power density to considerably increase from 158.4 mW cm-2 to 320.9 mW cm-2 and the ethane conversion to significantly rise from 12.6% to 30.9%, when the temperature increases from 650 °C to 750 °C. These changes agree well with the polarization resistance which dramatically decreases from 0.346 Ωcm2 to 0.112 Ωcm2. EDX measurement shows that no element diffusion exists (chemical compatibility) between anode (LSTA + Cr2O3+Cu) and electrolyte (BCZY). With these properties, the pure phase LSTA is evaluated as a high electro-catalytic activity anode material for ethane proton conducting solid oxide fuel cell (PC-SOFC).

  10. Proton therapy

    MedlinePlus

    ... direction of the tumor. A machine called a synchrotron or cyclotron creates and speeds up the protons. ... redness in the radiation area, and temporary hair loss. AFTER THE PROCEDURE Following proton therapy, you should ...

  11. Synthesis of methyl tert-butyl ether catalyzed by acidic ion-exchange resins. Influence of the proton activity

    SciTech Connect

    Panneman, H.J.; Beenackers, A.A.C.M.

    1995-12-01

    The catalytic activity of various strong acid ion-exchange resins on the synthesis of methyl tert-butyl ether (MtBE) from methanol and isobutene has been investigated. Relative to Amberlyst 15, Kastel CS 381 and Amberlyst CSP have similar rate constants, whereas Duolite ES 276 and Amberlyst XE 307 have significantly higher and Duolite C26 and Duolite C16P substantially lower rate constants. All resins show a great decrease in catalytic activity if part of the protons is exchanged by sodium ions. At 10% proton capacity the rate constants per equivalent acid are reduced by a factor of 9 (for Amberlyst Xe 307 and Kastel Cs 381) to more than a factor 20 for Amberlyst 15 and Duolite ES 276, resulting in 100--200 times lower MtBE production rates. Depending on the catalyst applied, mass transfer limitations start to occur between 50 and 80 C. Values of the effective diffusion coefficient of isobutene varied between 0.4 {times} 10{sup {minus}9} and 4.1 {times} 10{sup {minus}9} m{sup 2}/s at 80 C.

  12. Proton and gallium(III) binding properties of a biologically active salicylidene acylhydrazide.

    PubMed

    Hakobyan, Shoghik; Boily, Jean-François; Ramstedt, Madeleine

    2014-09-01

    Bacterial biofilm formation causes a range of problems in our society, especially in health care. Salicylidene acylhydrazides (hydrazones) are promising antivirulence drugs targeting secretion systems used during bacterial infection of host cells. When mixed with the gallium ion they become especially potent as bacterial and biofilm growth-suppressing agents, although the mechanisms through which this occurs are not fully understood. At the base of this uncertainty lies the nature of hydrazone-metal interactions. This study addresses this issue by resolving the equilibrium speciation of hydrazone-gallium aqueous solutions. The protonation constants of the target 2-oxo-2-[N-(2,4,6-trihydroxy-benzylidene)-hydrazino]-acetamide (ME0163) hydrazone species and of its 2,4,6-trihydroxybenzaldehyde and oxamic acid hydrazide building blocks were determined by UV-visible spectrophotometry to achieve this goal. These studies show that the hydrazone is an excessively strong complexing agent for gallium and that its antivirulence properties are predominantly ascribed to monomeric 1:1Ga-ME0163 complexes of various Ga hydrolysis and ME0163 protonation states. The chelation of Ga(III) to the hydrazone also increased the stability of the compounds against acid-induced hydrolysis, making this group of compounds very interesting for biological applications where the Fe-antagonist action of both Ga(III) and the hydrazone can be combined for enhanced biological effect.

  13. Enantioselective Protonation

    PubMed Central

    Mohr, Justin T.; Hong, Allen Y.; Stoltz, Brian M.

    2010-01-01

    Enantioselective protonation is a common process in biosynthetic sequences. The decarboxylase and esterase enzymes that effect this valuable transformation are able to control both the steric environment around the proton acceptor (typically an enolate) and the proton donor (typically a thiol). Recently, several chemical methods to achieve enantioselective protonation have been developed by exploiting various means of enantiocontrol in different mechanisms. These laboratory transformations have proven useful for the preparation of a number of valuable organic compounds. PMID:20428461

  14. Metals in the active site of native protein phosphatase-1.

    PubMed

    Heroes, Ewald; Rip, Jens; Beullens, Monique; Van Meervelt, Luc; De Gendt, Stefan; Bollen, Mathieu

    2015-08-01

    Protein phosphatase-1 (PP1) is a major protein Ser/Thr phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone. PMID:25890482

  15. Visualizing proton antenna in a high-resolution green fluorescent protein structure.

    PubMed

    Shinobu, Ai; Palm, Gottfried J; Schierbeek, Abraham J; Agmon, Noam

    2010-08-18

    "Proton-collecting antenna" are conjectured to consist of several carboxylates within hydrogen-bond (HB) networks on the surface of proteins, which funnel protons to the orifice of an internal proton wire leading to the protein's active site. Yet such constructions were never directly visualized. Here we report an X-ray structure of green fluorescent protein (GFP) of the highest resolution to date (0.9 A). It allows the identification of some pivotal hydrogen atoms pertinent to uncertainties concerning the protonation state of the chromophore. Applying a computer algorithm for mapping proton wires in proteins reveals the previously observed "active site wire" connecting Glu222 with the surface carboxylate Glu5. In addition, it is now possible to identify what appears to be a proton-collecting apparatus of GFP. It consists of a negative surface patch containing carboxylates, threonines, and water molecules, connected by a HB network to Glu5. Furthermore, we detect exit points via Asn146 and His148 to a hydrophobic surface region. The more extensive HB network of the present structure, as compared with earlier GFP structures, is not accidental. A systematic investigation of over 100 mutants shows a clear correlation between the observed water content of GFP X-ray structures and their resolution. With increasing water content, the proton wires become progressively larger. These findings corroborate the scenario in which the photodissociated proton from wild-type GFP can leak outside, whereafter another proton is recruited via the proton-collecting apparatus reported herein.

  16. Metavanadate at the active site of the phosphatase VHZ.

    PubMed

    Kuznetsov, Vyacheslav I; Alexandrova, Anastassia N; Hengge, Alvan C

    2012-09-01

    Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR spectroscopy to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, the crystal structures and NMR data suggest that the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases because of its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species and compared it with two classical trigonal-bipyramidal vanadate-phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex, the central, planar VO(3)(-) moiety has only one apical ligand, the nucleophilic Cys95, and a gap in electron density between V and S. A computational analysis showed that the V-S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence has shown to be able to bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure. PMID:22876963

  17. Zymogen Activation and Subcellular Activity of Subtilisin Kexin Isozyme 1/Site 1 Protease*

    PubMed Central

    da Palma, Joel Ramos; Burri, Dominique Julien; Oppliger, Joël; Salamina, Marco; Cendron, Laura; de Laureto, Patrizia Polverino; Seidah, Nabil Georges; Kunz, Stefan; Pasquato, Antonella

    2014-01-01

    The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B′/B followed by the herein newly identified C′/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B′/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1–P8) and P1′ are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates. PMID:25378398

  18. A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast.

    PubMed

    Pérez-Castiñeira, José R; Hernández, Agustín; Drake, Rocío; Serrano, Aurelio

    2011-07-15

    V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.

  19. Intracellular shunting of O2− contributes to charge compensation and preservation of neutrophil respiratory burst in the absence of voltage-gated proton channel activity

    PubMed Central

    Decleva, Eva; Menegazzi, Renzo; Fasolo, Alba; Defendi, Federica; Sebastianutto, Michele; Dri, Pietro

    2013-01-01

    Proton efflux via voltage-gated proton channels (Hv1) is considered to mediate the charge compensation necessary to preserve NADPH oxidase activity during the respiratory burst. Using the Hv1 inhibitor Zn2+, we found that the PMA-induced respiratory burst of human neutrophils is inhibited when assessed as extracellular production of O2− and H2O2, in accordance with literature studies, but, surprisingly, unaffected when measured as oxygen consumption or total (extracellular plus intracellular) H2O2 production. Furthermore, we show that inhibiting Hv1 with Zn2+ results in an increased production of intracellular ROS. Similar results, i.e. decreased extracellular and increased intracellular ROS production, were obtained using a human granulocyte-like cell line with severely impaired Hv1 expression. Acidic extracellular pH, which dampens proton efflux, also augmented intracellular production of H2O2. Zinc caused an increase in the rate but not in the extent of depolarization and cytosolic acidification indicating that mechanisms other than proton efflux take part in charge compensation. Our results suggest a hitherto unpredicted mechanism of charge compensation whereby, in the absence of proton efflux, part of O2− generated within gp91phox in the plasma membrane is shunted intracellularly down electrochemical gradient to dampen excessive depolarization. This would preserve NADPH oxidase activity under conditions such as the inflammatory exudate in which the acidic pH hinders charge compensation by proton efflux. PMID:23578765

  20. Electrochemical model of the function of the cytochrome c oxidase proton pump

    NASA Astrophysics Data System (ADS)

    Stojković, Branko P.; McMahon, Benjamin H.; Martin, R. L.; Gennis, Robert B.

    2000-03-01

    We combine quantum chemical calculations with an electrostatic model of the oxidase protein (in which the protein is seen as a non-uniform dielectric) to quantify the potential energy changes as the protein cycles through the four electron reduction of molecular oxygen to water. We find that the electric fields obtained directly from the quantum chemical calculations are adequate to explain the active translocation of four protons and joining of four protons and electrons at the oxygen, all against a 200 mV potential. The source of energy is the 500 mV per electron of redox energy released by bringing an electron from cytochrome c to the active binuclear site of the oxidase protein. Each electron entering the active site is accompanied by a proton taken up rapidly through the D channel. Two protons are pumped by the protons entering via the K channel, which are slower than D channel protons, but chemically stabilized at the active site, thus pushing the D channel protons ahead of them. Two other protons are forced out the high potential side of the oxidase when electrons move from the copper ligands and the heme onto the oxygen molecule during the 0=0 bond cleavage step.

  1. Activation cross-sections of proton induced reactions on vanadium in the 37-65 MeV energy range

    NASA Astrophysics Data System (ADS)

    Ditrói, F.; Tárkányi, F.; Takács, S.; Hermanne, A.

    2016-08-01

    Experimental excitation functions for proton induced reactions on natural vanadium in the 37-65 MeV energy range were measured with the activation method using a stacked foil irradiation technique. By using high resolution gamma spectrometry cross-section data for the production of 51,48Cr, 48V, 48,47,46,44m,44g,43Sc and 43,42K were determined. Comparisons with the earlier published data are presented and results predicted by different theoretical codes (EMPIRE and TALYS) are included. Thick target yields were calculated from a fit to our experimental excitation curves and compared with the earlier experimental yield data. Depth distribution curves to be used for thin layer activation (TLA) are also presented.

  2. Site-specific PEGylation of lidamycin and its antitumor activity.

    PubMed

    Li, Liang; Shang, Boyang; Hu, Lei; Shao, Rongguang; Zhen, Yongsu

    2015-05-01

    In this study, N-terminal site-specific mono-PEGylation of the recombinant lidamycin apoprotein (rLDP) of lidamycin (LDM) was prepared using a polyethyleneglycol (PEG) derivative (M w 20 kDa) through a reactive terminal aldehyde group under weak acidic conditions (pH 5.5). The biochemical properties of mPEG-rLDP-AE, an enediyne-integrated conjugate, were analyzed by SDS-PAGE, RP-HPLC, SEC-HPLC and MALDI-TOF. Meanwhile, in vitro and in vivo antitumor activity of mPEG-rLDP-AE was evaluated by MTT assays and in xenograft model. The results indicated that mPEG-rLDP-AE showed significant antitumor activity both in vitro and in vivo. After PEGylation, mPEG-rLDP still retained the binding capability to the enediyne AE and presented the physicochemical characteristics similar to that of native LDP. It is of interest that the PEGylation did not diminish the antitumor efficacy of LDM, implying the possibility that this derivative may function as a payload to deliver novel tumor-targeted drugs. PMID:26579455

  3. Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid

    SciTech Connect

    Shaya, D.; Hahn, Bum-Soo; Bjerkan, Tonje Marita; Kim, Wan Seok; Park, Nam Young; Sim, Joon-Soo; Kim, Yeong-Shik; Cygler, M.

    2008-03-19

    Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a {beta}-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: ({alpha}/{alpha}){sub 5} for CS (chondroitin lyases AC) and {beta}-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located {approx}12 {angstrom} from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.

  4. The Role of an Active Site Mg2+ in HDV Ribozyme Self-Cleavage: Insights from QM/MM Calculations

    PubMed Central

    Mlýnský, Vojtěch; Šponer, Jiří

    2014-01-01

    The hepatitis delta virus (HDV) ribozyme is a catalytic RNA motif embedded in the human pathogenic HDV RNA. It catalyzes self-cleavage of its sugar-phosphate backbone with direct participation of the active site cytosine C75. Biochemical and structural data support a general acid role of C75. Here, we used hybrid quantum mechanical/molecular mechanical (QM/MM) calculations to probe the reaction mechanism and changes in Gibbs energy along the ribozyme's reaction pathway with an N3-protonated C75H+ in the active site, which acts as the general acid, and a partially hydrated Mg2+ ion with one deprotonated, inner-shell coordinated water molecule that acts as the general base. We followed eight reaction paths with distinct position and coordination of the catalytically important active site Mg2+ ion. For six of them, we observed feasible activation barriers ranging from 14.2 to 21.9 kcal/mol, indicating that the specific position of the Mg2+ ion in the active site is predicted to strongly affect the kinetics of self-cleavage. The deprotonation of the U-1(2′-OH) nucleophile and the nucleophilic attack of the resulting U-1(2′-O−) on the scissile phosphodiester are found to be separate steps, as deprotonation precedes the nucleophilic attack. This sequential mechanism of the HDV ribozyme differs from the concerted nucleophilic activation and attack suggested for the hairpin ribozyme. We estimated the pKa of the U-1(2′-OH) group to range from 8.8 to 11.2, suggesting that the pKa is lowered by several units from that of a free ribose, comparable to and most likely smaller than the pKa of the solvated active site Mg2+ ion. Our results thus support the notion that the structure of the HDV ribozyme, and particularly the positioning of the active site Mg2+ ion, facilitates deprotonation and activation of the 2′-OH nucleophile. PMID:25412464

  5. Subunit CydX of Escherichia coli cytochrome bd ubiquinol oxidase is essential for assembly and stability of the di-heme active site.

    PubMed

    Hoeser, Jo; Hong, Sangjin; Gehmann, Gerfried; Gennis, Robert B; Friedrich, Thorsten

    2014-05-01

    Cytochrome bd ubiquinol oxidase uses the electron transport from ubiquinol to oxygen to establish a proton gradient across the membrane. The enzyme complex consists of subunits CydA and B and contains two b- and one d-type hemes as cofactors. Recently, it was proposed that a third subunit named CydX is essential for the function of the complex. Here, we show that CydX is indeed a subunit of purified Escherichia coli cytochrome bd oxidase and that the small protein is needed either for the assembly or the stability of the active site di-heme center and, thus, is essential for oxidase activity.

  6. Proton therapy in the clinic.

    PubMed

    DeLaney, Thomas F

    2011-01-01

    The clinical advantage for proton radiotherapy over photon approaches is the marked reduction in integral dose to the patient, due to the absence of exit dose beyond the proton Bragg peak. The integral dose with protons is approximately 60% lower than that with any external beam photon technique. Pediatric patients, because of their developing normal tissues and anticipated length of remaining life, are likely to have the maximum clinical gain with the use of protons. Proton therapy may also allow treatment of some adult tumors to much more effective doses, because of normal tissue sparing distal to the tumor. Currently, the most commonly available proton treatment technology uses 3D conformal approaches based on (a) distal range modulation, (b) passive scattering of the proton beam in its x- and y-axes, and (c) lateral beam-shaping. It is anticipated that magnetic pencil beam scanning will become the dominant mode of proton delivery in the future, which will lower neutron scatter associated with passively scattered beam lines, reduce the need for expensive beam-shaping devices, and allow intensity-modulated proton radiotherapy. Proton treatment plans are more sensitive to variations in tumor size and normal tissue changes over the course of treatment than photon plans, and it is expected that adaptive radiation therapy will be increasingly important for proton therapy as well. While impressive treatment results have been reported with protons, their cost is higher than for photon IMRT. Hence, protons should ideally be employed for anatomic sites and tumors not well treated with photons. While protons appear cost-effective for pediatric tumors, their cost-effectiveness for treatment of some adult tumors, such as prostate cancer, is uncertain. Comparative studies have been proposed or are in progress to more rigorously assess their value for a variety of sites. The utility of proton therapy will be enhanced by technological developments that reduce its cost

  7. Moving protons and electrons in biomimetic systems.

    PubMed

    Warren, Jeffrey J; Mayer, James M

    2015-03-17

    An enormous variety of biological redox reactions are accompanied by changes in proton content at enzyme active sites, in their associated cofactors, in substrates and/or products, and between protein interfaces. Understanding this breadth of reactivity is an ongoing chemical challenge. A great many workers have developed and investigated biomimetic model complexes to build new ways of thinking about the mechanistic underpinnings of such complex biological proton-coupled electron transfer (PCET) reactions. Of particular importance are those model reactions that involve transfer of one proton (H(+)) and one electron (e(-)), which is equivalent to transfer of a hydrogen atom (H(•)). In this Current Topic, we review key concepts in PCET reactivity and describe important advances in biomimetic PCET chemistry, with a special emphasis on research that has enhanced efforts to understand biological PCET reactions.

  8. 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.

  9. Hydrogen-bonded proton transfer in the protonated guanine-cytosine (GC+H)+ base pair.

    PubMed

    Lin, Yuexia; Wang, Hongyan; Gao, Simin; Schaefer, Henry F

    2011-10-13

    The single proton transfer at the different sites of the Watson-Crick (WC) guanine-cytosine (GC) DNA base pair are studied here using density functional methods. The conventional protonated structures, transition state (TS) and proton-transferred product (PT) structures of every relevant species are optimized. Each transition state and proton-transferred product structure has been compared with the corresponding conventional protonated structure to demonstrate the process of proton transfer and the change of geometrical structures. The relative energies of the protonated tautomers and the proton-transfer energy profiles in gas and solvent are analyzed. The proton-transferred product structure G(+H(+))-H(+)C(N3)(-H(+))(PT) has the lowest relative energy for which only two hydrogen bonds exist. Almost all 14 isomers of the protonated GC base pair involve hydrogen-bonded proton transfer following the three pathways, with the exception of structure G-H(+)C(O2). When the positive charge is primarily "located" on the guanine moiety (H(+)G-C, G-H(+)C(C4), and G-H(+)C(C6)), the H(1) proton transfers from the N(1) site of guanine to the N(3) site of cytosine. The structures G-H(+)C(C5) and G-H(+)C(C4) involve H(4a) proton transfer from the N(4) of cytosine to the O(6) site of guanine. H(2a) proton transfer from the N(2) site of guanine to the O(2) site of cytosine is found only for the structure G-H(+)C(C4). The structures to which a proton is added on the six-centered sites adjoining the hydrogen bonds are more prone to proton transfer in the gas phase, whereas a proton added on the minor groove and the sites adjoining the hydrogen bonds is favorable to the proton transfer in energy in the aqueous phase.

  10. N6-Methyldeoxyadenosine Marks Active Transcription Start Sites in Chlamydomonas

    PubMed Central

    Chen, Kai; Deng, Xin; Yu, Miao; Han, Dali; Hao, Ziyang; Liu, Jianzhao; Lu, Xingyu; Dore, Louis C; Weng, Xiaocheng; Ji, Quanjiang; Mets, Laurens; He, Chuan

    2015-01-01

    SUMMARY N6-methyldeoxyadenosine (6mA or m6A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria, and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution, and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms. PMID:25936837

  11. Detection limit for activation measurements in ultralow background sites

    NASA Astrophysics Data System (ADS)

    Trache, Livius; Chesneanu, D.; Margineanu, R.; Pantelica, A.; Ghita, D. G.; Burducea, I.; Straticiuc, M.; Tang, X. D.

