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J Breton

Publications and source records attributed to J Breton.

At least 55 records · Page 3Linked to original sources
PubMed1997

Electrostatic influence of QA reduction on the IR vibrational mode of the 10a-ester C==O of HA demonstrated by mutations at residues Glu L104 and Trp L100 in reaction centers from Rhodobacter sphaeroides.

The light-induced QA-/QA FTIR difference spectrum of the photoreduction of the primary quinone (QA) in reaction centers (RCs) from Rhodobacter sphaeroides exhibits a set of complex differential bands between 1750 and 1715 cm(-1). Several of these features correspond in frequency to bands that bleach in the HA-/HA FTIR difference spectra of the photoreduction of the bacteriopheophytin electron acceptor (HA). Since the 10a-ester C==O from HA and the side chains of protonated carboxylic acids would be expected to contribute in this spectral region, mutations were designed at Trp L100 and Glu L104, which have been proposed to form hydrogen bonds to the 10a-ester and the 9-keto carbonyls on ring V of HA, respectively. The QA/-/QA spectra measured in IH2O and 2H2O of RCs from wild type (WT) were compared to those of RCs with the mutation Trp to Phe at L100 [WF(L100)], Glu to Leu at L104 [EL(L104)], or both mutations [EL(L104)/WF(L100)]. The spectra of the mutants in the 1800-1400 cm(-1) frequency range exhibit only limited perturbations compared to those of WT, indicating the absence of significant structural changes due to the mutations. Part of a differential signal centered around 1732 cm(-1) in the spectrum of WT RCs is downshifted by approximately 7 cm(-1) in EL(L104), while it is upshifted by approximately 11 cm(-1) in WF(L100). This upshift of the differential signal is assigned to the frequency change of the 10a-ester C==O of HA induced by the rupture of the hydrogen bond with Trp L100. The 1H2O-minus-2H2O double-difference spectrum of WT RCs exhibits a characteristic differential signal positive at 1730 cm-1 and negative at 1724 cm-1 that is absent in the corresponding spectra of EL(L104) and of the double mutant, implicating Glu L104 in the QA-/QA spectral changes. This differential signal is strongly modified in frequency and amplitude in the 1H2O-minus-2H2O spectrum of WF(L100), indicating that it does not correspond to a direct response of the C==O mode of the Glu L104 side chain upon QA reduction. Instead, perturbation of the hydrogen bond of the 9-keto C==O with Glu L104 is proposed to induce a change of electron density on ring V of HA, thereby altering the frequency of the 10a-ester C==O that is in partial conjugation with ring V. The loss of the hydrogen bond to the 9-keto C==O of HA due to the Glu L104 to Leu mutation or the alteration of the strength of the hydrogen bond by 1H/2H exchange on Glu L104 appears to produce such effects. Thus, the QA-/QA spectra above 1700 cm-1 are dominated by contributions from the 10a-ester C==O of HA, with most of the differential signals assigned to a small frequency downshift of the 10a-ester C==O of HA in response to QA reduction. The complexity of the signals implies a structural heterogeneity of the conformation and hydrogen bonding of the 10a-ester C==O of HA, which may be related to the functional heterogeneity observed in electron transfer kinetics. The present FTIR results show that the reduction of QA can induce a pronounced electrostatic effect on molecular vibrations of chemical groups located about 10 A away from QA. They also demonstrate that, within experimental limits, the proton uptake observed at pH 7 upon QA photoreduction [McPherson, P. H., Okamura, M. Y., & Feher, G. (1988) Biochim. Biophys. Acta 934, 348-368] involves none of the exchangeable carboxylic groups of the RC.

Amino Acid Sequence↗
PubMed1996

Fourier transform infrared difference study of tyrosineD oxidation and plastoquinone QA reduction in photosystem II.