    2014-09-01

    We used 12C +13C fusion at the beam energies E = 6, 7 and 8 MeV to determine the sensitivity and the limits of activation method measurements in ultralow background sites. A 13C beam of 0.5 μA from the 3 MV Tandem accelerator of the Horia Hulubei National Institute of Physics and Nuclear Engineering - IFIN HH impinged on thick graphite targets. After about 24 hrs of irradiation targets were measured in two different laboratories: one with a heavy shielded Ge detector in the institute (at the surface) and one located underground in the microBequerel laboratory, in the salt mine of Slanic-Prahova, Romania. The 1369- and 2754 keV peaks from 24Na deactivation were clearly observed in the γ-ray spectra obtained for acquisitions lasting a few hours, or a few days. Determination of the detection limit in evaluating the cross sections for the target irradiated at Ec . m = 3 MeV indicates the fact that it is possible to measure gamma spectrum in underground laboratory down to Ec . m = 2 . 6 MeV. Cleaning the spectra with beta-gamma coincidences and increasing beam intensity 20 times will take as further down. The measurements are motivated by the study of the 12 C +12 C reaction at astrophysical energies.

  12. Disturbance opens recruitment sites for bacterial colonization in activated sludge.

    PubMed

    Vuono, David C; Munakata-Marr, Junko; Spear, John R; Drewes, Jörg E

    2016-01-01

    Little is known about the role of immigration in shaping bacterial communities or the factors that may dictate success or failure of colonization by bacteria from regional species pools. To address these knowledge gaps, the influence of bacterial colonization into an ecosystem (activated sludge bioreactor) was measured through a disturbance gradient (successive decreases in the parameter solids retention time) relative to stable operational conditions. Through a DNA sequencing approach, we show that the most abundant bacteria within the immigrant community have a greater probability of colonizing the receiving ecosystem, but mostly as low abundance community members. Only during the disturbance do some of these bacterial populations significantly increase in abundance beyond background levels and in few cases become dominant community members post-disturbance. Two mechanisms facilitate the enhanced enrichment of immigrant populations during disturbance: (i) the availability of resources left unconsumed by established species and (ii) the increased availability of niche space for colonizers to establish and displace resident populations. Thus, as a disturbance decreases local diversity, recruitment sites become available to promote colonization. This work advances our understanding of microbial resource management and diversity maintenance in complex ecosystems. PMID:25727891

  13. Active Site Characterization of Proteases Sequences from Different Species of Aspergillus.

    PubMed

    Morya, V K; Yadav, Virendra K; Yadav, Sangeeta; Yadav, Dinesh

    2016-09-01

    A total of 129 proteases sequences comprising 43 serine proteases, 36 aspartic proteases, 24 cysteine protease, 21 metalloproteases, and 05 neutral proteases from different Aspergillus species were analyzed for the catalytically active site residues using MEROPS database and various bioinformatics tools. Different proteases have predominance of variable active site residues. In case of 24 cysteine proteases of Aspergilli, the predominant active site residues observed were Gln193, Cys199, His364, Asn384 while for 43 serine proteases, the active site residues namely Asp164, His193, Asn284, Ser349 and Asp325, His357, Asn454, Ser519 were frequently observed. The analysis of 21 metalloproteases of Aspergilli revealed Glu298 and Glu388, Tyr476 as predominant active site residues. In general, Aspergilli species-specific active site residues were observed for different types of protease sequences analyzed. The phylogenetic analysis of these 129 proteases sequences revealed 14 different clans representing different types of proteases with diverse active site residues.

  14. A proposed definition of the 'activity' of surface sites on lactose carriers for dry powder inhalation.

    PubMed

    Grasmeijer, Floris; Frijlink, Henderik W; de Boer, Anne H

    2014-06-01

    A new definition of the activity of surface sites on lactose carriers for dry powder inhalation is proposed which relates to drug detachment during dispersion. The new definition is expected to improve the understanding of 'carrier surface site activity', which stimulates the unambiguous communication about this subject and may aid in the rational design and interpretation of future formulation studies. In contrast to the currently prevailing view on carrier surface site activity, it follows from the newly proposed definition that carrier surface site activity depends on more variables than just the physicochemical properties of the carrier surface. Because the term 'active sites' is ambiguous, it is recommended to use the term 'highly active sites' instead to denote carrier surface sites with a relatively high activity. PMID:24613490

  15. New activation cross section data on longer lived radio-nuclei produced in proton induced nuclear reaction on zirconium.

    PubMed

    Tárkányi, F; Ditrói, F; Takács, S; Hermanne, A; Al-Abyad, M; Yamazaki, H; Baba, M; Mohammadi, M A

    2015-03-01

    The excitation functions of (96)Nb, (95m)Nb, (95g)Nb, (92m)Nb, (91m)Nb, (90)Nb, (95)Zr, (89)Zr, (88)Zr, (86)Zr, (88)Y, (87m)Y, (87g)Y, (86)Y were measured up to 70MeV proton energy by using the stacked foil technique and the activation method. The new data were compared with the critically analyzed experimental data in the literature and with the TALYS based model results in TENDL-2013 library. The possible role of the investigated reactions in the production of medically relevant (90)Nb, (95m)Nb, (89)Zr, and (88)Y radionuclides is discussed. PMID:25579457

  16. MECHANICAL PROPERTIES AND MICROSTRUCTURE IN LOW ACTIVATION MARTENSITIC STEELS F82H AND OPTIMAX AFTER 800 MEV PROTON IRRADIATION

    SciTech Connect

    Y. DAI; ET AL

    1999-10-01

    Low-activation martensitic steels, F82H (mod.) and Optimax-A, have been irradiated with 800-MeV protons up to 5.9 dpa. The tensile properties and microstructure have been studied. The results show that radiation hardening increases continuously with irradiation dose. F82H has lesser irradiation hardening as compared to Optimax-A in the present work and DIN1.4926 from a previous study. The irradiation embrittlement effects are evident in the materials since the uniform elongation is reduced sharply to less than 2%. However, all the irradiated samples ruptured in a ductile-fracture mode. Defect clusters have been observed. The size and the density of defect clusters increase with the irradiation dose. Precipitates are amorphous after irradiation.

  17. Investigations on the role of proton-coupled electron transfer in hydrogen activation by [FeFe]-hydrogenase.

    PubMed

    Mulder, David W; Ratzloff, Michael W; Bruschi, Maurizio; Greco, Claudio; Koonce, Evangeline; Peters, John W; King, Paul W

    2014-10-29

    Proton-coupled electron transfer (PCET) is a fundamental process at the core of oxidation-reduction reactions for energy conversion. The [FeFe]-hydrogenases catalyze the reversible activation of molecular H2 through a unique metallocofactor, the H-cluster, which is finely tuned by the surrounding protein environment to undergo fast PCET transitions. The correlation of electronic and structural transitions at the H-cluster with proton-transfer (PT) steps has not been well-resolved experimentally. Here, we explore how modification of the conserved PT network via a Cys → Ser substitution at position 169 proximal to the H-cluster of Chlamydomonas reinhardtii [FeFe]-hydrogenase (CrHydA1) affects the H-cluster using electron paramagnetic resonance (EPR) and Fourier transform infrared (FTIR) spectroscopy. Despite a substantial decrease in catalytic activity, the EPR and FTIR spectra reveal different H-cluster catalytic states under reducing and oxidizing conditions. Under H2 or sodium dithionite reductive treatments, the EPR spectra show signals that are consistent with a reduced [4Fe-4S]H(+) subcluster. The FTIR spectra showed upshifts of νCO modes to energies that are consistent with an increase in oxidation state of the [2Fe]H subcluster, which was corroborated by DFT analysis. In contrast to the case for wild-type CrHydA1, spectra associated with Hred and Hsred states are less populated in the Cys → Ser variant, demonstrating that the exchange of -SH with -OH alters how the H-cluster equilibrates among different reduced states of the catalytic cycle under steady-state conditions. PMID:25286239

  18. Effects of proton radiation on evoked and spontaneous neuronal activity in the hippocampus of APP/PSEN1 transgenic mice

    PubMed Central

    Rudobeck, Emil; Szucs, Attila; Vlkolinsky, Roman

    2014-01-01

    Ground-based studies on space radiation indicate that exposure to charged particle radiation at relatively low doses may impair neuronal functions. Decrements in excitatory synaptic transmission and plasticity in the hippocampus have been previously reported in mouse brains irradiated with iron nuclei, but relatively little is known about the effects of protons on the synaptic activity in the hippocampus. Behavioral and neurophysiological decrements in irradiated subjects are also commonly observed in Alzheimer's disease (AD), and we hypothesized that irradiation with protons may exacerbate AD-like neurodegenerative pathology. We used transgenic (TG) mice with AD-like neurodegeneration to test whether whole-body irradiation with protons (150 MeV; 0.1, 0.5, 1 Gy) accelerates the onset of AD and/or exacerbates synaptic impairments. We measured evoked excitatory synaptic potentials, synaptic plasticity and spontaneous oscillations in hippocampal slices prepared from APP/PSEN1 double TG mice (males only) at 6 and 9 months post-irradiation. Our electrophysiological recordings indicate that most of the radiation-induced synaptic decrements can be observed at 9 months rather than 6 months post-irradiation and they are differently expressed in TG and wild-type (WT) mice. Presynaptic excitability evaluated by the amplitude of fiber volleys was significantly increased in non-irradiated TG mice when compared with non-irradiated WT mice, and irradiation did not cause further alterations. The post-synaptic, dendritic excitability, evaluated by the initial slope of the excitatory post-synaptic potentials (EPSPs), was reduced in TG mice exposed to 0.5 Gy, but increased relative to WT mice at the same dose. Interestingly, the suppressive effect on EPSP in TG mice was not observed at 1 Gy. Post-synaptic firing of action potentials, evaluated by the amplitude of population spikes (PS), was increased in non-irradiated TG mice when compared with WT controls. However, these increases

  19. Proton Therapy

    NASA Astrophysics Data System (ADS)

    Oelfke, Uwe

    Proton therapy is one of the most rapidly developing new treatment technologies in radiation oncology. This treatment approach has — after roughly 40 years of technical developments — reached a mature state that allows a widespread clinical application. We therefore review the basic physical and radio-biological properties of proton beams. The main physical aspect is the elemental dose distribution arising from an infinitely narrow proton pencil beam. This includes the physics of proton stopping powers and the concept of CSDA range. Furthermore, the process of multiple Coulomb scattering is discussed for the lateral dose distribution. Next, the basic terms for the description of radio-biological properties of proton beams like LET and RBE are briefly introduced. Finally, the main concepts of modern proton dose delivery concepts are introduced before the standard method of inverse treatment planning for hadron therapy is presented.

  20. Xalpha-DVM investigation of double water molecule interactions with active sites of alpha- and beta-subunits of hemoglobin

    NASA Astrophysics Data System (ADS)

    Yuryeva, Elmira I.

    In this work, the results of Xalpha-discrete variation method calculations of the electronic structure and interatomic parameters of chemical bonding between iron (II) and oxygen molecule with and without extra electrons and protons in active site (AS) of alpha- and beta-subunits of oxyhemoglobin are presented. The Skulachev model of O2 molecule existing in respiration medium in the 2H2O form was used. The introduction of extra electrons does not change considerably the interaction of the iron atom with the O2 oxygen molecule, but strengthens the repulsion in the Fe bond N bonds. In this case, the estimated effective charge of the iron atom is +1.8/1.5e for AS of alpha-/beta-subunits of oxyhemoglobin, and the magnetic moment of iron atoms becomes zero. The deoxygenation effect of the AS of the alpha- and beta-subunits of oxyhemoglobin is due to the ability of extra protons to break down covalent attraction between the iron atom and the nearest oxygen atom and also to weakening of the repulsive component of the covalent Fe bond N interactions.

  1. Asymmetric protonation of EmrE

    PubMed Central

    Morrison, Emma A.; Robinson, Anne E.; Liu, Yongjia

    2015-01-01

    The small multidrug resistance transporter EmrE is a homodimer that uses energy provided by the proton motive force to drive the efflux of drug substrates. The pKa values of its “active-site” residues—glutamate 14 (Glu14) from each subunit—must be poised around physiological pH values to efficiently couple proton import to drug export in vivo. To assess the protonation of EmrE, pH titrations were conducted with 1H-15N TROSY-HSQC nuclear magnetic resonance (NMR) spectra. Analysis of these spectra indicates that the Glu14 residues have asymmetric pKa values of 7.0 ± 0.1 and 8.2 ± 0.3 at 45°C and 6.8 ± 0.1 and 8.5 ± 0.2 at 25°C. These pKa values are substantially increased compared with typical pKa values for solvent-exposed glutamates but are within the range of published Glu14 pKa values inferred from the pH dependence of substrate binding and transport assays. The active-site mutant, E14D-EmrE, has pKa values below the physiological pH range, consistent with its impaired transport activity. The NMR spectra demonstrate that the protonation states of the active-site Glu14 residues determine both the global structure and the rate of conformational exchange between inward- and outward-facing EmrE. Thus, the pKa values of the asymmetric active-site Glu14 residues are key for proper coupling of proton import to multidrug efflux. However, the results raise new questions regarding the coupling mechanism because they show that EmrE exists in a mixture of protonation states near neutral pH and can interconvert between inward- and outward-facing forms in multiple different protonation states. PMID:26573622

  2. [Effects of palmitic acid on activity of uncoupling proteins and proton leak in in vitro cerebral mitochondria from the rats exposed to simulated high altitude hypoxia].

    PubMed

    Xu, Yu; Liu, Jun-Ze; Xia, Chen

    2008-02-25

    To reveal the roles of uncoupling proteins (UCPs) in disorder of mitochondrial oxidative phosphorylation induced by free fatty acid during hypoxic exposure, the effects of palmitic acid on activity of UCPs, proton leak and mitochondrial membrane potential in hypoxia-exposed rat brain mitochondria were observed in vitro. Adult Sprague-Dawley (SD) rats were set randomly into control, acute hypoxia and chronic hypoxia groups (n=8 in each group). The acute and chronic hypoxic rats were exposed to simulated 5000 m high altitude in a hypobaric chamber 23 h/d for 3 d and 30 d, respectively. The brain mitochondria were isolated by centrifugation. UCP content and activity were detected by [(3)H]-GTP binding method. The proton leak was measured by TPMP(+) electrode and oxygen electrode. The membrane potential of mitochondria was calculated by detecting the fluorescence from Rodamine 123. Hypoxic exposure resulted in an increase in UCP activity and content as well as proton leak, but a decrease in the membrane potential of rat brain mitochondria. Palmitic acid resulted in further increases in UCP activity and content as well as proton leak, and further decrease in membrane potential of brain mitochondria in vitro from hypoxia-exposed rats, but hypoxic exposure decreased the reactivity of cerebral mitochondria to palmitic acid, especially in the acute hypoxia group. There was a negative correlation between mitochondrial proton leak and K(d) value (representing derivative of UCP activity, P<0.01, r = -0.906), and a positive correlation between proton leak and B(max) (representing the maximal content of UCPs in mitochondrial inner membrane, P<0.01, r = 0.856). Cerebral mitochondrial membrane potential was negatively correlated with proton leak (P<0.01, r = -0.880). It is suggested that hypoxia-induced proton leak enhancement and membrane potential decrease are correlated with the increased activity of UCPs. Hypoxia can also decrease the sensitivity of cerebral mitochondria to

  3. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  4. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  5. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  6. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  7. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  8. Microsecond Molecular Simulations Reveal a Transient Proton Pathway in the Calcium Pump.

    PubMed

    Espinoza-Fonseca, L Michel; Ramírez-Salinas, G Lizbeth

    2015-06-10

    The calcium pump sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) counter-transports Ca(2+) and H(+) at the expense of ATP hydrolysis. SERCA uses separate proton and metal ion pathways during active transport to neutralize the highly charged transport site, thus preserving SERCA's structural stability during active Ca(2+) transport. Although separate metal ion and proton pathways have been identified during slow (millisecond) structural transitions of SERCA, the existence of simultaneous metal and proton pathways during fast (microsecond) structural transitions remains unknown. We have analyzed microsecond-long trajectories of E1·H(+)771, a protonated intermediate of the pump populated during SERCA regulation. We found a transiently established hydrophobic pore in the luminal side of the transmembrane helices 6, 8, and 9. This narrow (0.5-0.6 nm) pore connects the transport sites to the sarcoplasmic reticulum lumen through a chain of water molecules. Protein pKa calculations of the transport site residues and structural analysis of the water molecules showed that this pore is suitable for proton transport. This transient proton pathway ensures neutralization of the transport sites during the rapid structural transitions associated with regulation of the pump. We conclude that this transient proton pathway plays a central role in optimizing active Ca(2+) transport by SERCA. Our discovery provides insight into ion-exchange mechanisms through transient hydrophobic pores in P-type ATPases.

  9. GAS HYDRATES AT TWO SITES OF AN ACTIVE CONTINENTAL MARGIN.

    USGS Publications Warehouse

    Kvenvolden, K.A.

    1985-01-01

    Sediment containing gas hydrates from two distant Deep Sea Drilling Project sites (565 and 568), located about 670 km apart on the landward flank of the Middle America Trench, was studied to determine the geochemical conditions that characterize the occurrence of gas hydrates. Site 565 was located in the Pacific Ocean offshore the Nicoya Peninsula of Costa Rica in 3,111 m of water. The depth of the hole at this site was 328 m, and gas hydrates were recovered from 285 and 319 m. Site 568 was located about 670 km to the northwest offshore Guatemala in 2,031 m of water. At this site the hole penetrated to 418 m, and gas hydrates were encountered at 404 m.

  10. Control of active sites in selective flocculation: III -- Mechanism of site blocking

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    It has been shown in Parts I and II of this paper that heteroflocculation can be controlled by poisoning the sites for flocculant adsorption using a site blocking agent (SBA). An efficient SBA was determined to be the lower molecular weight fraction of the flocculant. In this paper, the underlying mechanism of SBA action is described. Also, the mathematical model detailed in Part I is used to determine the effect of different SBAs on apatite-dolomite separation efficiency. It has been demonstrated that the depression in flocculation is directly related to the site blocking parameter ([bar [Phi

  11. Dynamically achieved active site precision in enzyme catalysis.

    PubMed

    Klinman, Judith P

    2015-02-17

    CONSPECTUS: The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes' enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme-substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C-H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed.

  12. Carbonic anhydrase II binds to and increases the activity of the epithelial sodium-proton exchanger, NHE3.

    PubMed

    Krishnan, Devishree; Liu, Lei; Wiebe, Shane A; Casey, Joseph R; Cordat, Emmanuelle; Alexander, R Todd

    2015-08-15

    Two-thirds of sodium filtered by the renal glomerulus is reabsorbed from the proximal tubule via a sodium/proton exchanger isoform 3 (NHE3)-dependent mechanism. Since sodium and bicarbonate reabsorption are coupled, we postulated that the molecules involved in their reabsorption [NHE3 and carbonic anhydrase II (CAII)] might physically and functionally interact. Consistent with this, CAII and NHE3 were closely associated in a renal proximal tubular cell culture model as revealed by a proximity ligation assay. Direct physical interaction was confirmed in solid-phase binding assays with immobilized CAII and C-terminal NHE3 glutathione-S-transferase fusion constructs. To assess the effect of CAII on NHE3 function, we expressed NHE3 in a proximal tubule cell line and measured NHE3 activity as the rate of intracellular pH recovery, following an acid load. NHE3-expressing cells had a significantly greater rate of intracellular pH recovery than controls. Inhibition of endogenous CAII activity with acetazolamide significantly decreased NHE3 activity, indicating that CAII activates NHE3. To ascertain whether CAII binding per se activates NHE3, we expressed NHE3 with wild-type CAII, a catalytically inactive CAII mutant (CAII-V143Y), or a mutant unable to bind other transporters (CAII-HEX). NHE3 activity increased upon wild-type CAII coexpression, but not in the presence of the CAII V143Y or HEX mutant. Together these studies support an association between CAII and NHE3 that alters the transporter's activity.

  13. Acidosis Activation of the Proton-Sensing GPR4 Receptor Stimulates Vascular Endothelial Cell Inflammatory Responses Revealed by Transcriptome Analysis

    PubMed Central

    Dong, Lixue; Li, Zhigang; Leffler, Nancy R.; Asch, Adam S.; Chi, Jen-Tsan; Yang, Li V.

    2013-01-01

    Acidic tissue microenvironment commonly exists in inflammatory diseases, tumors, ischemic organs, sickle cell disease, and many other pathological conditions due to hypoxia, glycolytic cell metabolism and deficient blood perfusion. However, the molecular mechanisms by which cells sense and respond to the acidic microenvironment are not well understood. GPR4 is a proton-sensing receptor expressed in endothelial cells and other cell types. The receptor is fully activated by acidic extracellular pH but exhibits lesser activity at the physiological pH 7.4 and minimal activity at more alkaline pH. To delineate the function and signaling pathways of GPR4 activation by acidosis in endothelial cells, we compared the global gene expression of the acidosis response in primary human umbilical vein endothelial cells (HUVEC) with varying level of GPR4. The results demonstrated that acidosis activation of GPR4 in HUVEC substantially increased the expression of a number of inflammatory genes such as chemokines, cytokines, adhesion molecules, NF-κB pathway genes, and prostaglandin-endoperoxidase synthase 2 (PTGS2 or COX-2) and stress response genes such as ATF3 and DDIT3 (CHOP). Similar GPR4-mediated acidosis induction of the inflammatory genes was also noted in other types of endothelial cells including human lung microvascular endothelial cells and pulmonary artery endothelial cells. Further analyses indicated that the NF-κB pathway was important for the acidosis/GPR4-induced inflammatory gene expression. Moreover, acidosis activation of GPR4 increased the adhesion of HUVEC to U937 monocytic cells under a flow condition. Importantly, treatment with a recently identified GPR4 antagonist significantly reduced the acidosis/GPR4-mediated endothelial cell inflammatory response. Taken together, these results show that activation of GPR4 by acidosis stimulates the expression of a wide range of inflammatory genes in endothelial cells. Such inflammatory response can be suppressed by

  14. Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

    PubMed Central

    Hammond, S. E.; Hanna, P. C.

    1998-01-01

    The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase. PMID:9573135

  15. Monoclonal antibody against the active site of caeruloplasmin and the ELISA system detecting active caeruloplasmin.

    PubMed

    Hiyamuta, S; Ito, K

    1994-04-01

    Serum caeruloplasmin deficiency is a characteristic biochemical abnormality found in patients with Wilson's disease, but the mechanism of this disease is unknown. Although the phenylenediamine oxidase activity of serum caeruloplasmin is markedly low in patients with Wilson's disease, mRNA of caeruloplasmin exists to some extent. To investigate the deficiency of caeruloplasmin oxidase activity in Wilson's disease, we generated 14 monoclonal antibodies (MAbs) and selected ID1, which had the strongest reactivity, and ID2, which had neutralizing ability. We also established a system to measure active caeruloplasmin specifically using these MAbs. These MAbs and the system will be useful tools in analyzing the active site of caeruloplasmin in patients with Wilson's disease.

  16. Structural Diversity Within the Mononuclear and Binuclear Active Sites of N-Acetyl-D-Glucosamine-6-Phosphate Deacetylase

    SciTech Connect

    Hall,R.; Brown, S.; Fedorov, A.; Fedorov, E.; Xu, C.; Babbitt, P.; Almo, S.; Raushel, F.

    2007-01-01

    NagA catalyzes the hydrolysis of N-acetyl-D-glucosamine-6-phosphate to D-glucosamine-6-phosphate and acetate. X-ray crystal structures of NagA from Escherichia coli were determined to establish the number and ligation scheme for the binding of zinc to the active site and to elucidate the molecular interactions between the protein and substrate. The three-dimensional structures of the apo-NagA, Zn-NagA, and the D273N mutant enzyme in the presence of a tight-binding N-methylhydroxyphosphinyl-D-glucosamine-6-phosphate inhibitor were determined. The structure of the Zn-NagA confirms that this enzyme binds a single divalent cation at the beta-position in the active site via ligation to Glu-131, His-195, and His-216. A water molecule completes the ligation shell, which is also in position to be hydrogen bonded to Asp-273. In the structure of NagA bound to the tight binding inhibitor that mimics the tetrahedral intermediate, the methyl phosphonate moiety has displaced the hydrolytic water molecule and is directly coordinated to the zinc within the active site. The side chain of Asp-273 is positioned to activate the hydrolytic water molecule via general base catalysis and to deliver this proton to the amino group upon cleavage of the amide bond of the substrate. His-143 is positioned to help polarize the carbonyl group of the substrate in conjunction with Lewis acid catalysis by the bound zinc. The inhibitor is bound in the {alpha}-configuration at the anomeric carbon through a hydrogen bonding interaction of the hydroxyl group at C-1 with the side chain of His-251. The phosphate group of the inhibitor attached to the hydroxyl at C-6 is ion paired with Arg-227 from the adjacent subunit. NagA from Thermotoga maritima was shown to require a single divalent cation for full catalytic activity.

  17. Robotics and Automation Activities at the Savannah River Site: A Site Report for SUBWOG 39F

    SciTech Connect

    Teese, G.D.

    1995-09-28

    The Savannah River Site has successfully used robots, teleoperators, and remote video to reduce exposure to ionizing radiation, improve worker safety, and improve the quality of operations. Previous reports have described the use of mobile teleoperators in coping with a high level liquid waste spill, the removal of highly contaminated equipment, and the inspection of nuclear reactor vessels. This report will cover recent applications at the Savannah River, as well as systems which SRS has delivered to other DOE site customers.

  18. Control of active sites in selective flocculation: II -- Role of site blocking agents

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    Control of heteroflocculation using a lower molecular weight fraction of the flocculant as a site blocking agent is demonstrated in the apatite-dolomite-polyethylene oxide system. The most effective SBA (site blocking agent) was determined to be the highest molecular weight fraction of the flocculant itself which was not capable of flocculating any of the components of the mixture. In the presence of the SBA, flocculant adsorption decreased significantly on apatite particles, thereby inhibiting coflocculation.

  19. Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae

    PubMed Central

    Daniel, Bastian; Wallner, Silvia; Steiner, Barbara; Oberdorfer, Gustav; Kumar, Prashant; van der Graaff, Eric; Roitsch, Thomas; Sensen, Christoph W.; Gruber, Karl; Macheroux, Peter

    2016-01-01

    Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family. PMID:27276217

  20. Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae.

    PubMed

    Daniel, Bastian; Wallner, Silvia; Steiner, Barbara; Oberdorfer, Gustav; Kumar, Prashant; van der Graaff, Eric; Roitsch, Thomas; Sensen, Christoph W; Gruber, Karl; Macheroux, Peter

    2016-01-01

    Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family. PMID:27276217

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

    SciTech Connect

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

    2013-01-08

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

  2. Preliminary results, obtained by using a proton beam, for an active scanning system to installed on the KHIMA

    NASA Astrophysics Data System (ADS)

    Kim, Chang Hyeuk; Lee, Hwa-Ryun; Jang, Sea Duk; Kim, Hyunyong; Hahn, Garam; Kim, Jeong Hwan; Jang, Hong Suk; Park, Dong Wook; Hwang, Won Taek; Yang, Tae-Keun

    2015-08-01

    The active scanning technique is a pencil beam delivery method in particle therapy. The active scanning beam delivery system consists of a beam scanner, beam monitor, energy modulator, and related programs, such as the irradiation control and planning programs. A proposed prototype active scanning system was designed and installed on MC-50 at the Korea Institute of Radiological and Medical Science (KIRAMS) with a 45-MeV proton beam. The laminated magnetic yoke of the scanning magnet supported fast ramping. The beam intensity and the beam profile monitors were designed for measuring the beam's properties. Both the range shifter and the ridge filter modulate the incoming beam energy. The LabVIEW®-based beam-irradiation-control program operates the system in a sequential operation manner for use with the MC-50 cyclotron. In addition, an in-housecoded irradiation-planning program generates an optimal irradiation path. A scanning experiment was successfully completed to print the logo of the Korea Heavy Ion Medical Accelerator (KHIMA) on GaF film. Moreover, the beam's position accuracy was measured as 0.62 mm in the x-direction and as 0.83 mm in the y-direction.

  3. Effect of NaCl Salts on the Activation Energy of Excited-State Proton Transfer Reaction of Coumarin 183.

    PubMed

    Joung, Joonyoung F; Kim, Sangin; Park, Sungnam

    2015-12-17

    Coumarin 183 (C183) was used as a photoacid to study excited-state proton transfer (ESPT) reactions. Here, we studied the effect of ions on the ESPT of C183 in aqueous NaCl solutions using a steady-state fluorescence spectroscopy and time-correlated single photon counting (TCSPC) method. The acid dissociation equilibrium of excited-state C183 and the activation energy for the ESPT of C183 were determined as a function of NaCl concentration. The change in the equilibrium constant was found to be correlated with the solvation energy of deprotonated C183. Frequency-resolved TCSPC signals measured at several temperatures were analyzed by using a global fitting analysis method which enabled us to extract all the rate constants involving the ESPT reaction and the spectra of individual species. The activation energy for the ESPT reaction of C183 was highly dependent on NaCl concentration. Quantum chemical calculations were used to calculate the local hydrogen-bond (H-bond) configurations around C183 in aqueous NaCl solutions. It was found that the activation energy for the ESPT was determined by the local H-bond configurations around C183 which were significantly influenced by the dissolved ions.

  4. Proton uptake and pKa changes in the uncoupled Asn139Cys variant of cytochrome c oxidase.

    PubMed

    Johansson, Ann-Louise; Carlsson, Jens; Högbom, Martin; Hosler, Jonathan P; Gennis, Robert B; Brzezinski, Peter

    2013-02-01

    Cytochrome c oxidase (CytcO) is a membrane-bound enzyme that links electron transfer from cytochrome c to O(2) to proton pumping across the membrane. Protons are transferred through specific pathways that connect the protein surface with the catalytic site as well as the proton input with the proton output sides. Results from earlier studies have shown that one site within the so-called D proton pathway, Asn139, located ~10 Å from the protein surface, is particularly sensitive to mutations that uncouple the O(2) reduction reaction from the proton pumping activity. For example, none of the Asn139Asp (charged) or Asn139Thr (neutral) mutant CytcOs pump protons, although the proton-uptake rates are unaffected. Here, we have investigated the Asn139Cys and Asn139Cys/Asp132Asn mutant CytcOs. In contrast to other structural variants investigated to date, the Cys side chain may be either neutral or negatively charged in the experimentally accessible pH range. The data show that the Asn139Cys and Asn139Asp mutations result in the same changes of the kinetic and thermodynamic parameters associated with the proton transfer. The similarity is not due to introduction of charge at position 139, but rather introduction of a protonatable group that modulates the proton connectivity around this position. These results illuminate the mechanism by which CytcO couples electron transfer to proton pumping.

  5. Motion mitigation for lung cancer patients treated with active scanning proton therapy

    SciTech Connect

    Grassberger, Clemens; Dowdell, Stephen; Sharp, Greg; Paganetti, Harald

    2015-05-15

    Purpose: Motion interplay can affect the tumor dose in scanned proton beam therapy. This study assesses the ability of rescanning and gating to mitigate interplay effects during lung treatments. Methods: The treatments of five lung cancer patients [48 Gy(RBE)/4fx] with varying tumor size (21.1–82.3 cm{sup 3}) and motion amplitude (2.9–30.6 mm) were simulated employing 4D Monte Carlo. The authors investigated two spot sizes (σ ∼ 12 and ∼3 mm), three rescanning techniques (layered, volumetric, breath-sampled volumetric) and respiratory gating with a 30% duty cycle. Results: For 4/5 patients, layered rescanning 6/2 times (for the small/large spot size) maintains equivalent uniform dose within the target >98% for a single fraction. Breath sampling the timing of rescanning is ∼2 times more effective than the same number of continuous rescans. Volumetric rescanning is sensitive to synchronization effects, which was observed in 3/5 patients, though not for layered rescanning. For the large spot size, rescanning compared favorably with gating in terms of time requirements, i.e., 2x-rescanning is on average a factor ∼2.6 faster than gating for this scenario. For the small spot size however, 6x-rescanning takes on average 65% longer compared to gating. Rescanning has no effect on normal lung V{sub 20} and mean lung dose (MLD), though it reduces the maximum lung dose by on average 6.9 ± 2.4/16.7 ± 12.2 Gy(RBE) for the large and small spot sizes, respectively. Gating leads to a similar reduction in maximum dose and additionally reduces V{sub 20} and MLD. Breath-sampled rescanning is most successful in reducing the maximum dose to the normal lung. Conclusions: Both rescanning (2–6 times, depending on the beam size) as well as gating was able to mitigate interplay effects in the target for 4/5 patients studied. Layered rescanning is superior to volumetric rescanning, as the latter suffers from synchronization effects in 3/5 patients studied. Gating minimizes the

  6. Motion mitigation for lung cancer patients treated with active scanning proton therapy

    PubMed Central

    Grassberger, Clemens; Dowdell, Stephen; Sharp, Greg; Paganetti, Harald

    2015-01-01

    Purpose: Motion interplay can affect the tumor dose in scanned proton beam therapy. This study assesses the ability of rescanning and gating to mitigate interplay effects during lung treatments. Methods: The treatments of five lung cancer patients [48 Gy(RBE)/4fx] with varying tumor size (21.1–82.3 cm3) and motion amplitude (2.9–30.6 mm) were simulated employing 4D Monte Carlo. The authors investigated two spot sizes (σ ∼ 12 and ∼3 mm), three rescanning techniques (layered, volumetric, breath-sampled volumetric) and respiratory gating with a 30% duty cycle. Results: For 4/5 patients, layered rescanning 6/2 times (for the small/large spot size) maintains equivalent uniform dose within the target >98% for a single fraction. Breath sampling the timing of rescanning is ∼2 times more effective than the same number of continuous rescans. Volumetric rescanning is sensitive to synchronization effects, which was observed in 3/5 patients, though not for layered rescanning. For the large spot size, rescanning compared favorably with gating in terms of time requirements, i.e., 2x-rescanning is on average a factor ∼2.6 faster than gating for this scenario. For the small spot size however, 6x-rescanning takes on average 65% longer compared to gating. Rescanning has no effect on normal lung V20 and mean lung dose (MLD), though it reduces the maximum lung dose by on average 6.9 ± 2.4/16.7 ± 12.2 Gy(RBE) for the large and small spot sizes, respectively. Gating leads to a similar reduction in maximum dose and additionally reduces V20 and MLD. Breath-sampled rescanning is most successful in reducing the maximum dose to the normal lung. Conclusions: Both rescanning (2–6 times, depending on the beam size) as well as gating was able to mitigate interplay effects in the target for 4/5 patients studied. Layered rescanning is superior to volumetric rescanning, as the latter suffers from synchronization effects in 3/5 patients studied. Gating minimizes the irradiated volume

  7. Proton radiography and tomography with application to proton therapy

    PubMed Central

    Allinson, N M; Evans, P M

    2015-01-01

    Proton radiography and tomography have long promised benefit for proton therapy. Their first suggestion was in the early 1960s and the first published proton radiographs and CT images appeared in the late 1960s and 1970s, respectively. More than just providing anatomical images, proton transmission imaging provides the potential for the more accurate estimation of stopping-power ratio inside a patient and hence improved treatment planning and verification. With the recent explosion in growth of clinical proton therapy facilities, the time is perhaps ripe for the imaging modality to come to the fore. Yet many technical challenges remain to be solved before proton CT scanners become commonplace in the clinic. Research and development in this field is currently more active than at any time with several prototype designs emerging. This review introduces the principles of proton radiography and tomography, their historical developments, the raft of modern prototype systems and the primary design issues. PMID:26043157

  8. Proton transfer pathways in Photosystem II

    NASA Astrophysics Data System (ADS)

    Ishikita, Hiroshi

    2014-03-01

    Using quantum mechanics/molecular mechanics calculations and the 1.9-Å crystal structure of Photosystem II (Umena, Y., Kawakami, K., Shen, J.-R., and Kamiya, N. (2011) Nature 473, 55-60), we investigated the H-bonding environment of the redox active tyrosine, TyrD and obtained insights that help explain its slow redox kinetics and the stability of TyrD radical. The water molecule distal to TyrD, 4 Å away from the phenolic O of TyrD (OTyrD) , corresponds to the presence of the tyrosyl radical state. The water molecule proximal to TyrD, in H-bonding distance to OTyrD, corresponds to the presence of the unoxidised tyrosine. The H+ released upon oxidation of TyrD is transferred to the proximal water, which shifts to the distal position, triggering a concerted proton transfer pathway involving D2-Arg180 and a series of waters, through which the proton reaches the aqueous phase at D2-His61. The water movement linked to the ejection of the proton from the hydrophobic environment near TyrD makes oxidation slow and quasi-irreversible, explaining the great stability of the TyrD radical. A symmetry-related proton pathway associated with TyrZ is pointed out and this is associated with one of the Cl- sites. This may represent a proton pathway functional in the water oxidation cycle.

  9. Potential protonation sites in the Al2SiO5 polymorphs based on polarized FTIR spectroscopy and properties of the electron density distribution

    SciTech Connect

    Burt, Jason B.; Ross, Nancy L.; Gibbs, Gerald V.; Rossman, George R.; Rosso, Kevin M.

    2007-07-01

    Potential protonation sites within the three aluminosilicate polymorphs, kyanite, sillimanite, and andalusite, have been examined through analysis of (3,-3) bond critical point information in comparison with polarized FTIR spectroscopy of single crystals of kyanite and sillimanite from previous studies and examination with the polarized infrared spectrum of andalusite from this study. Seven peaks were observed, four strong peaks at 3440 cm-1, 3460 cm-1, 3530 cm-1, and 3600 cm-1 and three weak peaks at 3480 cm-1, 3520 cm-1, and 3650 cm-1, when the electric vector is parallel with a, six peaks, three strong at 3440 cm-1, 3460 cm-1, and 3530 cm-1 and three weak peaks at 3480 cm-1, 3520 cm-1, and 3560 cm-1, when the electric vector is parallel with b, and no peaks when the electric vector is parallel with c. The results indicate that hydrogen is located in the (001) plane of andalusite and sillimanite and the (11-1) plane in kyanite as determined from the polarized FTIR spectrum of the three minerals. The concentration of water in the samples of andalusite examined varied between 10 and 15 ppm H20 by weight. Examination of the (3,-3) critical points in comparison with the polarized FTIR indicates that hydrogen will prefer bonding to the O1 and O2 oxygen atoms in andalusite and the O2 and O4 oxygens in sillimanite, which correspond to the oxygen with the highest Laplacian value and the underbonded oxygen in the two structures. In kyanite, comparison of the FTIR spectrum and the bond critical points indicate that hydrogen will bond to the two four-coordinated oxygens, O4 and O6.

  10. Proton Transport

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; DeVincenzi, Donald L. (Technical Monitor)

    2001-01-01

    The transport of protons across membranes is an essential process for both bioenergetics of modern cells and the origins of cellular life. All living systems make use of proton gradients across cell walls to convert environmental energy into a high-energy chemical compound, adenosine triphosphate (ATP), synthesized from adenosine diphosphate. ATP, in turn, is used as a source of energy to drive many cellular reactions. The ubiquity of this process in biology suggests that even the earliest cellular systems were relying on proton gradient for harvesting environmental energy needed to support their survival and growth. In contemporary cells, proton transfer is assisted by large, complex proteins embedded in membranes. The issue addressed in this Study was: how the same process can be accomplished with the aid of similar but much simpler molecules that could have existed in the protobiological milieu? The model system used in the study contained a bilayer membrane made of phospholipid, dimyristoylphosphatidylcholine (DMPC) which is a good model of the biological membranes forming cellular boundaries. Both sides of the bilayer were surrounded by water which simulated the environment inside and outside the cell. Embedded in the membrane was a fragment of the Influenza-A M$_2$ protein and enough sodium counterions to maintain system neutrality. This protein has been shown to exhibit remarkably high rates of proton transport and, therefore, is an excellent model to study the formation of proton gradients across membranes. The Influenza M$_2$ protein is 97 amino acids in length, but a fragment 25 amino acids long. which contains a transmembrane domain of 19 amino acids flanked by three amino acids on each side. is sufficient to transport protons. Four identical protein fragments, each folded into a helix, aggregate to form small channels spanning the membrane. Protons are conducted through a narrow pore in the middle of the channel in response to applied voltage. This

  11. Changing hydration level in an internal cavity modulates the proton affinity of a key glutamate in cytochrome c oxidase.

    PubMed

    Goyal, Puja; Lu, Jianxun; Yang, Shuo; Gunner, M R; Cui, Qiang

    2013-11-19

    Cytochrome c oxidase contributes to the transmembrane proton gradient by removing two protons from the high-pH side of the membrane each time the binuclear center active site is reduced. One proton goes to the binuclear center, whereas the other is pumped to the low-pH periplasmic space. Glutamate 286 (Glu286) has been proposed to serve as a transiently deprotonated proton donor. Using unrestrained atomistic molecular dynamics simulations, we show that the size of and water distribution in the hydrophobic cavity that holds Glu286 is controlled by the protonation state of the propionic acid of heme a3, a group on the proton outlet pathway. Protonation of the propionate disrupts hydrogen bonding to two side chains, allowing a loop to swing open. Continuum electrostatics and atomistic free-energy perturbation calculations show that the resultant changes in hydration and electrostatic interactions lower the Glu proton affinity by at least 5 kcal/mol. These changes in the internal hydration level occur in the absence of major conformational transitions and serve to stabilize needed transient intermediates in proton transport. The trigger is not the protonation of the Glu of interest, but rather the protonation of a residue ∼10 Å away. Thus, unlike local water penetration to stabilize a new charge, this finding represents a specific role for water molecules in the protein interior, mediating proton transfers and facilitating ion transport.