Two redox active tyrosines are present in the homologous polypeptides D1 and D2 of photo-system II (PS II). TyrZ (D1-161) is involved in the electron transfer reactions resulting in oxygen evolution, while TyrD (D2-160) usually forms a dark-stable radical. In Mn-depleted PS II, TyrD. can be slowly reduced by exogenous reductants. Charge separation then results in the oxidation of TyrD and TyrZ and the reduction of the primary electron acceptor QA. The semiquinone QA- can be reoxidized by oxidants like ferricyanide. In the present work, experimental conditions leading to the generation of pure QA-/QA or TyrD./TyrD FTIR difference spectra have been optimized. Therefore, single-turnover flashes or short illuminations were performed on PS II samples in the presence of exogenous reductants or oxidants. The QA- and TyrD. radicals were generated with high yield and with a lifetime of several seconds or minutes allowing averaging of FTIR difference spectra with high signal to noise ratio. Both QA- formation and contributions at the electron donor side of PS II were monitored by EPR spectroscopy. In PS II samples at pH 6 in the presence of PMS, NH2OH, and DCMU, EPR measurements show that QA- is formed with high yield upon a 1 s illumination at 10 degrees C, while no radical from the electron donor side of PS II is detected. Therefore the QA-/QA FTIR spectrum obtained in these conditions shows only vibrational changes due to QA reduction in PS II. In contrast, a similar spectrum was recently interpreted in terms of dominant contributions from Chl+/Chl signals [MacDonald, G. M., Steenhuis, J. J., & Barry, B. A. (1995) J. Biol. Chem. 270, 8420-8428], although the contribution from the electron acceptor QA was not quantified. In particular, it is shown here that the large positive signal at 1478 cm-1 is due to the QA- state and not to a Chl+ mode. This band is not downshifted upon 15N-labeling of spinach PS II membranes within the +/- 1 cm-1 accuracy of the method and is therefore tentatively assigned to the v(C[symbol: see text]O) mode of the plastosemiquinone QA-. Also unchanged upon 15N-labeling, signals at 1644 and/or 1630 cm-1 are possible candidates for the v(C = O) mode(s) of neutral QA in PS II. The TyrD./TyrD FTIR spectrum is recorded at 4 degrees C on Tris-washed PS II membranes from spinach at pH 6 in the presence of phosphate, formate, and ferricyanide. EPR experiments performed on these samples show that almost all TyrD. is formed upon a 1 s illumination at 4 degrees C and that TyrD. is then reduced within 12 min in the dark. No contributions from TyrZ. or QA- are detected 2 s after illumination. It is thus possible to optimize experimental conditions to record the FTIR difference spectrum only due to TyrD photooxidation in PS II-enriched membranes of spinach. The TyrD./TyrD FTIR spectrum is compared to a cresol./cresol FTIR difference spectrum obtained by UV irradiation at 10 K of cresol at pH 8. The spectral analogies observed between the in vivo and in vitro spectra recorded either in H2O or in D2O suggest that IR modes of TyrD contribute at 1513 and 1252 cm-1. These frequencies are characteristic of a protonated tyrosine. A positive signal is observed at 1506 cm-1 for cresol. and at 1504 cm-1 for the TyrD. state. This suggests contribution of the TyrD. side chain at 1504 cm-1. A band at 1473 cm-1 was previously assigned to the v(CO) mode of TyrD. [MacDonald, G. M., Bixby, K. A., & Barry, B. A. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11024-11028]. In contrast, no positive signal is observed at 1473 cm-1 in the TyrD./TyrD FTIR difference spectrum presented here. The TyrD./TyrD spectrum also shows vibrational changes from peptide groups and amino acid side chains which are modified upon TyrD. formation. Proton release at the PS II protein surface upon TyrD. formation is deduced from differential signals at the v(PO) modes of phosphate.

Electron Spin Resonance Spectroscopy↗
PubMed1996

Probing native-like orientation of pigments in modified reaction centers from Rhodobacter sphaeroides R26 by linear dichroism.