  12. Proton Inventory and Dynamics in the Nia-S to Nia-C Transition of a [NiFe] Hydrogenase.

    PubMed

    Greene, Brandon L; Wu, Chang-Hao; Vansuch, Gregory E; Adams, Michael W W; Dyer, R Brian

    2016-03-29

    Hydrogenases (H2ases) represent one of the most striking examples of biological proton-coupled electron transfer (PCET) chemistry, functioning in facile proton reduction and H2 oxidation involving long-range proton and electron transport. Spectroscopic and electrochemical studies of the [NiFe] H2ases have identified several catalytic intermediates, but the details of their interconversion are still a matter of debate. Here we use steady state and time-resolved infrared spectroscopy, sensitive to the CO ligand of the active site iron, as a probe of the proton inventory as well as electron and proton transfer dynamics in the soluble hydrogenase I from Pyrococcus furiosus. Subtle shifts in infrared signatures associated with the Nia-C and Nia-S states as a function of pH revealed an acid-base equilibrium associated with an ionizable amino acid near the active site. Protonation of this residue was found to correlate with the photoproduct distribution that results from hydride photolysis of the Nia-C state, in which one of the two photoproduct states becomes inaccessible at low pH. Additionally, the ability to generate Nia-S via PCET from Nia-C was weakened at low pH, suggesting prior protonation of the proton acceptor. Kinetic and thermodynamic analysis of electron and proton transfer with respect to the various proton inventories was utilized to develop a chemical model for reversible hydride oxidation involving two intermediates differing in their hydrogen bonding character.

  13. Reversible inhibition of proton release activity and the anesthetic-induced acid-base equilibrium between the 480 and 570 nm forms of bacteriorhodopsin.

    PubMed Central

    Boucher, F; Taneva, S G; Elouatik, S; Déry, M; Messaoudi, S; Harvey-Girard, E; Beaudoin, N

    1996-01-01

    In purple membrane added with general anesthetics, there exists an acid-base equilibrium between two spectral forms of the pigment: bR570 and bR480 (apparent pKa = 7.3). As the purple 570 nm bacteriorhodopsin is reversibly transformed into its red 480 nm form, the proton pumping capability of the pigment reversibly decreases, as indicated by transient proton release measurements and proton translocation action spectra of mixture of both spectral forms. It happens in spite of a complete photochemical activity in bR480 that is mostly characterized by fast deprotonation and slow reprotonation steps and which, under continuous illumination, bleaches with a yield comparable to that of bR570. This modified photochemical activity has a correlated specific photoelectrical counterpart: a faster proton extrusion current and a slower reprotonation current. The relative areas of all photocurrent phases are reduced in bR480, most likely because its photochemistry is accompanied by charge movements for shorter distances than in the native pigment, reflecting a reversible inhibition of the pumping activity. PMID:8789112

  14. Proton exchange membrane fuel cell model for aging predictions: Simulated equivalent active surface area loss and comparisons with durability tests

    NASA Astrophysics Data System (ADS)

    Robin, C.; Gérard, M.; Quinaud, M.; d'Arbigny, J.; Bultel, Y.

    2016-09-01

    The prediction of Proton Exchange Membrane Fuel Cell (PEMFC) lifetime is one of the major challenges to optimize both material properties and dynamic control of the fuel cell system. In this study, by a multiscale modeling approach, a mechanistic catalyst dissolution model is coupled to a dynamic PEMFC cell model to predict the performance loss of the PEMFC. Results are compared to two 2000-h experimental aging tests. More precisely, an original approach is introduced to estimate the loss of an equivalent active surface area during an aging test. Indeed, when the computed Electrochemical Catalyst Surface Area profile is fitted on the experimental measures from Cyclic Voltammetry, the computed performance loss of the PEMFC is underestimated. To be able to predict the performance loss measured by polarization curves during the aging test, an equivalent active surface area is obtained by a model inversion. This methodology enables to successfully find back the experimental cell voltage decay during time. The model parameters are fitted from the polarization curves so that they include the global degradation. Moreover, the model captures the aging heterogeneities along the surface of the cell observed experimentally. Finally, a second 2000-h durability test in dynamic operating conditions validates the approach.

  15. Mutation at a Strictly Conserved, Active Site Tyrosine in the Copper Amine Oxidase Leads to Uncontrolled Oxygenase Activity

    SciTech Connect

    Chen, Zhi-wei; Datta, Saumen; DuBois, Jennifer L.; Klinman, Judith P.; Mathews, F. Scott

    2010-09-07

    The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ) and the subsequent oxidative deamination of substrate amines. Because the same active site pocket must facilitate both reactions, individual active site residues may serve multiple roles. We have examined the roles of a strictly conserved active site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically (Dubois and Klinman (2006) Biochemistry 45, 3178), and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy intermediates in cofactor biogenesis but also indicates the critical control of oxidation chemistry that can be conferred by a single active site residue.

  16. Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations

    PubMed Central

    Steinkellner, Georg; Gruber, Christian C.; Pavkov-Keller, Tea; Binter, Alexandra; Steiner, Kerstin; Winkler, Christoph; Łyskowski, Andrzej; Schwamberger, Orsolya; Oberer, Monika; Schwab, Helmut; Faber, Kurt; Macheroux, Peter; Gruber, Karl

    2014-01-01

    The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts. PMID:24954722

  17. NMR Observation of Mobile Protons in Proton-Implanted ZnO Nanorods.

    PubMed

    Park, Jun Kue; Kwon, Hyeok-Jung; Lee, Cheol Eui

    2016-03-18

    The diffusion properties of H(+) in ZnO nanorods are investigated before and after 20 MeV proton beam irradiation by using (1)H nuclear magnetic resonance (NMR) spectroscopy. Herein, we unambiguously observe that the implanted protons occupy thermally unstable site of ZnO, giving rise to a narrow NMR line at 4.1 ppm. The activation barrier of the implanted protons was found to be 0.46 eV by means of the rotating-frame spin-lattice relaxation measurements, apparently being interstitial hydrogens. High-energy beam irradiation also leads to correlated jump diffusion of the surface hydroxyl group of multiple lines at ~1 ppm, implying the presence of structural disorder at the ZnO surface.

  18. NMR Observation of Mobile Protons in Proton-Implanted ZnO Nanorods

    PubMed Central

    Park, Jun Kue; Kwon, Hyeok-Jung; Lee, Cheol Eui

    2016-01-01

    The diffusion properties of H+ in ZnO nanorods are investigated before and after 20 MeV proton beam irradiation by using 1H nuclear magnetic resonance (NMR) spectroscopy. Herein, we unambiguously observe that the implanted protons occupy thermally unstable site of ZnO, giving rise to a narrow NMR line at 4.1 ppm. The activation barrier of the implanted protons was found to be 0.46 eV by means of the rotating-frame spin-lattice relaxation measurements, apparently being interstitial hydrogens. High-energy beam irradiation also leads to correlated jump diffusion of the surface hydroxyl group of multiple lines at ~1 ppm, implying the presence of structural disorder at the ZnO surface. PMID:26988733

  19. NMR Observation of Mobile Protons in Proton-Implanted ZnO Nanorods.

    PubMed

    Park, Jun Kue; Kwon, Hyeok-Jung; Lee, Cheol Eui

    2016-01-01

    The diffusion properties of H(+) in ZnO nanorods are investigated before and after 20 MeV proton beam irradiation by using (1)H nuclear magnetic resonance (NMR) spectroscopy. Herein, we unambiguously observe that the implanted protons occupy thermally unstable site of ZnO, giving rise to a narrow NMR line at 4.1 ppm. The activation barrier of the implanted protons was found to be 0.46 eV by means of the rotating-frame spin-lattice relaxation measurements, apparently being interstitial hydrogens. High-energy beam irradiation also leads to correlated jump diffusion of the surface hydroxyl group of multiple lines at ~1 ppm, implying the presence of structural disorder at the ZnO surface. PMID:26988733

  20. Proton diffusion in the hexafluorophosphoric acid clathrate hydrate.

    PubMed

    Bedouret, Laura; Judeinstein, Patrick; Ollivier, Jacques; Combet, Jérôme; Desmedt, Arnaud

    2014-11-26

    The hexafluorophosphoric acid clathrate hydrate is known as a "super-protonic" conductor: its proton conductivity is of the order of 0.1 S/cm at ca. room temperature. The long-range proton diffusion and the associated mechanism have been analyzed with the help of incoherent quasi-elastic neutron scattering (QENS) and proton pulsed-field-gradient nuclear magnetic resonance ((1)H PFG-NMR). The system crystallizes into the so-called type I clathrate structure (SI) at low temperature and into the type VII structure (SVII) above ca. 230 K with a melting point close to room temperature. While, in the SI phase, no long-range proton diffusion is observed (at least faster than the present measurement capabilities, i.e., 10(-7) cm(2)·s(-1)) with respect to the probed time scale, both techniques evidence a long-range proton diffusion process in the SVII phase (3.85 × 10(-6) cm(2)·s(-1) at 275 K with an activation energy of 0.19 ± 0.04 eV). QENS experiments lead to modeling the microscopic mechanism of the long-range proton diffusion by means of a Chudley-Elliot jump diffusion model with a characteristic jump distance of 2.79 ± 0.17 Å. In other words, the long-range diffusion occurs through a Grotthus mechanism with proton jumping from one water-oxygen site to another. Moreover, the analysis of the proton diffusion for hydration numbers greater than 6 (i.e., in the SVII structure) reveals that the additional water molecules coexisting with the SVII structure act as a "structural defect" barrier for the proton diffusivity, responsible for the conductivity. PMID:24941122

  1. Using Proton Magnetic Resonance Imaging and Spectroscopy to Understand Brain "Activation"

    ERIC Educational Resources Information Center

    Baslow, Morris H.; Guilfoyle, David N.

    2007-01-01

    Upon stimulation, areas of the brain associated with specific cognitive processing tasks may undergo observable physiological changes, and measures of such changes have been used to create brain maps for visualization of stimulated areas in task-related brain "activation" studies. These perturbations usually continue throughout the period of the…

  2. An ionizable active-site tryptophan imparts catalase activity to a peroxidase core.

    PubMed

    Loewen, Peter C; Carpena, Xavi; Vidossich, Pietro; Fita, Ignacio; Rovira, Carme

    2014-05-21

    Catalase peroxidases (KatG's) are bifunctional heme proteins that can disproportionate hydrogen peroxide (catalatic reaction) despite their structural dissimilarity with monofunctional catalases. Using X-ray crystallography and QM/MM calculations, we demonstrate that the catalatic reaction of KatG's involves deprotonation of the active-site Trp, which plays a role similar to that of the distal His in monofunctional catalases. The interaction of a nearby mobile arginine with the distal Met-Tyr-Trp essential adduct (in/out) acts as an electronic switch, triggering deprotonation of the adduct Trp.

  3. Bromopyruvate, an active site-directed inactivator of E. coli 2-keto-4-hydroxyglutarate(KHG) aldolase, modifies glutamic acid residue-45

    SciTech Connect

    Vlahos, C.J.; Dekker, E.E.

    1987-05-01

    E. coli KHG-aldolase (2-keto-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate), a novel trimeric Class I aldolase, requires one active-site lysine residue (Lys 133)/subunit for Schiff-base formation as well as one arginine residue (Arg 49)/subunit for catalytic activity. The substrate analog, 3-bromopyruvate (BRPY), causes a time- and concentration-dependent loss of KHG-aldolase activity. This inactivation is regarded as active site-directed since: (a) BRPY modification results in complete loss of enzymatic activity; (b) saturation kinetics are exhibited, suggesting that a reversible complex is formed between the aldolase and BRPY prior to the rate-limiting inactivation step; (c) over 90% of the initial aldolase activity is protected by either substrate, pyruvate or KHG; (d) 1.1 mol of /sup 14/C-BRPY is bound/enzyme subunit. Peptide isolation and sequencing show that the incorporated radioactivity is associated with residue Glu-45. Denaturation of the enzyme with guanidine x HCl following treatment with excess /sup 14/C-BRPY allows for the incorporation of carbon-14 at Cys-159 and Cys-180 as well. The presence of pyruvate protects Glu-45 from being esterified but does not prevent the alkylation of the two cysteine residues. These results suggest that Glu-45 is essential for the catalytic activity of E. coli KHG-aldolase, most likely functioning as the active-site amphoteric proton donor/acceptor moiety that is involved in the overall mechanism of the reaction catalyzed by this enzyme.

  4. Beta-D-xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-Directed Mutagenesis, Alternate Substrates, and Active-site Inhibitor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium (SXA) is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D xylose. Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic ...

  5. Nuclear Site Security in the Event of Terrorist Activity

    SciTech Connect

    Thomson, M.L.; Sims, J.

    2008-07-01

    This paper, presented as a poster, identifies why ballistic protection should now be considered at nuclear sites to counter terrorist threats. A proven and flexible form of multi purpose protection is described in detail with identification of trial results that show its suitability for this role. (authors)

  6. Measurement of stray radiation within a scanning proton therapy facility: EURADOS WG9 intercomparison exercise of active dosimetry systems

    SciTech Connect

    Farah, J. Trompier, F.; Mares, V.; Schinner, K.; Wielunski, M.; Romero-Expósito, M.; Domingo, C.; Trinkl, S.; Dufek, V.; Klodowska, M.; Liszka, M.; Stolarczyk, L.; Olko, P.; Kubancak, J.; and others

    2015-05-15

    Purpose: To characterize stray radiation around the target volume in scanning proton therapy and study the performance of active neutron monitors. Methods: Working Group 9 of the European Radiation Dosimetry Group (EURADOS WG9—Radiation protection in medicine) carried out a large measurement campaign at the Trento Centro di Protonterapia (Trento, Italy) in order to determine the neutron spectra near the patient using two extended-range Bonner sphere spectrometry (BSS) systems. In addition, the work focused on acknowledging the performance of different commercial active dosimetry systems when measuring neutron ambient dose equivalents, H{sup ∗}(10), at several positions inside (8 positions) and outside (3 positions) the treatment room. Detectors included three TEPCs—tissue equivalent proportional counters (Hawk type from Far West Technology, Inc.) and six rem-counters (WENDI-II, LB 6411, RadEye™ NL, a regular and an extended-range NM2B). Meanwhile, the photon component of stray radiation was deduced from the low-lineal energy transfer part of TEPC spectra or measured using a Thermo Scientific™ FH-40G survey meter. Experiments involved a water tank phantom (60 × 30 × 30 cm{sup 3}) representing the patient that was uniformly irradiated using a 3 mm spot diameter proton pencil beam with 10 cm modulation width, 19.95 cm distal beam range, and 10 × 10 cm{sup 2} field size. Results: Neutron spectrometry around the target volume showed two main components at the thermal and fast energy ranges. The study also revealed the large dependence of the energy distribution of neutrons, and consequently of out-of-field doses, on the primary beam direction (directional emission of intranuclear cascade neutrons) and energy (spectral composition of secondary neutrons). In addition, neutron mapping within the facility was conducted and showed the highest H{sup ∗}(10) value of ∼51 μSv Gy{sup −1}; this was measured at 1.15 m along the beam axis. H{sup ∗}(10) values

  7. Preliminary siting activities for new waste handling facilities at the Idaho National Engineering Laboratory

    SciTech Connect

    Taylor, D.D.; Hoskinson, R.L.; Kingsford, C.O.; Ball, L.W.

    1994-09-01

    The Idaho Waste Processing Facility, the Mixed and Low-Level Waste Treatment Facility, and the Mixed and Low-Level Waste Disposal Facility are new waste treatment, storage, and disposal facilities that have been proposed at the Idaho National Engineering Laboratory (INEL). A prime consideration in planning for such facilities is the selection of a site. Since spring of 1992, waste management personnel at the INEL have been involved in activities directed to this end. These activities have resulted in the (a) identification of generic siting criteria, considered applicable to either treatment or disposal facilities for the purpose of preliminary site evaluations and comparisons, (b) selection of six candidate locations for siting,and (c) site-specific characterization of candidate sites relative to selected siting criteria. This report describes the information gathered in the above three categories for the six candidate sites. However, a single, preferred site has not yet been identified. Such a determination requires an overall, composite ranking of the candidate sites, which accounts for the fact that the sites under consideration have different advantages and disadvantages, that no single site is superior to all the others in all the siting criteria, and that the criteria should be assigned different weighing factors depending on whether a site is to host a treatment or a disposal facility. Stakeholder input should now be solicited to help guide the final selection. This input will include (a) siting issues not already identified in the siting, work to date, and (b) relative importances of the individual siting criteria. Final site selection will not be completed until stakeholder input (from the State of Idaho, regulatory agencies, the public, etc.) in the above areas has been obtained and a strategy has been developed to make a composite ranking of all candidate sites that accounts for all the siting criteria.

  8. V-ATPase Proton Pumping Activity Is Required for Adult Zebrafish Appendage Regeneration

    PubMed Central

    Monteiro, Joana; Aires, Rita; Becker, Jörg D.; Jacinto, António; Certal, Ana C.; Rodríguez-León, Joaquín

    2014-01-01

    The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration. PMID:24671205

  9. V-ATPase proton pumping activity is required for adult zebrafish appendage regeneration.

    PubMed

    Monteiro, Joana; Aires, Rita; Becker, Jörg D; Jacinto, António; Certal, Ana C; Rodríguez-León, Joaquín

    2014-01-01

    The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration.

  10. Investigation of activation cross-section data of proton induced nuclear reactions on rhenium.

    PubMed

    Ditrói, F; Tárkányi, F; Takács, S; Hermanne, A; Yamazaki, H; Baba, M; Mohammadi, A; Ignatyuk, A V

    2013-07-01

    In the frame of systematic investigations of activation cross-section data for different applications the excitation functions of (nat)Re(p,x)(185)Os, (183m)Os, (183g)Os, (182)Os, (181m)Os, (186g)Re, (184m)Re, (184g)Re, (183)Re, (182m)Re, (182g)Re and (181g)Re reactions were measured up to 70MeV. The data for the (nat)Re(p,x) (183m)Os, (183g)Os, (182)Os, (181g)Os,(186g)Re, (184m)Re,(182m)Re, (182g)Re, and (181)Re reactions are reported for the first time. The activation method, the stacked foil irradiation technique and γ-spectroscopy for activity detection were used. The experimental data were compared with predictions of three theoretical codes. From the measured cross-section thick target integral yields were also calculated and presented.

  11. Active Layer and Moisture Measurements for Intensive Site 0 and 1, Barrow, Alaska

    DOE Data Explorer

    John Peterson

    2015-04-17

    These are measurements of Active Layer Thickness collected along several lines beginning in September, 2011 to the present. The data were collected at several time periods along the Site0 L2 Line, the Site1 AB Line, and an ERT Monitoring Line near Area A in Site1.

  12. WE-D-17A-05: Measurement of Stray Radiation Within An Active Scanning Proton Therapy Facility: EURADOS WG9 Intercomparison Exercise of Active Dosimetry Systems

    SciTech Connect

    Farah, J; Trompier, F; Stolarczyk, L; Klodowska, M; Liszka, M; Olko, P; Algranati, C; Fellin, F; Schwarz, M; Domingo, C; Romero-Exposito, M; Dufek, V; Frojdh, E; George, S; Harrison, R; Kubancak, J; Ploc, O; Knezevic, Z; Majer, M; Miljanic, S; and others

    2014-06-15

    Purpose: Intercomparison of active dosemeters in the measurement of stray radiation at the Trento active-scanning proton therapy facility. Methods: EURADOS WG9 carried out a large intercomparison exercise to test different dosemeters while measuring secondary neutrons within a 230 MeV scanned proton therapy facility. Detectors included two Bonner Sphere Spectrometers (BSS), three tissue equivalent proportional counters (TEPCHawk) and six rem-counters (Wendi II, Berthold, RadEye, a regular and an extended-range Anderson and Braun NM2B counters). Measurements of neutron ambient dose equivalents, H*(10), were done at several positions inside (8 positions) and outside (3 positions) the treatment room while irradiating a water tank phantom with a 10 × 10 × 10 cc field. Results: A generally good agreement on H*(10) values was observed for the tested detectors. At distance of 2.25 m and angles 45°, 90° and 180° with respect to the beam axis, BSS and proportional counters agreed within 30%. Higher differences (up to 60%) were observed at the closest and farthest distances, i.e. at positions where detectors sensitivity, energy, fluence and angular response are highly dependent on neutron spectra (flux and energy). The highest neutron H*(10) value, ∼60 microSv/Gy, was measured at 1.15 m along the beam axis. H*(10) decreased significantly with the distance from the isocenter dropping to 1.1 microSv/Gy at 4.25 m and 90° from beam axis, ∼2 nanoSv/Gy at the entrance of the maze, 0.2 nanoSv/Gy at the door outside the room and below detection limit in the gantry control room and at an adjacent room. These values remain considerately lower than those of passively scattered proton beams. BSS and Hawk unfolded spectra provide valuable inputs when studying the response of each detector. Conclusion: TEPCs and BSS enable accurate measurements of stray neutrons while other rem-meters also give satisfactory results but require further improvements to reduce uncertainties.

  13. Structural mechanism of RuBisCO activation by carbamylation of the active site lysine

    PubMed Central

    Stec, Boguslaw

    2012-01-01

    Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a crucial enzyme in carbon fixation and the most abundant protein on earth. It has been studied extensively by biochemical and structural methods; however, the most essential activation step has not yet been described. Here, we describe the mechanistic details of Lys carbamylation that leads to RuBisCO activation by atmospheric CO2. We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O2 and CO2 bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO2 defines an elusive, preactivation complex that contains a metal cation Mg2+ surrounded by three H2O/OH molecules. Both structures suggest the mechanism for discriminating gaseous ligands by their quadrupole electric moments. We describe conformational changes that allow for intermittent binding of the metal ion required for activation. On the basis of these structures we propose the individual steps of the activation mechanism. Knowledge of all these elements is indispensable for engineering RuBisCO into a more efficient enzyme for crop enhancement or as a remedy to global warming. PMID:23112176

  14. Using catalytic atom maps to predict the catalytic functions present in enzyme active sites.

    PubMed

    Nosrati, Geoffrey R; Houk, K N

    2012-09-18

    Catalytic atom maps (CAMs) are minimal models of enzyme active sites. The structures in the Protein Data Bank (PDB) were examined to determine if proteins with CAM-like geometries in their active sites all share the same catalytic function. We combined the CAM-based search protocol with a filter based on the weighted contact number (WCN) of the catalytic residues, a measure of the "crowdedness" of the microenvironment around a protein residue. Using this technique, a CAM based on the Ser-His-Asp catalytic triad of trypsin was able to correctly identify catalytic triads in other enzymes within 0.5 Å rmsd of the CAM with 96% accuracy. A CAM based on the Cys-Arg-(Asp/Glu) active site residues from the tyrosine phosphatase active site achieved 89% accuracy in identifying this type of catalytic functionality. Both of these CAMs were able to identify active sites across different fold types. Finally, the PDB was searched to locate proteins with catalytic functionality similar to that present in the active site of orotidine 5'-monophosphate decarboxylase (ODCase), whose mechanism is not known with certainty. A CAM, based on the conserved Lys-Asp-Lys-Asp tetrad in the ODCase active site, was used to search the PDB for enzymes with similar active sites. The ODCase active site has a geometry similar to that of Schiff base-forming Class I aldolases, with lowest aldolase rmsd to the ODCase CAM at 0.48 Å. The similarity between this CAM and the aldolase active site suggests that ODCase has the correct catalytic functionality present in its active site for the generation of a nucleophilic lysine. PMID:22909276

  15. Using Catalytic Atom Maps to Predict the Catalytic Functions Present in Enzyme Active Sites

    PubMed Central

    Nosrati, Geoffrey R.; Houk, K. N.

    2012-01-01

    Catalytic Atom Maps (CAMs) are minimal models of enzyme active sites. The structures in the Protein Data Bank (PDB) were examined to determine if proteins with CAM-like geometries in their active sites all share the same catalytic function. We combined the CAM-based search protocol with a filter based on the weighted contact number (WCN) of the catalytic residues, a measure of the “crowdedness” of the microenvironment around a protein residue. Using this technique, a CAM based on the Ser-His-Asp catalytic triad of trypsin was able to correctly identify catalytic triads in other enzymes within 0.5 Å RMSD of the Catalytic Atom Map with 96% accuracy. A CAM based on the Cys-Arg-(Asp/Glu) active site residues from the tyrosine phosphatase active site achieved 89% accuracy in identifying this type of catalytic functionality. Both of these Catalytic Atom Maps were able to identify active sites across different fold types. Finally, the PDB was searched to locate proteins with catalytic functionality similar to that present in the active site of orotidine 5′-monophosphate decarboxylase (ODCase), whose mechanism is not known with certainty. A CAM, based on the conserved Lys-Asp-Lys-Asp tetrad in the ODCase active site, was used to search the PDB for enzymes with similar active sites. The ODCase active site has a geometry similar to that of Schiff base-forming Class I aldolases, with lowest aldolase RMSD to the ODCase CAM at 0.48 Å. The similarity between this CAM and the aldolase active site suggests that ODCase has the correct catalytic functionality present in its active site for the generation of a nucleophilic lysine. PMID:22909276

  16. A highly active anode functional layer for solid oxide fuel cells based on proton-conducting electrolyte BaZr0.1Ce0.7Y0.2O3-δ

    NASA Astrophysics Data System (ADS)

    Zhang, Xiuling; Qiu, Yu'e.; Jin, Feng; Guo, Feng; Song, Yulan; Zhu, Baoyong

    2013-11-01

    Extensive works have been performed to diminish cathode polarization for proton-conducting electrolyte based solid oxide fuel cells (SOFCs) while not much attention is paid to functional anode for improving electrochemical reaction at three-phase-boundaries (TPB). In this work, a highly active anode functional layer (FL) synthesized by a modified combustion method was employed to significantly elevate the cell performance at intermediate operation temperatures (550-650 °C). The effects of anode structure configuration, FL powder size and thickness on power outputs and electrode polarization were investigated. A maximum power density of 489 mW cm-2 and a low electrode polarization resistance of 0.37 Ω cm2 were achieved at 650 °C, indicating fuel gas transport and hydrogen oxidation reaction at TPB sites largely contribute to total cell resistance which could be effectively diminished by optimization of anodic interface environment with the adoption of highly active anode powders.

  17. Generation of proton aurora by magnetosonic waves

    PubMed Central

    Xiao, Fuliang; Zong, Qiugang; Wang, Yongfu; He, Zhaoguo; Su, Zhenpeng; Yang, Chang; Zhou, Qinghua

    2014-01-01

    Earth's proton aurora occurs over a broad MLT region and is produced by the precipitation of low-energy (2–10 keV) plasmasheet protons. Proton precipitation can alter chemical compositions of the atmosphere, linking solar activity with global climate variability. Previous studies proposed that electromagnetic ion cyclotron waves can resonate with protons, producing proton scattering precipitation. A long-outstanding question still remains whether there is another mechanism responsible for the proton aurora. Here, by performing satellite data analysis and diffusion equation calculations, we show that fast magnetosonic waves can produce trapped proton scattering that yields proton aurora. This provides a new insight into the mechanism of proton aurora. Furthermore, a ray-tracing study demonstrates that magnetosonic wave propagates over a broad MLT region, consistent with the global distribution of proton aurora. PMID:24898626

  18. Activation caused by proton beam losses in Accelerator Production of Tritium LINAC

    SciTech Connect

    Beard, C.A.; Eaton, S.L.; Daemen, L.L.; Waters, L.S.; Wilson, W.B.

    1996-03-01

    A variety of accelerator designs are being considered for the Accelerator Production of Tritium (APT) project at Alamos National Laboratory. Because activation of the structural components of the accelerator is considered a major radiation protection issue, we have developed a computational methodology to estimate quantitatively radionuclide inventories and gamma dose rates resulting from accelerator operation. The work presented here illustrates the use of our computational methodology by focusing on the 20 and 100 MeV sections of the Bridge-Coupled Drift Tube LINAC (BCDTL), and the 100 and 1000 MeV sections of the Coupled Cavity LINAC (CCL).

  19. Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

    SciTech Connect

    Miao, Yinglong; Baudry, Jerome Y

    2011-01-01

    Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.

  20. Parameterization of an Active Thermal Erosion Site, Caribou Creek, Alaska

    NASA Astrophysics Data System (ADS)

    Busey, R.; Bolton, W. R.; Cherry, J. E.; Hinzman, L. D.

    2012-12-01

    Thermokarst features are thought to be an important mechanism for landscape change in permafrost-dominated cold regions, but few such features have been incorporated into full featured landscape models. The root of this shortcoming is that historic observations are not detailed enough to parameterize a model, and the models typically do not include the relevant processes for thermal erosion. A new, dynamic thermokarst feature has been identified at the Caribou-Poker Creek Research Watershed (CPCRW) in the boreal forest of Interior Alaska. Located adjacent to a traditional use trail, this feature terminates directly in Caribou Creek. Erosion within the feature is driven predominantly by fluvial interflow. CPCRW is a Long-Term Ecological Research site underlain by varying degrees of relatively warm, discontinuous permafrost. This poster will describe the suite of measurements that have been undertaken to parameterize the ERODE model for this site, including thorough surveys, time lapse- and aerial photography, and 3-D structure from motion algorithms.

  1. Study of supported bilayer lipid membranes for use in chemo-electric energy conversion via active proton transport

    NASA Astrophysics Data System (ADS)

    Sarles, Stephen A.; Sundaresan, Vishnu B.; Leo, Donald J.

    2007-09-01

    Bilayer lipid membranes (BLMs) have been studied extensively due to functional and structural similarities to cell membranes, fostering research to understand ion-channel protein functions, measure bilayer mechanical properties, and identify self-assembly mechanisms. BLMs have traditionally been formed across single pores in substrates such as PTFE (Teflon). The incorporation of ion-channel proteins into the lipid bilayer enables the selective transfer of ions and fluid through the BLM. Processes of this nature have led to the measurement of ion current flowing across the lipid membrane and have been used to develop sensors that signal the presence of a particular reactant (glucose, urea, penicillin), improve drug recognition in cells, and develop materials capable of creating chemical energy from light. Recent research at Virginia Tech has shown that the incorporation of proton transporters in a supported BLM formed across an array of pores can convert chemical energy available in the adenosine triphosphate (ATP) into electricity. Experimental results from this work show that the system-named Biocell-is capable of developing 2µW/cm2 of membrane area with 15μl of ATPase. Efforts to increase the power output and conversion efficiency of this process while moving toward a packaged device present a unique engineering problem. The bilayer, as host to the active proton transporters, must therefore be formed evenly across a porous substrate, remain stable and yet fluid-like for protein interaction, and exhibit a large seal resistance. This article presents the ongoing work to characterize the Biocell using impedance analysis. Electrical impedance spectroscopy (EIS) is used to study the effect of adding ATPase proteins to POPS:POPE bilayer lipid membranes and correlate structural changes evident in the impedance data to the energy-conversion capability of various partial and whole Biocell assemblies. The specific membrane resistance of a pure BLM drops from 40-120k

  2. Blogs and Social Network Sites as Activity Systems: Exploring Adult Informal Learning Process through Activity Theory Framework

    ERIC Educational Resources Information Center

    Heo, Gyeong Mi; Lee, Romee

    2013-01-01

    This paper uses an Activity Theory framework to explore adult user activities and informal learning processes as reflected in their blogs and social network sites (SNS). Using the assumption that a web-based space is an activity system in which learning occurs, typical features of the components were investigated and each activity system then…

  3. Solar neutrinos, solar flares, solar activity cycle and the proton decay

    NASA Technical Reports Server (NTRS)

    Raychaudhuri, P.

    1985-01-01

    It is shown that there may be a correlation between the galactic cosmic rays and the solar neutrino data, but it appears that the neutrino flux which may be generated during the large solar cosmic ray events cannot in any way effect the solar neutrino data in Davis experiment. Only initial stage of mixing between the solar core and solar outer layers after the sunspot maximum in the solar activity cycle can explain the higher (run number 27 and 71) of solar neutrino data in Davis experiment. But solar flare induced atmospheric neutrino flux may have effect in the nucleon decay detector on the underground. The neutrino flux from solar cosmic rays may be a useful guide to understand the background of nucleon decay, magnetic monopole search, and the detection of neutrino flux in sea water experiment.

  4. Zn{sup 2+} induces apoptosis in human highly metastatic SHG-44 glioma cells, through inhibiting activity of the voltage-gated proton channel Hv1

    SciTech Connect

    Wang, Yifan; Zhang, Shangrong; Li, Shu Jie

    2013-08-23

    Highlights: •Hv1 is expressed in highly metastatic glioma cell. •Zn{sup 2+} ions induces apoptosis in highly metastatic glioma cells. •Zn{sup 2+} ions markedly inhibit proton secretion. •Zn{sup 2+} ions reduce the gelatinase activity. •Inhibition of Hv1 activity via Zn{sup 2+} ions can effectively retard the cancer growth. -- Abstract: In contrast to the voltage-gated K{sup +} channels, the voltage-gated proton channel Hv1 contains a voltage-sensor domain but lacks a pore domain. Here, we showed that Hv1 is expressed in the highly metastatic glioma cell SHG-44, but lowly in the poorly metastatic glioma cell U-251. Inhibition of Hv1 activity by 140 μM zinc chloride induces apoptosis in the human highly metastatic glioma cells. Zn{sup 2+} ions markedly inhibit proton secretion, and reduce the gelatinase activity in the highly metastatic glioma cells. In vivo, the glioma tumor sizes of the implantation of the SHG-44 xenografts in nude mice that were injected zinc chloride solution, were dramatically smaller than that in the controlled groups. The results demonstrated that the inhibition of Hv1 activity via Zn{sup 2+} ions can effectively retard the cancer growth and suppress the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity. Our results suggest that Zn{sup 2+} ions may be used as a potential anti-glioma drug for glioma therapy.

  5. Early Site Permit Demonstration Program: Recommendations for communication activities and public participation in the Early Site Permit Demonstration Program

    SciTech Connect

    Not Available

    1993-01-27

    On October 24, 1992, President Bush signed into law the National Energy Policy Act of 1992. The bill is a sweeping, comprehensive overhaul of the Nation`s energy laws, the first in more than a decade. Among other provisions, the National Energy Policy Act reforms the licensing process for new nuclear power plants by adopting a new approach developed by the US Nuclear Regulatory Commission (NRC) in 1989, and upheld in court in 1992. The NRC 10 CFR Part 52 rule is a three-step process that guarantees public participation at each step. The steps are: early site permit approval; standard design certifications; and, combined construction/operating licenses for nuclear power reactors. Licensing reform increases an organization`s ability to respond to future baseload electricity generation needs with less financial risk for ratepayers and the organization. Costly delays can be avoided because design, safety and siting issues will be resolved before a company starts to build a plant. Specifically, early site permit approval allows for site suitability and acceptability issues to be addressed prior to an organization`s commitment to build a plant. Responsibility for site-specific activities, including communications and public participation, rests with those organizations selected to try out early site approval. This plan has been prepared to assist those companies (referred to as sponsoring organizations) in planning their communications and public involvement programs. It provides research findings, information and recommendations to be used by organizations as a resource and starting point in developing their own plans.

  6. Active Site Structure and Peroxidase Activity of Oxidatively Modified Cytochrome c Species in Complexes with Cardiolipin.

    PubMed

    Capdevila, Daiana A; Oviedo Rouco, Santiago; Tomasina, Florencia; Tortora, Verónica; Demicheli, Verónica; Radi, Rafael; Murgida, Daniel H

    2015-12-29

    We report a resonance Raman and UV-vis characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity with respect to those of the wild-type protein. In spite of the structural and activity differences between the protein variants free in solution, binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation. The spectroscopic results indicate that the bis-His form is in equilibrium with small amounts of high-spin species, thus suggesting a labile distal His ligand as the basis for the CL-induced increase in enzymatic activity observed for all protein variants. For Cyt-c nitrated at Tyr74 and sulfoxidized at Met80, the measured apparent binding affinities for CL are ∼4 times larger than for wild-type Cyt-c. On the basis of these results, we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein.

  7. Identification of ice nucleation active sites on feldspar dust particles.

    PubMed

    Zolles, Tobias; Burkart, Julia; Häusler, Thomas; Pummer, Bernhard; Hitzenberger, Regina; Grothe, Hinrich

    2015-03-19

    Mineral dusts originating from Earth's crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts. Here we investigated in closer detail the reasons for its activity and the difference in the activity of the different feldspars. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. K-feldspar showed by far the highest ice nucleation activity. Finally, we give a potential explanation of this effect, finding alkali-metal ions having different hydration shells and thus an influence on the ice nucleation activity of feldspar surfaces. PMID:25584435

  8. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles

    PubMed Central

    2015-01-01

    Mineral dusts originating from Earth’s crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts. Here we investigated in closer detail the reasons for its activity and the difference in the activity of the different feldspars. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. K-feldspar showed by far the highest ice nucleation activity. Finally, we give a potential explanation of this effect, finding alkali-metal ions having different hydration shells and thus an influence on the ice nucleation activity of feldspar surfaces. PMID:25584435

  9. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  10. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.

  11. Active site densities, oxygen activation and adsorbed reactive oxygen in alcohol activation on npAu catalysts.

    PubMed

    Wang, Lu-Cun; Friend, C M; Fushimi, Rebecca; Madix, Robert J

    2016-07-01

    The activation of molecular O2 as well as the reactivity of adsorbed oxygen species is of central importance in aerobic selective oxidation chemistry on Au-based catalysts. Herein, we address the issue of O2 activation on unsupported nanoporous gold (npAu) catalysts by applying a transient pressure technique, a temporal analysis of products (TAP) reactor, to measure the saturation coverage of atomic oxygen, its collisional dissociation probability, the activation barrier for O2 dissociation, and the facility with which adsorbed O species activate methanol, the initial step in the catalytic cycle of esterification. The results from these experiments indicate that molecular O2 dissociation is associated with surface silver, that the density of reactive sites is quite low, that adsorbed oxygen atoms do not spill over from the sites of activation onto the surrounding surface, and that methanol reacts quite facilely with the adsorbed oxygen atoms. In addition, the O species from O2 dissociation exhibits reactivity for the selective oxidation of methanol but not for CO. The TAP experiments also revealed that the surface of the npAu catalyst is saturated with adsorbed O under steady state reaction conditions, at least for the pulse reaction. PMID:27376884

  12. Proton uptake associated with the reduction of the primary quinone Q(A) influences the binding site of the secondary quinone Q(B) in Rhodopseudomonas viridis photosynthetic reaction centers.

    PubMed

    Zachariae, U; Lancaster, C R

    2001-06-01

    Previously, two binding sites for the secondary quinone Q(B) in the photosynthetic reaction center (RC) from Rhodopseudomonas viridis were identified by X-ray crystallography, a 'proximal' binding site close to the non-heme iron, and a 'distal' site, displaced by 4.2 A along the path of the isoprenoid tail [C.R.D. Lancaster and H. Michel, Structure 5 (1997) 1339-1359]. The quinone ring planes in the two sites differ by roughly a 180 degrees rotation around the isoprenoid tail. Here we present molecular dynamics simulations, which support the theory of a spontaneous transfer of Q(B) between the distal site and the proximal site. In contrast to earlier computational studies on RCs, the molecular dynamics simulations of Q(B) migration resulted in a proximal Q(B) binding pattern identical to that of the crystallographic findings. Also, we demonstrate that the preference towards the proximal Q(B) location is not necessarily attributed to reduction of Q(B) to the semiquinone, but already to the preceding reduction of the primary quinone Q(A) and resulting protonation changes in the protein. Energy mapping of the Q(B) binding pocket indicates that the quinone ring rotation required for completion of the transfer between the two sites is improbable at the distal or proximal binding sites due to high potential barriers, but may be possible at a newly identified position near the distal binding site.

  13. [Cp2TiCH2CHMe(SiMe3)]+, an alkyl-titanium complex which (a) exists in equilibrium between a β-agostic and a lower energy γ-agostic isomer and (b) undergoes hydrogen atom exchange between α-, β-, and γ-sites via a combination of conventional β-hydrogen elimination-reinsertion and a nonconventional CH bond activation process which involves proton tunnelling.