Site-specific pigment modifications are useful to investigate structure-function relationships in photosynthesis. In reaction centers bearing modified (bacterio)pheophytins, changed electron transfer kinetics have been related to the changed redox potentials of the pigments introduced (Huber, H. et al. (1995) Chemical Physics, Special Issue, vol 197 (Hochstrasser, R.M. and Hofacker, G.L. eds.) pp. 297-305; [1]). In order to analyze potentially interfering structural changes induced in these reaction centers by the exchange procedure, in particular mispositioning or misorientation of the pigments, low-temperature linear dichroism spectra have been measured for reaction centers from Rhodobacter sphaeroides containing modified bacteriopheophytins and bacteriochlorophylls at the sites HA,B and BA,B, respectively. They show that all modified pigments are oriented similar to the native ones, and that they do not affect significantly the linear dichroism of the monomeric bacteriocholorophylls and bacteriopheophytins or of the primary donor.

Bacteriochlorophylls↗
PubMed1996

Conformational changes induced by phosphorylation in the CP29 subunit of photosystem II.

Light energy absorbed by the chloroplast membranes of higher plants is dissipated by nonradiative de-excitation in order to protect against photodamage. In photosystem II, which is the photosynthetic component most sensitive to photoinhibition, three pigment binding subunits, called CP24, CP26, and CP29, have been proposed to act in the regulation of the chlorophyll excited states concentration. In heavy stress conditions, CP29 becomes phosphorylated in its stroma-exposed portion, and this process is reversed by returning to normal conditions. In this study, we have used the pigments bound to the intramembrane hydrophobic portion of the protein as intrinsic probes to detect conformational changes induced by phosphorylation. We isolated the phosphorylated and unphosphorylated forms of the protein and showed that, although they have the same pigment complement, spectral differences can be consistently detected by absorption, linear dichroism, and circular dichroism spectroscopy. Alkaline phosphatase treatment of the phosphoprotein restores both the electrophoretic mobility and the spectral properties of the unphosphorylated CP29. The results of this study show that phosphorylation of CP29 can modulate the spectral properties of this photosystem II subunit and provide a possible mechanism for the regulation of excitation energy supply to the reaction center.

Alkaline Phosphatase↗
PubMed1996

Energy and electron transfer upon selective femtosecond excitation of pigments in membranes of Heliobacillus mobilis.

Excitation energy transfer steps in membranes of Heliobacillus mobilis were directly monitored by transient absorption spectroscopy with a time resolution of 30 fs under selective excitation within the inhomogeneously broadened bacteriochlorophyll g QY band. The initial anisotropy was found to be > 0.4, indicating that the pigments are excitonically coupled. After initial decay of this anisotropy in < 50 fs, major sub-picosecond components associated with spectral equilibration were identified, corresponding to uphill energy transfer with a 300 fs time constant (812 nm excitation) and downhill energy transfer with 100 and 500 fs components (770 nm excitation). These equilibrations are ascribed predominantly to single excitation transfer steps, as anisotropy measurements showed that equilibration within spectrally similar pigments occurs on the same time scale as spectral equilibration, a situation which contrasts with that in photosystem I. Downhill energy transfer occurs to a significant extent directly to an energetically heterogeneous population of excited states as well as in a sequential way via gradually lower-lying pools of bacteriochlorophyll g. This finding supports a description in which all pigments, including the bluemost absorbing, are spatially organized in a random way rather than in clusters of spectrally similar species. Spectral equilibration is not entirely completed prior to formation of the primary radical pair P798 + A0-, which was found to proceed in a multiexponential way (time constants of 5 and 30 ps). No indication for the formation of radical species other than P798 + A0- on the time scale up to 100 ps was found.

Bacteria↗
PubMed1996

Identification of residues of Rhodobacter capsulatus ferredoxin I important for its interaction with nitrogenase.