    PubMed

    Dunlop-Brière, Alexandre F; Baird, Michael C; Budzelaar, Peter H M

    2013-11-20

    The compound [Cp2Ti(Me)(CD2Cl2)][B(C6F5)4] reacts with trimethylvinylsilane (TMVS) to form the 1,2-insertion product [Cp2TiCH2CHMe(SiMe3)](+) (III), which exists in solution as equilibrating β- and γ-agostic isomers. In addition, while free rotation of the β-methyl group results in a single, averaged γ-H atom resonance at higher temperatures, decoalescence occurs below ~200 K, and the resonance of the γ-agostic hydrogen atom at δ ~ -7.4 is observed. Reaction of [Cp2Ti(CD3)(CD2Cl2)](+) with TMVS results in the formation of [Cp2TiCH2CH(CD3)(SiMe3)](+), which converts, via reversible β-elimination, to an equilibrium mixture of specifically [Cp2TiCH2CH(CD3)(SiMe3)](+) and [Cp2TiCD2CD(CH3)(SiMe3)](+). Complementing this conventional process, exchange spectroscopy experiments show that the β-H atom of [Cp2TiCH2CHMe(SiMe3)](+) undergoes exchange with the three hydrogen atoms of the β-methyl group (β-H/γ-H exchange) but not with the two α-H atoms. This exchange process is completely shut down when [Cp2TiCH2CH(CD3)(SiMe3)](+) is used, suggesting an H/D kinetic isotope effect much larger (apparently >16,000) than the maximum possible for an over-the-barrier process. It is proposed that β-H/γ-H exchange is facilitated by quantum mechanical proton tunnelling in which a hydrogen atom of the 2-methyl group of the alkene-hydride deinsertion product [Cp2TiH{CH2═CMe(SiMe3)}](+) undergoes reversible exchange with the hydride ligand via the allyl dihydrogen species [Cp2TiH2{(η(3)-CH2C(SiMe3)CH2}](+). Complementing these findings, DFT calculations were carried out to obtain energies and NMR parameters for all relevant species and thence to obtain better insight into the agostic preference(s) of complex III and the observed exchange processes. In all cases where comparisons between experimental and calculated data were possible, agreement was excellent. PMID:24147934

  14. Site-directed mutagenesis of Arginine282 suggests how protons and peptides are co-transported by rabbit PepT1.

    PubMed

    Pieri, Myrtani; Hall, Dashiell; Price, Richard; Bailey, Patrick; Meredith, David

    2008-01-01

    The mammalian proton-coupled peptide transporter PepT1 is the major route of uptake for dietary nitrogen, as well as the oral absorption of a number of drugs, including beta-lactam antibiotics and angiotensin-converting enzyme inhibitors. Here we have used site-directed mutagenesis to investigate further the role of conserved charged residues in transmembrane domains. Mutation of rabbit PepT1 arginine282 (R282, transmembrane domain 7) to a positive (R282K) or physiologically titratable residue (R282H), resulted in a transporter with wild-type characteristics when expressed in Xenopus laevis oocytes. Neutral (R282A, R282Q) or negatively charged (R282D, R282E) substitutions gave a transporter that was not stimulated by external acidification (reducing pH(out) from 7.4 to 5.5) but transported at the same rate as the wild-type maximal rate (pH(out) 5.5); however, only the R282E mutation was unable to concentrate substrate above the extracellular level. All of the R282 mutants showed trans-stimulation of efflux comparable to the wild-type, except R282E-PepT1 which was faster. A conserved negatively charged residue, aspartate341 (D341) in transmembrane domain 8 was implicated in forming a charge pair with R282, as R282E/D341R- and R282D/D341R-PepT1 had wild-type transporter characteristics. Despite their differences in ability to accumulate substrate, both R282E- and R282D-PepT1 showed an increased charge:peptide stoichiometry over the wild-type 1:1 ratio for the neutral dipeptide Gly-l-Gln, measured using two-electrode voltage clamp. This extra charge movement was linked to substrate transport, as 4-aminobenzoic acid, which binds but is not translocated, did not induce membrane potential depolarisation in R282E-expressing oocytes. A model is proposed for the substrate binding/translocation process in PepT1.

  15. Visualizing the kinetic power stroke that drives proton-coupled Zn(II) transport

    PubMed Central

    Gupta, Sayan; Chai, Jin; Cheng, Jie; D'Mello, Rhijuta; Chance, Mark R.; Fu, Dax

    2014-01-01

    The proton gradient is a principal energy source for respiration-dependent active transport, but the structural mechanisms of proton-coupled transport processes are poorly understood. YiiP is a proton-coupled zinc transporter found in the cytoplasmic membrane of E. coli, and the transport-site of YiiP receives protons from water molecules that gain access to its hydrophobic environment and transduces the energy of an inward proton gradient to drive Zn(II) efflux1,2. This membrane protein is a well characterized member3-7 of the protein family of cation diffusion facilitators (CDFs) that occurs at all phylogenetic levels8-10. X-ray mediated hydroxyl radical labeling of YiiP and mass spectrometric analysis showed that Zn(II) binding triggered a highly localized, all-or-none change of water accessibility to the transport-site and an adjacent hydrophobic gate. Millisecond time-resolved dynamics revealed a concerted and reciprocal pattern of accessibility changes along a transmembrane helix, suggesting a rigid-body helical reorientation linked to Zn(II) binding that triggers the closing of the hydrophobic gate. The gated water access to the transport-site enables a stationary proton gradient to facilitate the conversion of zinc binding energy to the kinetic power stroke of a vectorial zinc transport. The kinetic details provide energetic insights into a proton-coupled active transport reaction. PMID:25043033

  16. Proton Reaction Data Library for Nuclear Activation (Medium Energy Nuclear Data Library.)

    2002-03-01

    Version 00 GROUPXS does file handling and processing of the double-differential continuum-emission cross sections stored in the new MF6 format of ENDF/VI. It treats the energy-angle data that are supposed to be represented by a Legendre-polynomial expansion in the center-of-mass system and can do the following: (1) Conversion of MF6 data from center-of-mass system to the laboratory system, with the possibility to continue the calculation with the options (2), (3), and (4). (2) Conversion ofmore » Legendre-polynomial representation into point-wise angular data, in MF6 format. (3) Conversion of data from MF6 into MF4 + MF5 (ENDF-V). (4) Calculation of group constants, scattering matrices and transfer matrices for arbitrary group structures with a fusion micro-flux weighting spectrum (PN-approximation). The code treats only continuum reaction types that are stored in the MF6 format with the restrictions as specified for the European Fusion File (EFF1). These restrictions are not inconvenient for the purpose of fusion neutronics calculations and they facilitate relatively simple processing . This neutron reaction data library can be used for nuclear activation and transmutation applications at energies up to 100 MeV.« less

  17. Positional effects of hydroxy groups on catalytic activity of proton-responsive half-sandwich Cp*Iridium(III) complexes

    SciTech Connect

    Suna, Yuki; Fujita, Etsuko; Ertem, Mehmed Z.; Wang, Wan-Hui; Kambayashi, Hide; Manaka, Yuichi; Muckerman, James T.; Himeda, Yuichiro

    2014-11-12

    Proton-responsive half-sandwich Cp*Ir(III) complexes possessing a bipyridine ligand with two hydroxy groups at the 3,3'-, 4,4'-, 5,5'- or 6,6'-positions (3DHBP, 4DHBP, 5DHBP, or 6DHBP) were systematically investigated. UV-vis titration data provided average pK a values of the hydroxy groups on the ligands. Both hydroxy groups were found to deprotonate in the pH 4.6–5.6 range for the 4–6DHBP complexes. One of the hydroxy groups of the 3DHBP complex exhibited the low pKa value of < 0.4 because the deprotonation is facilitated by the strong intramolecular hydrogen bond formed between the generated oxyanion and the remaining hydroxy group, which in turn leads to an elevated pKa value of ~13.6 for the second deprotonation step. The crystal structures of the 4– and 6DHBP complexes obtained from basic aqueous solutions revealed their deprotonated forms. The intramolecular hydrogen bond in the 3DHBP complex was also observed in the crystal structures. The catalytic activities of these complexes in aqueous phase reactions, at appropriate pH, for hydrogenation of carbon dioxide (pH 8.5), dehydrogenation of formic acid (pH 1.8), transfer hydrogenation reactions using formic acid/formate as a hydrogen source (pH 7.2 and 2.6) were investigated to compare the positional effects of the hydroxy groups. The 4– and 6DHBP complexes exhibited remarkably enhanced catalytic activities under basic conditions because of the resonance effect of the strong electrondonating oxyanions, whereas the 5DHBP complex exhibited negligible activity despite the presence of electron-donating groups. The 3DHBP complex exhibited relatively high catalytic activity at low pH owing to the one strong electron-donating oxyanion group stabilized by the intramolecular hydrogen bond. DFT calculations were employed to study the mechanism of CO₂ hydrogenation by the 4DHBP and 6DHBP complexes, and comparison of the activation free energies of the H₂ heterolysis and CO

  18. Positional effects of hydroxy groups on catalytic activity of proton-responsive half-sandwich Cp*Iridium(III) complexes

    DOE PAGES

    Suna, Yuki; Fujita, Etsuko; Ertem, Mehmed Z.; Wang, Wan-Hui; Kambayashi, Hide; Manaka, Yuichi; Muckerman, James T.; Himeda, Yuichiro

    2014-11-12

    Proton-responsive half-sandwich Cp*Ir(III) complexes possessing a bipyridine ligand with two hydroxy groups at the 3,3'-, 4,4'-, 5,5'- or 6,6'-positions (3DHBP, 4DHBP, 5DHBP, or 6DHBP) were systematically investigated. UV-vis titration data provided average pK a values of the hydroxy groups on the ligands. Both hydroxy groups were found to deprotonate in the pH 4.6–5.6 range for the 4–6DHBP complexes. One of the hydroxy groups of the 3DHBP complex exhibited the low pKa value of < 0.4 because the deprotonation is facilitated by the strong intramolecular hydrogen bond formed between the generated oxyanion and the remaining hydroxy group, which in turn leadsmore » to an elevated pKa value of ~13.6 for the second deprotonation step. The crystal structures of the 4– and 6DHBP complexes obtained from basic aqueous solutions revealed their deprotonated forms. The intramolecular hydrogen bond in the 3DHBP complex was also observed in the crystal structures. The catalytic activities of these complexes in aqueous phase reactions, at appropriate pH, for hydrogenation of carbon dioxide (pH 8.5), dehydrogenation of formic acid (pH 1.8), transfer hydrogenation reactions using formic acid/formate as a hydrogen source (pH 7.2 and 2.6) were investigated to compare the positional effects of the hydroxy groups. The 4– and 6DHBP complexes exhibited remarkably enhanced catalytic activities under basic conditions because of the resonance effect of the strong electrondonating oxyanions, whereas the 5DHBP complex exhibited negligible activity despite the presence of electron-donating groups. The 3DHBP complex exhibited relatively high catalytic activity at low pH owing to the one strong electron-donating oxyanion group stabilized by the intramolecular hydrogen bond. DFT calculations were employed to study the mechanism of CO₂ hydrogenation by the 4DHBP and 6DHBP complexes, and comparison of the activation free energies of the H₂ heterolysis and CO₂ insertion steps

  19. Synthesis, characterization and antiglaucoma activity of a novel proton transfer compound and a mixed-ligand Zn(II) complex.

    PubMed

    Yenikaya, Cengiz; Sari, Musa; Bülbül, Metin; Ilkimen, Halil; Celik, Hülya; Büyükgüngör, Orhan

    2010-01-15

    A novel proton transfer compound, pyridin-2-ylmethanaminium 2,4-dichloro-5-sulfamoylbenzoate (1), and a mixed-ligand Zn(II) complex, bis(2,4-dichloro-5-sulfamoylbenzoate)(2-aminomethylpyridine)aquazinc(II) monohydrate (2), have been synthesized from the same free ligands, which are 2,4-dichloro-5-sulfamoylbenzoic acid (Hsba) and 2-aminomethylpyridine (amp). They have been characterized by elemental, spectral ((1)H NMR, IR and UV-vis.) and thermal analyses. Additionally, magnetic measurement and single crystal X-ray diffraction technique were applied to compound 2. In the complex, Zn(II) ion exhibits a distorted octahedral configuration coordinated by O1 and O1(i) atoms of two mono dentante sba anions and N1, N2, N2(i) atoms of bidentante amp anion and a water molecule (O1w). The free ligands Hsba and amp, and the products 1 and 2, and acetazolamide (AAZ) as the control compound, were also evaluated for their in vitro inhibitor effects on human Carbonic Anhydrase isoenzymes (hCA I and hCA II) purified from erythrocyte cell by affinity chromatography for their hydratase and esterase activities. The IC(50) values of products 1 and 2 for hydratase activity are 0.26 and 0.13microM for hCA I and 0.30 and 0.15microM for hCA II, respectively. The IC(50) values of the same inhibitors for esterase activity are 0.32 and 0.045microM for hCA I and 0.29 and 0.23microM for hCA II, respectively. In relation to esterase activities, the inhibition equilibrium constants (K(i)) were also determined and found 0.25 and 0.058microM on hCA I and 0.22 and 0.24microM on hCA II for 1 and 2, respectively. The comparison of the inhibition studies of newly synthesized compounds 1 and 2 to parent compounds Hsba and amp and to AAZ indicated that 1 and 2 have effective inhibitory activity on hCA I and II, and might be used potential inhibitors. PMID:20006931

  20. The nitric-oxide reductase from Paracoccus denitrificans uses a single specific proton pathway.

    PubMed

    ter Beek, Josy; Krause, Nils; Reimann, Joachim; Lachmann, Peter; Ädelroth, Pia

    2013-10-18

    The NO reductase from Paracoccus denitrificans reduces NO to N2O (2NO + 2H(+) + 2e(-) → N2O + H2O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O2-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O2 reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O2. We further show that electron transfer during single-turnover reduction of O2 is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.

  1. Possible active site of the sweet-tasting protein thaumatin.

    PubMed

    Slootstra, J W; De Geus, P; Haas, H; Verrips, C T; Meloen, R H

    1995-10-01

    Epitopes on thaumatin and monellin were studied using the PEPSCAN-technology. The antibodies used were raised against thaumatin. Only antibodies that, in an ELISA, both recognized thaumatin and monellin were used in the PEPSCAN-analyses. On thaumatin two major overlapping epitopes were identified. On monellin no epitopes could be identified. The identified epitope region on thaumatin shares structural features with various peptide and protein sweeteners. It contains an aspartame-like site which is formed by Asp21 and Phe80, tips of the two extruding loops KGDAALDAGGR19-29 and CKRFGRPP77-84, which are spatially positioned next to each other. Furthermore, sub-sequences of the KGDAALDAGGR19-29 loop are similar to peptide-sweeteners such as L-Asp-D-Ala-L-Ala-methyl ester and L-Asp-D-Ala-Gly-methyl ester. Since the aspartame-like Asp21-Phe80 site and the peptide-sweetener-like sequences are also not present in non-sweet thaumatin-like proteins it is postulated that the KGDAALDAGGR19-29- and CKRFGRPP77-84 loop contain important sweet-taste determinants. This region has previously not been implicated as a sweet-taste determinant of thaumatin.

  2. Vision 20/20: Proton therapy

    SciTech Connect

    Smith, Alfred R.

    2009-02-15

    The first patients were treated with proton beams in 1955 at the Lawrence Berkeley Laboratory in California. In 1970, proton beams began to be used in research facilities to treat cancer patients using fractionated treatment regimens. It was not until 1990 that proton treatments were carried out in hospital-based facilities using technology and techniques that were comparable to those for modern photon therapy. Clinical data strongly support the conclusion that proton therapy is superior to conventional radiation therapy in a number of disease sites. Treatment planning studies have shown that proton dose distributions are superior to those for photons in a wide range of disease sites indicating that additional clinical gains can be achieved if these treatment plans can be reliably delivered to patients. Optimum proton dose distributions can be achieved with intensity modulated protons (IMPT), but very few patients have received this advanced form of treatment. It is anticipated widespread implementation of IMPT would provide additional improvements in clinical outcomes. Advances in the last decade have led to an increased interest in proton therapy. Currently, proton therapy is undergoing transitions that will move it into the mainstream of cancer treatment. For example, proton therapy is now reimbursed, there has been rapid development in proton therapy technology, and many new options are available for equipment, facility configuration, and financing. During the next decade, new developments will increase the efficiency and accuracy of proton therapy and enhance our ability to verify treatment planning calculations and perform quality assurance for proton therapy delivery. With the implementation of new multi-institution clinical studies and the routine availability of IMPT, it may be possible, within the next decade, to quantify the clinical gains obtained from optimized proton therapy. During this same period several new proton therapy facilities will be built

  3. Structure of the 2-Aminopurine-Cytosine Base Pair Formed in the Polymerase Active Site of the RB69 Y567A-DNA Polymerase

    SciTech Connect

    Reha-Krantz, Linda J.; Hariharan, Chithra; Subuddhi, Usharani; Xia, Shuangluo; Zhao, Chao; Beckman, Jeff; Christian, Thomas; Konigsberg, William

    2011-11-21

    The adenine base analogue 2-aminopurine (2AP) is a potent base substitution mutagen in prokaryotes because of its enhanceed ability to form a mutagenic base pair with an incoming dCTP. Despite more than 50 years of research, the structure of the 2AP-C base pair remains unclear. We report the structure of the 2AP-dCTP base pair formed within the polymerase active site of the RB69 Y567A-DNA polymerase. A modified wobble 2AP-C base pair was detected with one H-bond between N1 of 2AP and a proton from the C4 amino group of cytosine and an apparent bifurcated H-bond between a proton on the 2-amino group of 2-aminopurine and the ring N3 and O2 atoms of cytosine. Interestingly, a primer-terminal region rich in AT base pairs, compared to GC base pairs, facilitated dCTP binding opposite template 2AP. We propose that the increased flexibility of the nucleotide binding pocket formed in the Y567A-DNA polymerase and increased 'breathing' at the primer-terminal junction of A+T-rich DNA facilitate dCTP binding opposite template 2AP. Thus, interactions between DNA polymerase residues with a dynamic primer-terminal junction play a role in determining base selectivity within the polymerase active site of RB69 DNA polymerase.

  4. Assessment of activation products in the Savannah River Site environment

    SciTech Connect

    Carlton, W.H.; Denham, M.

    1996-07-01

    This document assesses the impact of radioactive activation products released from SRS facilities since the first reactor became operational late in 1953. The isotopes reported here are those whose release resulted in the highest dose to people living near SRS: {sup 32}P, {sup 51}Cr, {sup 60}C, and {sup 65}Zn. Release pathways, emission control features, and annual releases to the aqueous and atmospheric environments are discussed. No single incident has resulted in a major acute release of activation products to the environment. The releases were the result of normal operations of the reactors and separations facilities. Releases declined over the years as better controls were established and production was reduced. The overall radiological impact of SRS activation product atmospheric releases from 1954 through 1994 on the offsite maximally exposed individual can be characterized by a total dose of 0.76 mrem. During the same period, such an individual received a total dose of 14,400 mrem from non-SRS sources of ionizing radiation present in the environment. SRS activation product aqueous releases between 1954 and 1994 resulted in a total dose of 54 mrem to the offsite maximally exposed individual. The impact of SRS activation product releases on offsite populations also has been evaluated.

  5. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

    PubMed Central

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-01-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

  6. Characterization of an Active Thermal Erosion Site, Caribou Creek, Alaska

    NASA Astrophysics Data System (ADS)

    Busey, R.; Bolton, W. R.; Cherry, J. E.; Hinzman, L. D.

    2013-12-01

    The goal of this project is to estimate volume loss of soil over time from this site, provide parameterizations on erodibility of ice rich permafrost and serve as a baseline for future landscape evolution simulations. Located in the zone of discontinuous permafrost, the interior region of Alaska (USA) is home to a large quantity of warm, unstable permafrost that is both high in ice content and has soil temperatures near the freezing point. Much of this permafrost maintains a frozen state despite the general warming air temperature trend in the region due to the presence of a thick insulating organic mat and a dense root network in the upper sub-surface of the soil column. At a rapidly evolving thermo-erosion site, located within the Caribou-Poker Creeks Research Watershed (part of the Bonanza Creek LTER) near Chatanika, Alaska (N65.140, W147.570), the protective organic layer and associated plants were disturbed by an adjacent traditional use trail and the shifting of a groundwater spring. These triggers have led to rapid geomorphological change on the landscape as the soil thaws and sediment is transported into the creek at the valley bottom. Since 2006 (approximately the time of initiation), the thermal erosion has grown to 170 meters length, 3 meters max depth, and 15 meters maximum width. This research combines several data sets: DGPS survey, imagery from an extremely low altitude pole-based remote sensing (3 to 5 meters above ground level), and imagery from an Unmanned Aerial System (UAS) at about 60m altitude.

  7. Observations of protons not exceeding 1 MeV/nuc and ions during the September 1974 series of flares. [solar activity events

    NASA Technical Reports Server (NTRS)

    Ipavich, F. M.; Gloeckler, G.; Fan, C. Y.; Hovestadt, D.

    1975-01-01

    Results are presented on observations of energetic particles made during an active solar period in September 1974, concentrating in particular on an ESP (Energetic Storm Particle) event observed in association with an interplanetary shock wave on 21 September. It is shown that the observed variations in the proton to alpha particle ratios and spectral indices can be explained either by pile-up or by acceleration models of ESP events.

  8. Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

    PubMed

    Miner, Kyle D; Kurtz, Donald M

    2016-02-16

    HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites.

  9. Exploring the proton pump and exit pathway for pumped protons in cytochrome ba3 from Thermus thermophilus.

    PubMed

    Chang, Hsin-Yang; Choi, Sylvia K; Vakkasoglu, Ahmet Selim; Chen, Ying; Hemp, James; Fee, James A; Gennis, Robert B

    2012-04-01

    The heme-copper oxygen reductases are redox-driven proton pumps. In the current work, the effects of mutations in a proposed exit pathway for pumped protons are examined in the ba(3)-type oxygen reductase from Thermus thermophilus, leading from the propionates of heme a(3) to the interface between subunits I and II. Recent studies have proposed important roles for His376 and Asp372, both of which are hydrogen-bonded to propionate-A of heme a(3), and for Glu126(II) (subunit II), which is hydrogen-bonded to His376. Based on the current results, His376, Glu126(II), and Asp372 are not essential for either oxidase activity or proton pumping. In addition, Tyr133, which is hydrogen-bonded to propionate-D of heme a(3), was also shown not to be essential for function. However, two mutations of the residues hydrogen-bonded to propionate-A, Asp372Ile and His376Asn, retain high electron transfer activity and normal spectral features but, in different preparations, either do not pump protons or exhibit substantially diminished proton pumping. It is concluded that either propionate-A of heme a(3) or possibly the cluster of groups centered about the conserved water molecule that hydrogen-bonds to both propionates-A and -D of heme a(3) is a good candidate to be the proton loading site.

  10. A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations.

    PubMed

    O'Farrell, Norah; Kreiner, Michaela; Moore, Barry D; Parker, Marie-Claire

    2006-11-01

    We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions. Two types of enzyme preparation were investigated, lyophilised enzymes and protein-coated microcrystals (PCMC).

  11. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

    PubMed

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  12. Marine Biology Field Trip Sites. Ocean Related Curriculum Activities.

    ERIC Educational Resources Information Center

    Pauls, John

    The ocean affects all of our lives. Therefore, awareness of and information about the interconnections between humans and oceans are prerequisites to making sound decisions for the future. Project ORCA (Ocean Related Curriculum Activities) has developed interdisciplinary curriculum materials designed to meet the needs of students and teachers…

  13. Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity.

    PubMed

    Bright, Nicholas A; Davis, Luther J; Luzio, J Paul

    2016-09-12

    The endocytic delivery of macromolecules from the mammalian cell surface for degradation by lysosomal acid hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochemistry to identify which endocytic compartments are acid hydrolase active. We found that endolysosomes are the principal organelles in which acid hydrolase substrates are cleaved. Endolysosomes also accumulated acidotropic probes and could be distinguished from terminal storage lysosomes, which were acid hydrolase inactive and did not accumulate acidotropic probes. Using live-cell microscopy, we have demonstrated that fusion events, which form endolysosomes, precede the onset of acid hydrolase activity. By means of sucrose and invertase uptake experiments, we have also shown that acid-hydrolase-active endolysosomes and acid-hydrolase-inactive, terminal storage lysosomes exist in dynamic equilibrium. We conclude that the terminal endocytic compartment is composed of acid-hydrolase-active, acidic endolysosomes and acid hydrolase-inactive, non-acidic, terminal storage lysosomes, which are linked and function in a lysosome regeneration cycle. PMID:27498570

  14. Outside-binding site mutations modify the active site's shapes in neuraminidase from influenza A H1N1.

    PubMed

    Tolentino-Lopez, Luis; Segura-Cabrera, Aldo; Reyes-Loyola, Paola; Zimic, Mirko; Quiliano, Miguel; Briz, Veronica; Muñoz-Fernández, Angeles; Rodríguez-Pérez, Mario; Ilizaliturri-Flores, Ian; Correa-Basurto, Jose

    2013-01-01

    The recent occurrence of 2009 influenza A (H1N1) pandemic as well as others has raised concern of a far more dangerous outcome should this virus becomes resistant to current drug therapies. The number of clinical cases that are resistant to oseltamivir (Tamiflu®) is larger than the limited number of neuraminidase (NA) mutations (H275Y, N295S, and I223R) that have been identified at the active site and that are associated to oseltamivir resistance. In this study, we have performed a comparative analysis between a set of NAs that have the most representative mutations located outside the active site. The recently crystallized NA-oseltamivir complex (PDB ID: 3NSS) was used as a wild-type structure. After selecting the target NA sequences, their three-dimensional (3D) structure was built using 3NSS as a template by homology modeling. The 3D NA models were refined by molecular dynamics (MD) simulations. The refined models were used to perform a docking study, using oseltamivir as a ligand. Furthermore, the docking results were refined by free-energy analysis using the MM-PBSA method. The analysis of the MD simulation results showed that the NA models reached convergence during the first 10 ns. Visual inspection and structural measures showed that the mutated NA active sites show structural variations. The docking and MM-PBSA results from the complexes showed different binding modes and free energy values. These results suggest that distant mutations located outside the active site of NA affect its structure and could be considered to be a new source of resistance to oseltamivir, which agrees with reports in the clinical literature.

  15. Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine.

    PubMed

    Wilson, B A; Reich, K A; Weinstein, B R; Collier, R J

    1990-09-18

    Glutamic acid-148, an active-site residue of diphtheria toxin identified by photoaffinity labeling with NAD, was replaced with aspartic acid, glutamine, or serine by directed mutagenesis of the F2 fragment of the toxin gene. Wild-type and mutant F2 proteins were synthesized in Escherichia coli, and the corresponding enzymic fragment A moieties (DTA) were derived, purified, and characterized. The Glu----Asp (E148D), Glu----Gln (E148Q), and Glu----Ser (E148S) mutations caused reductions in NAD:EF-2 ADP-ribosyltransferase activity of ca. 100-, 250-, and 300-fold, respectively, while causing only minimal changes in substrate affinity. The effects of the mutations on NAD-glycohydrolase activity were considerably different; only a 10-fold reduction in activity was observed for E148S, and the E148D and E148Q mutants actually exhibited a small but reproducible increase in NAD-glycohydrolytic activity. Photolabeling by nicotinamide-radiolabeled NAD was diminished ca. 8-fold in the E148D mutant and was undetectable in the other mutants. The results confirm that Glu-148 plays a crucial role in the ADP-ribosylation of EF-2 and imply an important function for the side-chain carboxyl group in catalysis. The carboxyl group is also important for photochemical labeling by NAD but not for NAD-glycohydrolase activity. The pH dependence of the catalytic parameters for the ADP-ribosyltransferase reaction revealed a group in DTA-wt that titrates with an apparent pKa of 6.2-6.3 and is in the protonated state in the rate-determining step.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Proton affinities and photoelectron spectra of phenylalanine and N-methyl- and N,N-dimethylphenylalanine. Correlation of lone pair ionization energies with proton affinities and implications for N-methylation as a method to effect site specific protonation of peptides

    SciTech Connect

    Campbell, S.; Marzluff, E.M.; Rodgers, M.T.; Beauchamp, J.L. ); Rempe, M.E.; Schwinck, K.F.; Lichtenberger, D.L. )

    1994-06-15

    A Fourier transform ion cyclotron resonance (FT-ICR) technique for measuring gas-phase proton affinities is presented which utilizes collisional dissociation of proton-bound clusters by off-resonance translational excitation. A simplified RRKM analysis relates unimolecular dissociation rates to proton affinities. This technique is used to measure values for the proton affinities of phenylalanine and N-methyl- and N,N-dimethylphenylalanine of 220.3, 223.6, and 224.5 kcal/mol, respectively (relative to the proton affinity of NH[sub 3] = 204.0 kcal/mol). The proton affinity measured for phenylalanine is in excellent agreement with reported literature values. The photoelectron spectra of these three molecules are also presented and analyzed. Assignments of bands to specific ionization processes are aided by comparison with model compounds such as methyl-substituted amines and 2-phenylethylamines. These data are employed to examine the correlation of adiabatic nitrogen lone pair ionization energies with gas-phase proton affinities for phenylalanine, N-methylphenylalanine, and N,N-dimethylphenylalanine in comparison to correlations for other amino acids and selected aliphatic amines. 41 refs., 7 figs., 2 tabs.

  17. Identification of inhibitors against the potential ligandable sites in the active cholera toxin.

    PubMed

    Gangopadhyay, Aditi; Datta, Abhijit

    2015-04-01

    The active cholera toxin responsible for the massive loss of water and ions in cholera patients via its ADP ribosylation activity is a heterodimer of the A1 subunit of the bacterial holotoxin and the human cytosolic ARF6 (ADP Ribosylation Factor 6). The active toxin is a potential target for the design of inhibitors against cholera. In this study we identified the potential ligandable sites of the active cholera toxin which can serve as binding sites for drug-like molecules. By employing an energy-based approach to identify ligand binding sites, and comparison with the results of computational solvent mapping, we identified two potential ligandable sites in the active toxin which can be targeted during structure-based drug design against cholera. Based on the probe affinities of the identified ligandable regions, docking-based virtual screening was employed to identify probable inhibitors against these sites. Several indole-based alkaloids and phosphates showed strong interactions to the important residues of the ligandable region at the A1 active site. On the other hand, 26 top scoring hits were identified against the ligandable region at the A1 ARF6 interface which showed strong hydrogen bonding interactions, including guanidines, phosphates, Leucopterin and Aristolochic acid VIa. This study has important implications in the application of hybrid structure-based and ligand-based methods against the identified ligandable sites using the identified inhibitors as reference ligands, for drug design against the active cholera toxin.

  18. Encroachment of Human Activity on Sea Turtle Nesting Sites

    NASA Astrophysics Data System (ADS)

    Ziskin, D.; Aubrecht, C.; Elvidge, C.; Tuttle, B.; Baugh, K.; Ghosh, T.

    2008-12-01

    The encroachment of anthropogenic lighting on sea turtle nesting sites poses a serious threat to the survival of these animals [Nicholas, 2001]. This danger is quantified by combining two established data sets. The first is the Nighttime Lights data produced by the NOAA National Geophysical Data Center [Elvidge et al., 1997]. The second is the Marine Turtle Database produced by the World Conservation Monitoring Centre (WCMC). The technique used to quantify the threat of encroachment is an adaptation of the method described in Aubrecht et al. [2008], which analyzes the stress on coral reef systems by proximity to nighttime lights near the shore. Nighttime lights near beaches have both a direct impact on turtle reproductive success since they disorient hatchlings when they mistake land-based lights for the sky-lit surf [Lorne and Salmon, 2007] and the lights are also a proxy for other anthropogenic threats. The identification of turtle nesting sites with high rates of encroachment will hopefully steer conservation efforts to mitigate their effects [Witherington, 1999]. Aubrecht, C, CD Elvidge, T Longcore, C Rich, J Safran, A Strong, M Eakin, KE Baugh, BT Tuttle, AT Howard, EH Erwin, 2008, A global inventory of coral reef stressors based on satellite observed nighttime lights, Geocarto International, London, England: Taylor and Francis. In press. Elvidge, CD, KE Baugh, EA Kihn, HW Kroehl, ER Davis, 1997, Mapping City Lights with Nighttime Data from the DMSP Operational Linescan System, Photogrammatic Engineering and Remote Sensing, 63:6, pp. 727-734. Lorne, JK, M Salmon, 2007, Effects of exposure to artificial lighting on orientation of hatchling sea turtles on the beach and in the ocean, Endangered Species Research, Vol. 3: 23-30. Nicholas, M, 2001, Light Pollution and Marine Turtle Hatchlings: The Straw that Breaks the Camel's Back?, George Wright Forum, 18:4, p77-82. Witherington, BE, 1999, Reducing Threats To Nesting Habitat, Research and Management Techniques for

  19. Fluoroquinolones: A micro-species equilibrium in the protonation of amphoteric compounds.

    PubMed

    Nurchi, Valeria M; Crisponi, Guido; Lachowicz, Joanna I; Zoroddu, Maria Antonietta; Peana, Massimiliano; Medici, Serenella; Veclani, Daniele; Tolazzi, Marilena; Melchior, Andrea

    2016-10-10

    The knowledge of the speciation of fluoroquinolones is of great actuality for the implications on the activity, bioavailability and pharmacokinetics. Literature reports a number of contrasting evaluations on the existence of tautomeric forms of mono-protonated species, described by a set of protonation micro-constants. Here the protonation sequence and the related protonation constants of four representative molecules are evaluated by a combined potentiometric-spectrophotometric method. The experimental observations necessary to differentiate between a protonation scheme represented by macro-constants alone, and the one that requires the introduction of a micro-protonation scheme, are clearly delineated based on a careful analysis of experimental data and of simulated models. The role of the medium was investigated and UV-vis spectra in water- methanol solution were analyzed. The existence of the zwitterionic species alone at physiological pH in water, and an increase of the relative amount of the neutral species with the lipophilicity of the medium were remarked. This surely affects the bioavailability of FQs, with the increase of the neutral species when the molecules approach the local lipophilic environment close to the cellular membranes. NMR studies allowed the attribution of the protonation sites of the different forms. Quantum chemical evaluation of all the possible existent forms with different protonation degrees and in different sites strongly substantiates the experimental results. The study of the relevant frontier molecular orbitals completed the detailed theoretical characterization of the species. PMID:27586021

  20. Reduction of Urease Activity by Interaction with the Flap Covering the Active Site

    PubMed Central

    Macomber, Lee; Minkara, Mona S.; Hausinger, Robert P.; Merz, Kenneth M.

    2015-01-01

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  1. The general base in the thymidylate synthase catalyzed proton abstraction.

    PubMed

    Ghosh, Ananda K; Islam, Zahidul; Krueger, Jonathan; Abeysinghe, Thelma; Kohen, Amnon

    2015-12-14

    The enzyme thymidylate synthase (TSase), an important chemotherapeutic drug target, catalyzes the formation of 2'-deoxythymidine-5'-monophosphate (dTMP), a precursor of one of the DNA building blocks. TSase catalyzes a multi-step mechanism that includes the abstraction of a proton from the C5 of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP). Previous studies on ecTSase proposed that an active-site residue, Y94 serves the role of the general base abstracting this proton. However, since Y94 is neither very basic, nor connected to basic residues, nor located close enough to the pyrimidine proton to be abstracted, the actual identity of this base remains enigmatic. Based on crystal structures, an alternative hypothesis is that the nearest potential proton-acceptor of C5 of dUMP is a water molecule that is part of a hydrogen bond (H-bond) network comprised of several water molecules and several protein residues including H147, E58, N177, and Y94. Here, we examine the role of the residue Y94 in the proton abstraction step by removing its hydroxyl group (Y94F mutant). We investigated the effect of the mutation on the temperature dependence of intrinsic kinetic isotope effects (KIEs) and found that these KIEs are more temperature dependent than those of the wild-type enzyme (WT). These results suggest that the phenolic -OH of Y94 is a component of the transition state for the proton abstraction step. The findings further support the hypothesis that no single functional group is the general base, but a network of bases and hydroxyls (from water molecules and tyrosine) sharing H-bonds across the active site can serve the role of the general base to remove the pyrimidine proton.

  2. Spectroscopic definition of the copper active sites in mordenite: selective methane oxidation.

    PubMed

    Vanelderen, Pieter; Snyder, Benjamin E R; Tsai, Ming-Li; Hadt, Ryan G; Vancauwenbergh, Julie; Coussens, Olivier; Schoonheydt, Robert A; Sels, Bert F; Solomon, Edward I

    2015-05-20

    Two distinct [Cu-O-Cu](2+) sites with methane monooxygenase activity are identified in the zeolite Cu-MOR, emphasizing that this Cu-O-Cu active site geometry, having a ∠Cu-O-Cu ∼140°, is particularly formed and stabilized in zeolite topologies. Whereas in ZSM-5 a similar [Cu-O-Cu](2+) active site is located in the intersection of the two 10 membered rings, Cu-MOR provides two distinct local structures, situated in the 8 membered ring windows of the side pockets. Despite their structural similarity, as ascertained by electronic absorption and resonance Raman spectroscopy, the two Cu-O-Cu active sites in Cu-MOR clearly show different kinetic behaviors in selective methane oxidation. This difference in reactivity is too large to be ascribed to subtle differences in the ground states of the Cu-O-Cu sites, indicating the zeolite lattice tunes their reactivity through second-sphere effects. The MOR lattice is therefore functionally analogous to the active site pocket of a metalloenzyme, demonstrating that both the active site and its framework environment contribute to and direct reactivity in transition metal ion-zeolites.

  3. Magnetic resonance spectral characterization of the heme active site of Coprinus cinereus peroxidase

    SciTech Connect

    Lukat, G.S.; Rodgers, K.R.; Jabro, M.N.; Goff, H.M. )

    1989-04-18

    Examination of the peroxidase isolated from the inkcap Basidiomycete Coprinus cinereus shows that the 42,000-dalton enzyme contains a protoheme IX prosthetic group. Reactivity assays and the electronic absorption spectra of native Coprinus peroxidase and several of its ligand complexes indicate that this enzyme has characteristics similar to those reported for horseradish peroxidase. In this paper, the authors characterize the H{sub 2}O{sub 2}-oxidized forms of Coprinus peroxidase compounds I, II, and III by electronic absorption and magnetic resonance spectroscopies. Electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) studies of this Coprinus peroxidase indicate the presence of high-spin Fe(III) in the native protein and a number of differences between the heme site of Coprinus peroxidase and horseradish peroxidase. Carbon-13 (of the ferrous CO adduct) and nitrogen-15 (of the cyanide complex) NMR studies together with proton NMR studies of the native and cyanide-complexed Caprinus peroxidase are consistent with coordination of a proximal histidine ligand. The EPR spectrum of the ferrous NO complex is also reported. Protein reconstitution with deuterated hemin has facilitated the assignment of the heme methyl resonances in the proton NMR spectrum.

  4. Magnetic resonance spectral characterization of the heme active site of Coprinus cinereus peroxidase.

    PubMed

    Lukat, G S; Rodgers, K R; Jabro, M N; Goff, H M

    1989-04-18

    Examination of the peroxidase isolated from the inkcap Basidiomycete Coprinus cinereus shows that the 42,000-dalton enzyme contains a protoheme IX prosthetic group. Reactivity assays and the electronic absorption spectra of native Coprinus peroxidase and several of its ligand complexes indicate that this enzyme has characteristics similar to those reported for horseradish peroxidase. In this paper, we characterize the H2O2-oxidized forms of Coprinus peroxidase compounds I, II, and III by electronic absorption and magnetic resonance spectroscopies. Electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) studies of this Coprinus peroxidase indicate the presence of high-spin Fe(III) in the native protein and a number of differences between the heme site of Coprinus peroxidase and horseradish peroxidase. Carbon-13 (of the ferrous CO adduct) and nitrogen-15 (of the cyanide complex) NMR studies together with proton NMR studies of the native and cyanide-complexed Coprinus peroxidase are consistent with coordination of a proximal histidine ligand. The EPR spectrum of the ferrous NO complex is also reported. Protein reconstitution with deuterated hemin has facilitated the assignment of the heme methyl resonances in the proton NMR spectrum.

  5. School Pharmacist/School Environmental Hygienic Activities at School Site.

    PubMed

    Muramatsu, Akiyoshi

    2016-01-01

    The "School Health and Safety Act" was enforced in April 2009 in Japan, and "school environmental health standards" were established by the Minister of Education, Culture, Sports, Science and Technology. In Article 24 of the Enforcement Regulations, the duties of the school pharmacist have been clarified; school pharmacists have charged with promoting health activities in schools and carrying out complete and regular checks based on the "school environmental health standards" in order to protect the health of students and staff. In supported of this, the school pharmacist group of Japan Pharmaceutical Association has created and distributed digital video discs (DVDs) on "check methods of school environmental health standards" as support material. We use the DVD to ensure the basic issues that school pharmacists deal with, such as objectives, criteria, and methods for each item to be checked, advice, and post-measures. We conduct various workshops and classes, and set up Q&A committees so that inquiries from members are answered with the help of such activities. In addition, school pharmacists try to improve the knowledge of the school staff on environmental hygiene during their in-service training. They also conduct "drug abuse prevention classes" at school and seek to improve knowledge and recognition of drugs, including "dangerous drugs". PMID:27252053

  6. Rate-equilibria relationships in intramolecular proton transfer in human carbonic anhydrase III.

    PubMed

    Silverman, D N; Tu, C; Chen, X; Tanhauser, S M; Kresge, A J; Laipis, P J

    1993-10-12

    Maximal turnover rates for the dehydration of HCO3- catalyzed by the zinc metalloenzyme carbonic anhydrase III are limited by a proton transfer to zinc-bound hydroxide in the active site. We have used site-directed mutagenesis to place a proton donor, histidine, at position 64 and used 18O exchange between CO2 and water measured by mass spectrometry to determine the rates of intramolecular proton transfer to the zinc-bound hydroxide. In a series of site-specific mutants, the values of pKa of the zinc-bound water ranged from approximately 5 to 9. The rate constants for proton transfer obeyed a Brønsted correlation and showed sharp curvature characteristic of facile proton transfers. Application of Marcus rate theory shows that this proton transfer has the small intrinsic energy barrier (near 1.5 kcal/mol) characteristic of rapid proton transfer between nitrogen and oxygen acids and bases, but has an observed overall energy barrier (near 10 kcal/mol), indicating the involvement of accompanying, energy requiring processes such as solvent reorganization or conformational change. PMID:8399223

  7. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors.