In Rhodobacter capsulatus, ferredoxin I (FdI) serves as natural electron donor to nitrogenase. In order to probe amino acid residues possibly involved in the interaction with dinitrogenase reductase, FdI was subjected to site-specific mutagenesis. A three-dimensional structure of FdI was designed by computer modelling and used for selecting target residues. Mutant ferredoxins bearing substitutions of surface residues, as well as a variant having a Met2 --> Tyr replacement in the vicinity of one cluster, have been constructed. All FdI variants were expressed to similar levels both in Escherichia coli and in a FdI-deleted mutant of the natural host. Once purified, the mutant ferredoxins exhibited molecular and spectroscopic properties almost identical to wild-type FdI. Determination of the reduction potential of FdI by cyclic voltammetry gave an E'o of -510 mV (pH 7.6) for both clusters, which is one of the lowest values reported for a 2[4Fe-4S] ferredoxin. Only the [Tyr2]FdI variant showed a significant difference in redox potential (delta E'o = -15 mV). Based on in vitro assays, a [Glu27, Glu28]FdI double mutant exhibited a twofold decrease in the electron transfer rate to dinitrogenase reductase while the affinity of this mutant for the enzyme was barely affected. On the other hand, an Asp36 --> His substitution resulted in a sevenfold increase of the apparent Km for dinitrogenase reductase. Unlike FdI and the other mutant ferredoxins, the [His36]FdI variant also failed to form a cross-linked complex with dinitrogenase reductase upon incubation with a carbodiimide. It is concluded that Asp36 in FdI probably participates in the interaction between the two protein partners. Nevertheless, all the FdI mutants proved competent in restoring a wild-type phenotype when expressed in a FdI-deleted mutant background, indicating that none of the studied residues was absolutely critical for electron transfer to nitrogenase.

Amino Acid Sequence↗
PubMed1996

Vibrational dephasing of long- and short-lived primary donor excited states in mutant reaction centers of Rhodobacter sphaeroides.

Femtosecond spectroscopy was used to study vibrational dynamics in the first singlet excited state (P*) of the primary donor of bacterial reaction centers (RC)in which primary electron transfer dynamics have been altered by single amino acid modifications. We studied intracytoplasmic RC-only membranes containing Rhodobacter sphaeroides wild-type RCs and RCs bearing mutations in the vicinity of P, where Tyr M210 was modified to His, Leu, and Trp and where Phe L181 was modified to Tyr. These mutations do not change the frequencies of the main low-frequency activated modes, which is consistent with a description in which these modes involve extended regions of the protein. Electron transfer in FL181Y, YM210H, and wild-type RCs at 10 K occurs in approximately 1 ps or less, and damping of the coherences occurs simultaneously with the decay of the P* excited state. These results, and a comparison with YM210L RCs, show that in wild-type RCs the damping is primarily determined by the depletion of P* and not by vibrational dephasing induced by interactions with the bath or nonharmonic coupling. In the YM210L and W mutants, electron transfer occurs on a time scale of hundreds of picoseconds at 10 K. Analysis of the longer-lasting vibrational dynamics in these mutants sets a new lower limit for the intrinsic vibrational dephasing time of 1.2 ps for some modes, but of approximately 2 ps for most activated modes.

Electron Transport↗
PubMed1996

Human keratinocytes lack the components to produce leukotriene B4.

The cellular origin of leukotriene B4 (LTB4), a potent pro-inflammatory molecule present in psoriatic lesions, has yet to be determined. In the present study, cultured human keratinocytes were evaluated for their ability to produce LTB4. Keratinocytes stimulated under a variety of conditions did not produce detectable amounts of LTB4, as measured by enzyme immunoassay and liquid chromatographic techniques. Prostaglandin E2 and 15-hydroxyeicosatetraenoic acid were the only eicosanoids detected. The capacity of keratinocytes to synthesize 5-lipoxygenase (5-LO) products, or lack thereof, was further evaluated by preparing subcellular fractions and examining them for the presence of 5-LO activity and the proteins responsible for LTB4 production. Using Western blot analysis, we detected no bands that migrated with the 78-kDa 5-LO enzyme. Subcellular fractions were also examined for the presence of the 5-LO-activating protein (FLAP). This protein, which is essential to 5-LO activity, could not be detected in any keratinocyte preparation examined. Consistent with the absence of proteins, the mRNAs for 5-LO and FLAP were undetectable by reverse transcriptase polymerase chain reactions analysis. These results demonstrate that human keratinocytes lack the crucial proteins necessary for LTB4 production.

5-Lipoxygenase-Activating Proteins↗
PubMed1995

Fourier transforms infrared difference spectroscopy of secondary quinone acceptor photoreduction in proton transfer mutants of Rhodobacter sphaeroides.

In order to investigate the changes of protonation or environment of carboxylic residues occurring upon photoreduction of the secondary quinone acceptor (QB) in the reaction center (RC) of the photosynthetic bacteria Rhodobacter sphaeroides 2.4.1., we have performed light-induced Fourier transform infrared (FTIR) spectroscopy on RCs from wild-type (Wt) and several site-directed mutants. The FTIR QB-/QB spectra have been obtained at pH 7 upon single-saturating flash excitation for native RCs and RC mutants containing either a single-site mutation, with Gln at L212 (EQ L212), Asn at L213 (DN L213), or Asn at L210 (DN L210), or a double-site mutation with both Gln at L212 and Asn at L213 (EQ L212 + DN L213). The assignment of an IR band to the protonation/deprotonation of a particular carboxylic side chain was analyzed by combining the effects of site-directed mutagenesis and 1H/2H isotope exchange. A positive band at 1728 cm-1 in the QB-/QB spectra was observed in Wt, DN L213, and DN L210 and was absent in the mutants EQ L212 and EQ L212 + DN L213. The intensity of the 1728 cm-1 band was significantly reduced in 2H2O, and a new feature appears at 1717 +/- 1 cm-1. Furthermore, the amplitude of the 1728 cm-1 band was similar in native and DN L210 RCs but was increased in DN L213. This band is attributed to partial proton uptake by Glu L212 estimated to be 0.3-0.4 H+/QB- in native and DN L210 RCs and O.5-0.6 H+/QB- in DN L213 RCs. In contrast, the FTIR QB-/QB spectra show no evidence for change of protonation or environment of Asp L213 upon QB- formation. The increased protonation of Glu L212 in DN L213 RCs is explained by a decreased Glu L212 pKa value due to the loss of a negatively charged Asp L213. Part of a small differential signal at 1732 (+)/1740 (-) cm-1 that is affected by 1H/2H exchange is tentatively assigned to an environmental shift of the protonated Asp L210. A negative signal at 1685 cm-1 is propose to arise from the absorption change of the amide I carbonyl mode of Glu L212.(ABSTRACT TRUNCATED AT 400 WORDS)

Electron Transport↗
PubMed1995

Binding sites of quinones in photosynthetic bacterial reaction centers investigated by light-induced FTIR difference spectroscopy: symmetry of the carbonyl interactions and close equivalence of the QB vibrations in Rhodobacter sphaeroides and Rhodopseudomonas viridis probed by isotope labeling.

The photoreduction of the secondary quinone acceptor QB in reaction centers (RCs) of the photosynthetic bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis has been investigated by light-induced FTIR difference spectroscopy of RCs reconstituted with several isotopically labeled ubiquinones. The labels used were 18O on both carbonyls and 13C either uniformly or selectively at the 1- or the 4-position, i.e., on either one of the two carbonyls. The QB-/QB spectra of RCs reconstituted with the isotopically labeled and unlabeled quinones as well as the double differences calculated from these spectra exhibit distinct isotopic shifts for a number of bands attributed to vibrations of QB and QB-. The vibrational modes of the quinone in the QB site are compared to those of ubiquinone in vitro, leading to band assignments for the C = O and C = C vibrations of the neutral QB and for the C***O and C***C of the semiquinone. The C = O frequency of each of the carbonyls of the unlabeled quinone is revealed at 1641 cm-1 for both species. This demonstrates symmetrical and weak hydrogen bonding of the two C = O groups to the protein at the QB site. In contrast, the C = C vibrations are not equivalent for selective labeling at C1 or at C4, although they both contribute to the approximately 1617-cm-1 band in the QB-/QB spectra of the two species. Compared to the vibrations of isolated ubiquinone, the C = C mode of QB does not involve displacement of the C4 carbon atom, while the motion of C1 is not hindered. Further analysis of the the spectra suggests that the protein at the binding site imposes a specific constraint on the methoxy and/or the methyl group proximal to the C4 carbonyl. The FTIR observations provide compelling evidence for almost identical conformation and identical interactions of the ubiquinone in the QB binding site of Rb. sphaeroides and Rp. viridis in contrast to the X-ray structures, which yield different descriptions for the hydrogen-bonding pattern of QB binding. In the semiquinone state, the bonding interactions of the C***O groups are also symmetrical and the C***C are inequivalent at C1 and C4. However, the interactions are almost the same in the RCs of both species.

Binding Sites↗
PubMed1995

Photoelectric characterization of forward electron transfer to iron-sulfur centers in photosystem I.

The photoelectric response of oriented PS I membranes from the cyanobacterium Synechocystis 6803 has been investigated in the nanosecond time range. Besides an unresolved rapidly rising phase, there is a further positive electrogenic phase with a rise time constant of 220 +/- 20 ns. The amplitude of the 220-ns phase is 66 +/- 10% that of the subnanosecond phase. The fast phase contains two kinetic components faster than 100 ps, which have recently been resolved and attributed to primary charge separation (P+Ao-formation) and subsequent electron transfer to A1, respectively (Hecks, B., Wulf, K., Breton, J., Leibl, W., & Trissl, H.-W. (1994) Biochemistry 33, 8619-8624). The 220-ns phase is lost under conditions where iron-sulfur centers FA, FB, and Fx are prereduced, and its kinetics match the reoxidation kinetics of A1- as verified by absorbance change measurements at 380 nm. Therefore, this electrogenic phase is attributed to electron transfer to the iron-sulfur centers that function as further electron acceptors in the PS I reaction center. Gradual removal of FA and FB by urea treatment reveals that the amplitude of the 220-ns phase is linearly correlated with the fraction of FA,B present. However, complete removal of FA,B does not lead to a complete loss of the nanosecond phase but reduces its amplitude by more than a factor of 2 to yield an amplitude of 25-30% relative to the initial picosecond rise, with only a slight change in kinetics. The residual amplitude is further reduced when a large fraction of Fx is removed.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyanobacteria↗
PubMed1995

Detection of traces of a trisulphide derivative in the preparation of a recombinant truncated interleukin-6 mutein.

A new mutein of interleukin-6, called delta 22-IL-6 Cys 3,4, characterized by the deletion of the first 22 amino acids at the N-terminal end and by the substitution of the first two cysteines (Cys23 and Cys29) with serine residues, was produced in Escherichia coli and was found to maintain the structural and functional properties of the human native form. A partially purified preparation still showed in isoelectric focusing a minor acidic component (pI 6.10) and a more basic component (pI 6.70), the native form having a pI of 6.56. This preparation was further fractionated in a multi-compartment electrolyser with isoelectric membranes, which allowed the collection of the more alkaline species for characterization. Mass spectra of the pI 6.70 form gave an additional mass of 32 atomic mass units (amu), suggesting the addition of two oxygen atoms (a potential oxidation of two methionine residues to sulphoxide). However, the five methionine residues in this higher pI form were identified after enzymatic hydrolysis and peptide mapping and were found to be in a reduced state. In addition, the pI 6.70 form was quickly converted into the native form by mild reductive treatment. On digestion and fingerprinting, the peptide from residues 50 to 65 of the pI 6.70 species (containing the only two cysteine residues of the molecule) exhibited a more hydrophobic behaviour in reversed-phase high-performance liquid chromatography and retained a mass increase of 32 amu. These experimental findings more likely suggest the addition of an extra sulphur atom to the only disulphide bridge to give an unusual protein trisulphide molecule.

Amino Acid Sequence↗
PubMed1995

Prolonged half-life in the circulation of a chemical conjugate between a pro-urokinase derivative and human serum albumin.

Pro-urokinase is a natural plasminogen activator that displays a clot-lysis activity through a fibrin-dependent mechanism. It seems to be a promising agent for the treatment of coronary thrombosis. Like tissue-type plasminogen activator and two-chain urokinase-type plasminogen activator, pro-urokinase has a very short half-life in circulation. It has been described that conjugation of serum albumin with pro-urokinase in plasma may occur that could protect this protein from degradation. In this study we describe the insertion of an extra cysteine residue in the N-terminal end of des-(C11-K135)-pro-urokinase (delta 125-proUK), a pro-urokinase deletion mutant lacking amino acids 11-135. We have expressed and purified the new mutein [H5K, S9C, N10T] des-(C11-K135)-pro-urokinase (Cys-delta 125-pro-urokinase) and chemically conjugated it with serum albumin via the extra cysteine of Cys-delta-pro-urokinase. The purified conjugate obtained has a lower specific amidolytic activity (72,000 U/mg) than unconjugated Cys-delta 125-pro-urikinase (240,000 U/mg) due to its higher molecular mass and has a similar fibrinolytic activity in a clot lysis test to that of delta 125-pro-urokinase. We established an ELISA to measure the concentration of the conjugate in plasma and to follow the pharmacokinetics of the conjugate in monkeys after bolus injection. The conjugate displays significant lysis of human plasma clots in vivo and a dramatic increase of the half-life in the circulation, with respect to pro-urokinase and delta 125-pro-urokinase. Therefore, preliminary biological characterisation of this conjugate indicates that it could be a good candidate to inject as a bolus, compared with the infusion regimen needed with pro-urokinase.

Amino Acid Sequence↗
PubMed1995

Determination of the [Fe4S4]Cys4 cluster geometry of Desulfovibrio africanus ferredoxin I by 1H NMR spectroscopy.

1D and 2D 1H NMR studies of the Fe4S4 cluster containing ferredoxin I from Desulfovibrio africanus have been carried out with the aim of determining the geometry of the cluster linkages with the 4 Cys side chains that bind the cluster. This required the Cys beta CH resonances of the oxidised protein to be sequence-specifically and stereo-specifically assigned, and this was accomplished by a combination of TOCSY and NOE measurements, allied to model building based on X-ray structures of related ferredoxins. An analysis of the estimated hyperfine shifts of the Cys beta CH resonances with a Karplus-type equation relating the shifts to iron-sulfur-beta carbon-beta proton dihedral angles, taken together with the relative relaxation rates of the two beta CH2 resonances, estimated from their linewidths, then allowed the iron-sulfur-beta-carbon-alpha-carbon dihedral angles to be determined. A novel representation of the NMR data is presented which shows that the cluster dihedral angles are uniquely determined by the NMR data. The analysis reveals that the dihedral angles for D. africanus ferredoxin I are similar to the corresponding angles of other ferredoxins even though there are differences in their 1H NMR spectra. The sequence-specific and stereospecific assignments have been extended by analogy to the related Fe4S4-containing D. gigas ferredoxin I, and the stereospecific assignments to the Fe4S4-containing Thermococcus litoralis ferredoxin.

Chemical Phenomena↗
PubMed1995

Protonation of Glu L212 following QB- formation in the photosynthetic reaction center of Rhodobacter sphaeroides: evidence from time-resolved infrared spectroscopy.

The protonation events that occur upon QA-QB-->QAQB- electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides were investigated by time-resolved infrared spectroscopy using tunable diode lasers as previously described [Mäntele, W., Hienerwadel, R., Lenz, F., Riedel, E. J., Grisar, R., & Tacke, M. (1990) Spectrosc. Int. 2, 29-35; Hienerwadel, R., Thibodeau, D. L., Lenz, F., Nabedryk, E., Breton, J., Kreutz, W., & Mäntele, W. (1992) Biochemistry 31, 5799-5808]. In the mid-infrared region between 1695 and 1780 cm-1, transient signals associated with QA-QB-->QAQB- electron transfer were observed and characterized. The dominant transient absorbance changes are three positive signals at 1732, 1725, and 1706 cm-1 and two negative signals at 1716 and at 1698 cm-1. The 1725 cm-1-signal disappears upon 1H-->2H exchange as expected for an accessible COOH group and is absent in Glu L212 Gln mutant reaction centers. On this basis, we propose an assignment of this signal to the COOH group of Glu L212. The other signals could correspond to intensity changes and/or shifts of other carboxylic residues, although contributions from ester C = O groups of bacteriopheophytins cannot be ruled out. In native reaction centers at pH 7 and at 4 degrees C, biphasic kinetics of the transient components were observed at most frequencies. The major signal at 1725 cm-1 exhibits a fast kinetic component of t 1/2 = 0.18 ms (25% of the total amplitude) and a slow one of t1/2 = 1 ms (75% of the total amplitude). A global fit analysis of the signals between 1695 and 1780 cm-1 revealed that the spectral distributions of the fast and the slow components are different. Biphasic kinetics with comparable half-times were also observed for the Glu L212 to Gln mutant. The simplest model to explain these results is that the fast phase represents electron transfer and the slow phase represents proton transfer and/or conformational changes coupled to electron transfer. The difference spectra of the slow component from native reaction centers show that the 1725 cm-1 band corresponds to an absorbance increase and not to a shift of an existing band. The signal is therefore proposed to arise from the protonation of Glu L212. The amplitude of the 1725 cm-1 signal varies distinctly with pH as expected for protonation of a COO- group. With increasing pH, the amplitude of the slow component increases while that of the fast component decreases slightly.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1995

Structure, stability and biological properties of a N-terminally truncated form of recombinant human interleukin-6 containing a single disulfide bond.

A mutant species of the 185-residue chain of human interleukin-6 lacking 22-residues at its N-terminus and with a Cys-->Ser substitution at positions 45 and 51 was produced in Escherichia coli. The 163-residue protein des-(A1-S22)-[C45S, C51S]interleukin-6, containing a single disulfide bridge, formed inclusion bodies. Mutant interleukin-6 was solubilized in 6 M guanidine hydrochloride, subjected to oxidative refolding and purified to homogeneity by ammonium sulfate precipitation and hydrophobic chromatography. The purity of the mutant species was established by electrophoresis, isoelectrofocusing and reverse-phase HPLC and its structural identity was checked by N-terminal sequencing of both the intact protein and several of its proteolytic fragments. Electrospray mass spectrometry analysis of mutant interleukin-6 gave a molecular mass of 18,695 +/- 2 Da in excellent agreement with the calculated value. Circular dichroic, fluorescence emission and second-derivative ultraviolet absorption spectra indicated that mutant interleukin-6 maintains the overall secondary and tertiary structure, as well as stability characteristics, of the recombinant wild-type human interleukin-6. The urea-induced unfolding of mutant interleukin-6, monitored by circular dichroic measurements in the far-ultraviolet region, occurs as a highly cooperative process with a midpoint of denaturation at 5.5 M urea. The data of the reversible unfolding of mutant interleukin-6 mediated by urea were used to calculate a value of 20.9 +/- 0.4 kJ.mol-1 for the thermodynamic stability of the protein at 25 degrees C in the absence of denaturant. The biological activity of mutant interleukin-6 was evaluated in vitro by the hybridoma proliferation assay, and in vivo by measuring thrombopoiesis in monkeys. Dose/response effects of the mutant were comparable or even higher than those of the wild-type protein. Overall the results of this study show that mutant interleukin-6 is a biologically active cytokine, which could find practical use as a therapeutic agent.

Amino Acid Sequence↗
PubMed1995

Characterization of proteins by sequential isoelectric focusing on immobilized pH gradients and electrospray mass spectrometry.

The coupling of isoelectric focusing on immobilized pH gradients (IPG) with electrospray-mass spectrometry (ES-MS) was applied to the characterization of proteins according to two different and important properties, such as net surface charge and molecular mass. From a technical point of view, these methods are complementary, since ES-MS requires ion-free samples as usually supplied by isoelectric focusing on IPGs. This report describes the experiments carried out on model proteins to demonstrate the feasibility of the sequential application of these two techniques for the characterization of proteins. A minimum of 5 micrograms protein was needed for good signal by mass spectrometry. The following proteins were studied: myoglobin, truncated interleukin-6-mutein, recombinant cytochrome c551 and insulin-like growth factor I. Extraction from the IPG matrix was carried out in 70% acetonitrile/30% water/0.05% trifluoroacetic acid either by passive diffusion or by centrifugation through a 0.22 micron Amicon membrane, with protein recoveries of 80-85%.

Animals↗