    PubMed

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M; Kenny, Paul J; Lindstrom, Jon

    2015-05-29

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets.

  8. The Proton

    NASA Astrophysics Data System (ADS)

    Canal, Carlos Garcia; Sassot, Rodolfo

    2003-10-01

    In this talk we present a collection of selected topics concerning the structure of the proton and the fundamental interactions as seen inside it. These topics have been thoroughly covered by high energy experiments with ever increasing precision in recent years and beautifully illustrate our present knowledge of the standard model.

  9. Proton Radiobiology

    PubMed Central

    Tommasino, Francesco; Durante, Marco

    2015-01-01

    In addition to the physical advantages (Bragg peak), the use of charged particles in cancer therapy can be associated with distinct biological effects compared to X-rays. While heavy ions (densely ionizing radiation) are known to have an energy- and charge-dependent increased Relative Biological Effectiveness (RBE), protons should not be very different from sparsely ionizing photons. A slightly increased biological effectiveness is taken into account in proton treatment planning by assuming a fixed RBE of 1.1 for the whole radiation field. However, data emerging from recent studies suggest that, for several end points of clinical relevance, the biological response is differentially modulated by protons compared to photons. In parallel, research in the field of medical physics highlighted how variations in RBE that are currently neglected might actually result in deposition of significant doses in healthy organs. This seems to be relevant in particular for normal tissues in the entrance region and for organs at risk close behind the tumor. All these aspects will be considered and discussed in this review, highlighting how a re-discussion of the role of a variable RBE in proton therapy might be well-timed. PMID:25686476

  10. Isolated metal active site concentration and stability control catalytic CO2 reduction selectivity.

    PubMed

    Matsubu, John C; Yang, Vanessa N; Christopher, Phillip

    2015-03-01

    CO2 reduction by H2 on heterogeneous catalysts is an important class of reactions that has been studied for decades. However, atomic scale details of structure-function relationships are still poorly understood. Particularly, it has been suggested that metal particle size plays a unique role in controlling the stability of CO2 hydrogenation catalysts and the distribution of active sites, which dictates reactivity and selectivity. These studies often have not considered the possible role of isolated metal active sites in the observed dependences. Here, we utilize probe molecule diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) with known site-specific extinction coefficients to quantify the fraction of Rh sites residing as atomically dispersed isolated sites (Rhiso), as well as Rh sites on the surface of Rh nanoparticles (RhNP) for a series of TiO2 supported Rh catalysts. Strong correlations were observed between the catalytic reverse water gas shift turn over frequency (TOF) and the fraction of Rhiso sites and between catalytic methanation TOF and the fraction of RhNP sites. Furthermore, it was observed that reaction condition-induced disintegration of Rh nanoparticles, forming Rhiso active sites, controls the changing reactivity with time on stream. This work demonstrates that isolated atoms and nanoparticles of the same metal on the same support can exhibit uniquely different catalytic selectivity in competing parallel reaction pathways and that disintegration of nanoparticles under reaction conditions can play a significant role in controlling stability.

  11. Isolated metal active site concentration and stability control catalytic CO2 reduction selectivity.

    PubMed

    Matsubu, John C; Yang, Vanessa N; Christopher, Phillip

    2015-03-01

    CO2 reduction by H2 on heterogeneous catalysts is an important class of reactions that has been studied for decades. However, atomic scale details of structure-function relationships are still poorly understood. Particularly, it has been suggested that metal particle size plays a unique role in controlling the stability of CO2 hydrogenation catalysts and the distribution of active sites, which dictates reactivity and selectivity. These studies often have not considered the possible role of isolated metal active sites in the observed dependences. Here, we utilize probe molecule diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) with known site-specific extinction coefficients to quantify the fraction of Rh sites residing as atomically dispersed isolated sites (Rhiso), as well as Rh sites on the surface of Rh nanoparticles (RhNP) for a series of TiO2 supported Rh catalysts. Strong correlations were observed between the catalytic reverse water gas shift turn over frequency (TOF) and the fraction of Rhiso sites and between catalytic methanation TOF and the fraction of RhNP sites. Furthermore, it was observed that reaction condition-induced disintegration of Rh nanoparticles, forming Rhiso active sites, controls the changing reactivity with time on stream. This work demonstrates that isolated atoms and nanoparticles of the same metal on the same support can exhibit uniquely different catalytic selectivity in competing parallel reaction pathways and that disintegration of nanoparticles under reaction conditions can play a significant role in controlling stability. PMID:25671686

  12. Site specific rationale for technical impracticability of active groundwater restoration at a former manufactured gas plant site

    SciTech Connect

    Logan, C.M.; Walden, R.H.; MacFarlane, I.D.

    1995-12-31

    The National Contingency Plan (40 CFR Part 300 ) requires that remedial strategies must, at minimum, protect human health and the environment and meet applicable and relevant or appropriate requirements (ARARs). Where groundwater is impacted, maximum contaminant levels (MCLs) and maximum contaminant level goals (MCLGs) set under the Safe Drinking Water Act are often used as ARARs, whether or not the aquifer is a reasonably anticipated future source of drinking water. The US Environmental Protection Agency now recognizes the difficulty of groundwater restoration at sites where dense nonaqueous phase liquids are present, particularly in certain complex hydrogeological settings (EPA 1993). However, demonstration of impracticability generally does not occur until active remediation (e.g., pump and treat) has been shown to be ineffective. A case study of a former manufactured gas plant (MGP) is used to demonstrate how physical and chemical properties of the aquifer and coal tar, the major waste product from MGP sites, influence the feasibility of active restoration. Field characterization investigations, laboratory studies, and groundwater modeling are integrated into a demonstration following EPA guidelines. Laboratory studies included microbiological characterization and natural biodegradation and suggest that intrinsic bioremediation is occurring at this site. This work will be useful as EPA continues to develop presumptive remedies for cleanup under Superfund.

  13. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-11-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  14. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-01-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  15. Extending the Diffuse Layer Model of Surface Acidity Behavior: III. Estimating Bound Site Activity Coefficients

    EPA Science Inventory

    Although detailed thermodynamic analyses of the 2-pK diffuse layer surface complexation model generally specify bound site activity coefficients for the purpose of accounting for those non-ideal excess free energies contributing to bound site electrochemical potentials, in applic...

  16. Activity of site-specific endonucleases on complexes of plasmid DNA with multiwalled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Egorova, V. P.; Krylova, H. V.; Lipnevich, I. V.; Veligura, A. A.; Shulitsky, B. G.; Asayonok, A. A.; Vaskovtsev, E. V.

    2016-08-01

    We have synthesized and investigated structural and functional properties of plasmid DNA complexes with multi-walled carbon nanotubes (MWCNTs) for detection of changes in structural state of the plasmid DNA at its recognition by site-specific endonuclease. It has been also established that the site-specific endonuclease is functionally active on the surface of MWCNTs.

  17. 77 FR 5560 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ... project proposals on those leases) in identified Wind Energy Areas (WEAs) on the OCS offshore New Jersey... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on the... site assessment plans (SAPs) on those leases. BOEM may issue one or more commercial wind energy...

  18. The balance of flexibility and rigidity in the active site residues of hen egg white lysozyme

    NASA Astrophysics Data System (ADS)

    Qi, Jian-Xun; Jiang, Fan

    2011-05-01

    The crystallographic temperature factors (B factor) of individual atoms contain important information about the thermal motion of the atoms in a macromolecule. Previously the theory of flexibility of active site has been established based on the observation that the enzyme activity is sensitive to low concentration denaturing agents. It has been found that the loss of enzyme activity occurs well before the disruption of the three-dimensional structural scaffold of the enzyme. To test the theory of conformational flexibility of enzyme active site, crystal structures were perturbed by soaking in low concentration guanidine hydrochloride solutions. It was found that many lysozyme crystals tested could still diffract until the concentration of guanidine hydrochloride reached 3 M. It was also found that the B factors averaged over individually collected data sets were more accurate. Thus it suggested that accurate measurement of crystal temperature factors could be achieved for medium-high or even medium resolution crystals by averaging over multiple data sets. Furthermore, we found that the correctly predicted active sites included not only the more flexible residues, but also some more rigid residues. Both the flexible and the rigid residues in the active site played an important role in forming the active site residue network, covering the majority of the substrate binding residues. Therefore, this experimental prediction method may be useful for characterizing the binding site and the function of a protein, such as drug targeting.

  19. A new set of regulatory molecules in plants: A plant phospholipid similar to platelet-activating factor stimulates protein kinase and proton-translocating ATPase in membrane vesicles.

    PubMed

    Scherer, G F; Martiny-Baron, G; Stoffel, B

    1988-08-01

    1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, an ether phospholipid from mammals known as platelet-activating factor (PAF), specifically stimulates proton transport in zucchini (Cucurbita pepo L.) microsomes (G.F.E. Scherer, 1985, Biochem. Biophys. Res. Commm. 133, 1160-1167). When plant lipids were analyzed by two-dimensional thin-layer chromatography a lipid was found with chromatographic properties very similar to the PAF (G.F.E. Scherer and B. Stoffel, 1987, Planta, 172, 127-130). This lipid was isolated from zucchini hypocotyls, red beet root, lupin root, maize seedlings and crude soybean phospholipids. It had biological activity similar to that of the PAF, based on phosphorus content, and stimulated the steady-state ΔpH in zucchini hypocotyl microsomes about twofold. Other phospholipids, monoglyceride, diglyceride, triglyceride, oleic acid, phorbol ester, and 1-O-alkylglycerol did not stimulate proton transport. When microsomes were washed the PAF was ineffective but when soluble protein was added the PAF stimulation of H(+) transport was reconstituted. The soluble protein responsible for the PAF-dependent stimulation of transport activity could be partially purified by diethylaminoethyl Sephacel column chromatography. In the same fractions where the PAF-dependent transport-stimulatory protien was found, a protein kinase was active. This protein kinase was stimulated twofold either by the PAF or by Ca(2+). When Ca(2+) was present the PAF did not stimulate protein-kinase activity. When either the PAF, protein kinase, or both were added to membranes isolated on a linear sucrose gradient, ATPase activity was stimulated up to 30%. Comparison with marker enzymes indicated the possibility that tonoplast and plasma-membrane H(+)-ATPase might be stimulated by the PAF and protein kinase. We speculate that a PAF-dependent protein kinase is involved in the regulation of proton transport in plants in vitro and in vivo.

  20. 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

  1. Dose-rate effects of protons on in vivo activation of nuclear factor-kappa B and cytokines in mouse bone marrow cells

    SciTech Connect

    Rithidech, K.N.; Rusek, A.; Reungpatthanaphong, P.; Honikel, L.; Simon, S.R.

    2010-05-28

    The objective of this study was to determine the kinetics of nuclear factor-kappa B (NF-{kappa}B) activation and cytokine expression in bone marrow (BM) cells of exposed mice as a function of the dose rate of protons. The cytokines included in this study are pro-inflammatory [i.e., tumor necrosis factor-alpha (TNF-{alpha}), interleukin-1beta (IL-1{beta}), and IL-6] and anti-inflammatory cytokines (i.e., IL-4 and IL-10). We gave male BALB/cJ mice a whole-body exposure to 0 (sham-controls) or 1.0 Gy of 100 MeV protons, delivered at 5 or 10 mGy min{sup -1}, the dose and dose rates found during solar particle events in space. As a reference radiation, groups of mice were exposed to 0 (sham-controls) or 1 Gy of {sup 137}Cs {gamma} rays (10 mGy min{sup -1}). After irradiation, BM cells were collected at 1.5, 3, 24 h, and 1 month for analyses (five mice per treatment group per harvest time). The results indicated that the in vivo time course of effects induced by a single dose of 1 Gy of 100 MeV protons or {sup 137}Cs {gamma} rays, delivered at 10 mGy min{sup -1}, was similar. Although statistically significant levels of NF-{kappa}B activation and pro-inflammatory cytokines in BM cells of exposed mice when compared to those in the corresponding sham controls (Student's t-test, p < 0.05 or < 0.01) were induced by either dose rate, these levels varied over time for each protein. Further, only a dose rate of 5 mGy min{sup -1} induced significant levels of anti-inflammatory cytokines. The results indicate dose-rate effects of protons.

  2. Acetylene hydratase: a non-redox enzyme with tungsten and iron-sulfur centers at the active site.

    PubMed

    Kroneck, Peter M H

    2016-03-01

    In living systems, tungsten is exclusively found in microbial enzymes coordinated by the pyranopterin cofactor, with additional metal coordination provided by oxygen and/or sulfur, and/or selenium atoms in diverse arrangements. Prominent examples are formate dehydrogenase, formylmethanofuran dehydrogenase, and aldehyde oxidoreductase all of which catalyze redox reactions. The bacterial enzyme acetylene hydratase (AH) stands out of its class as it catalyzes the conversion of acetylene to acetaldehyde, clearly a non-redox reaction and a reaction distinct from the reduction of acetylene to ethylene by nitrogenase. AH harbors two pyranopterins bound to W, and a [4Fe-4S] cluster. W is coordinated by four dithiolene sulfur atoms, one cysteine sulfur, and one oxygen ligand. AH activity requires a strong reductant suggesting W(IV) as the active oxidation state. Two different types of reaction pathways have been proposed. The 1.26 Å structure reveals a water molecule coordinated to W which could gain a partially positive net charge by the adjacent protonated Asp-13, enabling a direct attack of C2H2. To access the W-Asp site, a substrate channel was evolved distant from where it is found in other members of the DMSOR family. Computational studies of this second shell mechanism led to unrealistically high energy barriers, and alternative pathways were proposed where C2H2 binds directly to W. The architecture of the catalytic cavity, the specificity for C2H2 and the results from site-directed mutagenesis do not support this first shell mechanism. More investigations including structural information on the binding of C2H2 are needed to present a conclusive answer.

  3. Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site.

    PubMed

    Zhu, Wen-Jing; Li, Miao; Wang, Xiao-Yun

    2007-12-01

    Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (DeltaH degrees ) was 12.38kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK. PMID:17765964

  4. 1993 annual report of hazardous waste activities for the Oak Ridge K-25 site

    SciTech Connect

    Not Available

    1994-02-01

    This report is a detailed listing of all of the Hazardous Waste activities occurring at Martin Marietta`s K-25 site. Contained herein are hazardous waste notification forms, waste stream reports, generator fee forms and various TSDR reports.

  5. Electrocatalytic Hydrogen Production by [Ni(7PPh2NH)2]2+: Removing the Distinction Between Endo- and Exo- Protonation Sites

    SciTech Connect

    Brown, Houston JS; Wiese, Stefan; Roberts, John A.; Bullock, R. Morris; Helm, Monte L.

    2015-04-03

    A new Ni(II) complex, [Ni(7PPh2NH)2]2+ (7PPh2NH = 3,6-triphenyl-1-aza-3,6-diphosphacycloheptane) has been synthesized, and its electrochemical properties are reported. The 7PPh2NH ligand features an NH, ensuring properly positioned protonated amine groups (N–H+) for electrocatalysis, regardless of whether protonation occurs exo- or endo- to the metal center. The compound is an electrocatalyst for H2 production in the presence of organic acids (pKa range 10–13 in CH3CN) with turnover frequencies ranging from 160–770 s-1 at overpotentials between 320–470 mV, as measured at the half peak potential of the catalytic wave. In stark contrast to [Ni(PR2NR'2)2]2+ and other [Ni(7PPh2NR')]2+ complexes, catalytic turnover frequencies for H2 production by [Ni(7PPh2NH)2]2+ do not show catalytic rate enhancement upon the addition of H2O. This finding supports the assertion that [Ni(7PPh2NH)2]2+ eliminates the distinction between the endo- and exo-protonation isomers. This research was supported as part of the Center for Molecular Electrocatalysis, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences. Pacific Northwest National Laboratory is operated by Battelle for the U.S. Department of Energy.

  6. Proton-coupled electron transfer dynamics in the catalytic mechanism of a [NiFe]-hydrogenase.

    PubMed

    Greene, Brandon L; Wu, Chang-Hao; McTernan, Patrick M; Adams, Michael W W; Dyer, R Brian

    2015-04-01

    The movement of protons and electrons is common to the synthesis of all chemical fuels such as H2. Hydrogenases, which catalyze the reversible reduction of protons, necessitate transport and reactivity between protons and electrons, but a detailed mechanism has thus far been elusive. Here, we use a phototriggered chemical potential jump method to rapidly initiate the proton reduction activity of a [NiFe] hydrogenase. Coupling the photochemical initiation approach to nanosecond transient infrared and visible absorbance spectroscopy afforded direct observation of interfacial electron transfer and active site chemistry. Tuning of intramolecular proton transport by pH and isotopic substitution revealed distinct concerted and stepwise proton-coupled electron transfer mechanisms in catalysis. The observed heterogeneity in the two sequential proton-associated reduction processes suggests a highly engineered protein environment modulating catalysis and implicates three new reaction intermediates; Nia-I, Nia-D, and Nia-SR(-). The results establish an elementary mechanistic understanding of catalysis in a [NiFe] hydrogenase with implications in enzymatic proton-coupled electron transfer and biomimetic catalyst design.

  7. Anisotropic Covalency Contributions to Superexchange Pathways in Type One Copper Active Sites

    PubMed Central

    2015-01-01

    Type one (T1) Cu sites deliver electrons to catalytic Cu active sites: the mononuclear type two (T2) Cu site in nitrite reductases (NiRs) and the trinuclear Cu cluster in the multicopper oxidases (MCOs). The T1 Cu and the remote catalytic sites are connected via a Cys-His intramolecular electron-transfer (ET) bridge, which contains two potential ET pathways: P1 through the protein backbone and P2 through the H-bond between the Cys and the His. The high covalency of the T1 Cu–S(Cys) bond is shown here to activate the T1 Cu site for hole superexchange via occupied valence orbitals of the bridge. This covalency-activated electronic coupling (HDA) facilitates long-range ET through both pathways. These pathways can be selectively activated depending on the geometric and electronic structure of the T1 Cu site and thus the anisotropic covalency of the T1 Cu–S(Cys) bond. In NiRs, blue (π-type) T1 sites utilize P1 and green (σ-type) T1 sites utilize P2, with P2 being more efficient. Comparing the MCOs to NiRs, the second-sphere environment changes the conformation of the Cys-His pathway, which selectively activates HDA for superexchange by blue π sites for efficient turnover in catalysis. These studies show that a given protein bridge, here Cys-His, provides different superexchange pathways and electronic couplings depending on the anisotropic covalencies of the donor and acceptor metal sites. PMID:25310460

  8. Probing impact of active site residue mutations on stability and activity of Neisseria polysaccharea amylosucrase.

    PubMed

    Daudé, David; Topham, Christopher M; Remaud-Siméon, Magali; André, Isabelle

    2013-12-01

    The amylosucrase from Neisseria polysaccharea is a transglucosidase from the GH13 family of glycoside-hydrolases that naturally catalyzes the synthesis of α-glucans from the widely available donor sucrose. Interestingly, natural molecular evolution has modeled a dense hydrogen bond network at subsite -1 responsible for the specific recognition of sucrose and conversely, it has loosened interactions at the subsite +1 creating a highly promiscuous subsite +1. The residues forming these subsites are considered to be likely involved in the activity as well as the overall stability of the enzyme. To assess their role, a structure-based approach was followed to reshape the subsite -1. A strategy based on stability change predictions, using the FoldX algorithm, was considered to identify the best candidates for site-directed mutagenesis and guide the construction of a small targeted library. A miniaturized purification protocol was developed and both mutant stability and substrate promiscuity were explored. A range of 8 °C between extreme melting temperature values was observed and some variants were able to synthesize series of oligosaccharides with distributions differing from that of the parental enzyme. The crucial role of subsite -1 was thus highlighted and the biocatalysts generated can now be considered as starting points for further engineering purposes.

  9. 'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.

    PubMed

    Martin, David P; Blachly, Patrick G; Marts, Amy R; Woodruff, Tessa M; de Oliveira, César A F; McCammon, J Andrew; Tierney, David L; Cohen, Seth M

    2014-04-01

    The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins.

  10. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

    SciTech Connect

    Scott, A.I.; Roessner, C.A.; Stolowich, N.J.; Karuso, P.; Williams, H.J.; Grant, S.K.; Gonzalez, M.D.; Hoshino, T. )

    1988-10-18

    The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes es