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H Michel

Publications and source records attributed to H Michel.

At least 19 recordsLinked to original sources
PubMed2003

Asymmetric binding of the high-affinity Q(H)(*)(-) ubisemiquinone in quinol oxidase (bo3) from Escherichia coli studied by multifrequency electron paramagnetic resonance spectroscopy.

Ubiquinone-2 (UQ-2) selectively labeled with (13)C (I =(1)/(2)) at either the position 1- or the 4-carbonyl carbon is incorporated into the ubiquinol oxidase bo(3) from Escherichia coli in which the native quinone (UQ-8) has been previously removed. The resulting stabilized anion radical in the high-affinity quinone-binding site (Q(H)(*)(-)) is investigated using multifrequency (9, 34, and 94 GHz) electron paramagnetic resonance (EPR) spectroscopy. The corresponding spectra reveal dramatic differences in (13)C hyperfine couplings indicating a strongly asymmetric spin density distribution over the quinone headgroup. By comparison with previous results on labeled ubisemiquinones in proteins as well as in organic solvents, it is concluded that Q(H)(*)(-) is most probably bound to the protein via a one-sided hydrogen bond or a strongly asymmetric hydrogen-bonding network. This observation is discussed with regard to the function of Q(H) in the enzyme and contrasted with the information available on other protein-bound semiquinone radicals.

Binding Sites↗
PubMed2002

Palliative home care: improving co-operation between the specialist team and the family doctor.

The aim of this study is to ascertain family doctors' opinions of the home care services dispensed by a specialist palliative care team. Fifty-six doctors responded to the questionnaire they had been sent. Ninety-four per cent of them were satisfied with the team's intervention. The highest satisfaction ratings were those for the provision of specialist medical equipment, technical competence and medical support. The lowest ratings were for information coming from the team and for recognition of the practitioner as the primary carer. Among these items, information, regard for the doctor's role and medical support are closely correlated to the practitioners' overall index of satisfaction. The palliative care team answered a mirror questionnaire. It found its availability, its speed of intervention, its technical competence, and its regard for the doctor's role very satisfactory. It was less satisfied with the information, medical support and psychosocial support it provided. Its centres of interest also differed, with the team clearly attributing greater importance to psychosocial support than to technical competence, regard for the role, and information to the practitioner. In conclusion, a survey of this type is a useful and easy way of identifying the aspects that need to be improved on by the palliative care team so as to improve its co-operation with the family physician. But it is of interest only if it forms part of an overall approach whereby, having taken the survey results on board, the team uses them to modify its intervention procedures.

Adult↗
PubMed2002

Plutonium and americium inventories in atmospheric fallout and sediment cores from Blelham Tarn, Cumbria (UK).

The objective of this paper is to report on the results of a study of 238Pu, 239 + 240Pu and 241Am inventories onto Blelham Tarn in Cumbria (UK). The atmospheric fallout inventory was obtained by analysing soil cores and the results are in good agreement with the literature: 101 Bq m(-2) for 239 + 240Pu; 4.5 Bq m(-2) for 238Pu and 37 Bq m(-2) for 241Am. The sediment core inventory for the whole lake is compared to the atmospheric fallout inventory. The sediment activity is 60-80% higher than the estimated fallout activity, showing a catchment area contribution and in particular the stream input.

Americium↗
PubMed2001

X-ray structure analyses of photosynthetic reaction center variants from Rhodobacter sphaeroides: structural changes induced by point mutations at position L209 modulate electron and proton transfer.

The structures of the reaction center variants Pro L209 --> Tyr, Pro L209 --> Phe, and Pro L209 --> Glu from the photosynthetic purple bacterium Rhodobacter sphaeroides have been determined by X-ray crystallography to 2.6-2.8 A resolution. These variants were constructed to interrupt a chain of tightly bound water molecules that was assumed to facilitate proton transfer from the cytoplasm to the secondary quinone Q(B) [Baciou, L., and Michel, H. (1995) Biochemistry 34, 7967-7972]. However, the amino acid exchanges Pro L209 --> Tyr and Pro L209 --> Phe do not interrupt the water chain. Both aromatic side chains are oriented away from this water chain and interact with three surrounding polar side chains (Asp L213, Thr L226, and Glu H173) which are displaced by up to 2.6 A. The conformational changes induced by the bulky aromatic rings of Tyr L209 and Phe L209 lead to unexpected displacements of Q(B) compared to the wild-type protein. In the structure of the Pro L209 --> Tyr variant, Q(B) is shifted by approximately 4 A and is now located at a position similar to that reported for the wild-type reaction center after illumination [Stowell, M. H. B., et al. (1997) Science 276, 812-816]. In the Pro L209 --> Phe variant, the electron density map reveals an intermediate Q(B) position between the binding sites of the wild-type protein in the dark and the Pro L209 --> Tyr protein. In the Pro L209 --> Glu reaction center, the carboxylic side chain of Glu L209 is located within the water chain, and the binding site of Q(B) remains unchanged compared to the wild-type structure.

Amino Acid Substitution↗
PubMed2001

QH*- ubisemiquinone radical in the bo3-type ubiquinol oxidase studied by pulsed electron paramagnetic resonance and hyperfine sublevel correlation spectroscopy.

The high-affinity QH ubiquinone-binding site in the bo(3) ubiquinol oxidase from Escherichia coli has been characterized by an investigation of the native ubiquinone radical anion QH(*-) by pulsed electron paramagnetic resonance (EPR) spectroscopy. One- and two-dimensional electron spin-echo envelope modulation (ESEEM) spectra reveal strong interactions of the unpaired electron of QH(*-) with a nitrogen nucleus from the surrounding protein matrix. From analysis of the experimental data, the (14)N nuclear quadrupolar parameters have been determined: kappa = e(2)qQ/4h = 0.93 MHz and eta = 0.50. This assignment is confirmed by hyperfine sublevel correlation (HYSCORE) spectroscopy. On the basis of a comparison of these data with those obtained previously for other membrane-protein bound semiquinone radicals and model systems, this nucleus is assigned to a protein backbone nitrogen. This result is discussed with regard to the location and potential function of QH in the enzyme.

Anions↗
PubMed2001

Analysis of a putative voltage-gated prokaryotic potassium channel.

Most of the completely sequenced prokaryotic genomes contain genes of potassium channel homologues, but there is still not much known about the role of these proteins in prokaryotes. Here we describe the large-scale overproduction and purification of a prokaryotic voltage-gated potassium channel homologue, Kch, from Escherichia coli. After successful overproduction of the protein, a specific increase in the potassium permeability of the cells was found. Kch could be purified in large amounts using classical purification methods to prevent aggregation of the protein. The physiological state of the protein was revealed to be a homotetramer and the protein was shown to be localized to the cytoplasmic membrane of the cells. In the course of the localization studies, we found a specific increase in the density of the cytoplasmic membrane on Kch production. This was linked to the observed increase in the protein to lipid ratio in the membranes. Another observed change in the membrane composition was an increase in the cardiolipin to phosphatidylglycerol ratio, which may indicate a specific cardiolipin requirement of Kch. On the basis of some of our results, we discuss a function for Kch in the maintenance of the membrane potential in E. coli.

Cell Membrane↗
PubMed2001

Solubilization and purification of the human ETB endothelin receptor produced by high-level fermentation in Pichia pastoris.

In the present report, the successful solubilization and purification of the ETB receptor heterologously produced in the methylotrophic yeast P. pastoris is described for the first time. In comparison to the baculovirus system where successful production, solubilization and purification have already been reported, handling and up-scaling of recombinant P. pastoris cells was much easier and less time consuming. Recombinant P. pastoris clones producing two different ETB receptor constructs were grown in a fermenter to a density of about 360 g/l. After induction with methanol, a production level of maximally 45 pmol/mg was obtained, a value which is in the range of that reported for baculovirus-infected insect cells. A method for the large-scale preparation of membranes was established. Solubilization of the recombinant ETB receptor was achieved with the detergent n-dodecyl-/beta-D-maltopyranoside. The stability of the solubilized and ligand-bound receptor was examined in detail. Subsequently, two purification methods for two different receptor constructs were tested and a large-scale procedure for isolation of recombinant receptor was established. In general, the purification methods described herein will be adaptable to other G protein-coupled receptors heterologously produced in heterologous expression systems including P. pastoris.

Binding Sites↗
PubMed2000

Structural basis of the drastically increased initial electron transfer rate in the reaction center from a Rhodopseudomonas viridis mutant described at 2.00-A resolution.

It has previously been shown that replacement of the residue His L168 with Phe (HL168F) in the Rhodopseudomonas viridis reaction center (RC) leads to an unprecedented drastic acceleration of the initial electron transfer rate. Here we describe the determination of the x-ray crystal structure at 2.00-A resolution of the HL168F RC. The electron density maps confirm that a hydrogen bond from the protein to the special pair is removed by this mutation. Compared with the wild-type RC, the acceptor of this hydrogen bond, the ring I acetyl group of the "special pair" bacteriochlorophyll, D(L), is rotated, and its acetyl oxygen is found 1.1 A closer to the bacteriochlorophyll-Mg(2+) of the other special pair bacteriochlorophyll, D(M). The rotation of this acetyl group and the increased interaction between the D(L) ring I acetyl oxygen and the D(M)-Mg(2+) provide the structural basis for the previously observed 80-mV decrease in the D(+)/D redox potential and the drastically increased rate of initial electron transfer to the accessory bacteriochlorophyll, B(A). The high quality of the electron density maps also allowed a reliable discussion of the mode of binding of the triazine herbicide terbutryn at the binding site of the secondary quinone, Q(B).

Bacteriochlorophylls↗
PubMed2000

Structure at 2.3 A resolution of the cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae co-crystallized with an antibody Fv fragment.

BACKGROUND: The cytochrome bc(1) complex is part of the energy conversion machinery of the respiratory and photosynthetic electron transfer chains. This integral membrane protein complex catalyzes electron transfer from ubiquinol to cytochrome c. It couples the electron transfer to the electrogenic translocation of protons across the membrane via a so-called Q cycle mechanism. RESULTS: The cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae was crystallized together with a bound antibody Fv fragment. The structure was determined at 2.3 A resolution using multiple isomorphous replacement, and refined to a crystallographic R factor of 22.2% (R(free) = 25.4%). The complex is present as a homodimer. Each 'monomer' of the refined model includes 2178 amino acid residues of subunits COR1, QCR2, COB, CYT1, RIP1, QCR6, QCR7, QCR8 and QCR9 of the cytochrome bc(1) complex and of the polypeptides V(H) and V(L) of the Fv fragment, the cofactors heme b(H), heme b(L), heme c(1), the [2Fe-2S] cluster and 346 water molecules. The Fv fragment binds to the extrinsic domain of the [2Fe-2S] Rieske protein and is essential for formation of the crystal lattice. CONCLUSIONS: The approach to crystallize membrane proteins as complexes with specific antibody fragments appears to be of general importance. The structure of the yeast cytochrome bc(1) complex reveals in detail the binding sites of the natural substrate coenzyme Q6 and the inhibitor stigmatellin. Buried water molecules close to the binding sites suggest possible pathways for proton uptake and release. A comparison with other cytochrome bc(1) complexes shows features that are specific to yeast.

Animals↗
PubMed2000

Tracing the D-pathway in reconstituted site-directed mutants of cytochrome c oxidase from Paracoccus denitrificans.

Heme-copper terminal oxidases use the free energy of oxygen reduction to establish a transmembrane proton gradient. While the molecular mechanism of coupling electron transfer to proton pumping is still under debate, recent structure determinations and mutagenesis studies have provided evidence for two pathways for protons within subunit I of this class of enzymes. Here, we probe the D-pathway by mutagenesis of the cytochrome c oxidase of the bacterium Paracoccus denitrificans; amino acid replacements were selected with the rationale of interfering with the hydrophilic lining of the pathway, in particular its assumed chain of water molecules. Proton pumping was assayed in the reconstituted vesicle system by a stopped-flow spectroscopic approach, allowing a reliable assessment of proton translocation efficiency even at low turnover rates. Several mutations at positions above the cytoplasmic pathway entrance (Asn 131, Asn 199) and at the periplasmic exit region (Asp 399) led to complete inhibition of proton pumping; one of these mutants, N131D, exhibited an ideal decoupled phenotype, with a turnover comparable to that of the wild-type enzyme. Since sets of mutations in other positions along the presumed course of the pathway showed normal proton translocation stoichiometries, we conclude that the D-pathway is too wide in most areas above positions 131/199 to be disturbed by single amino acid replacements.

Bacterial Proteins↗
PubMed2000

Heavy and light chain primary structures control IgG3 nephritogenicity in an experimental model for cryocrystalglobulinemia.

Crystal formation by monoclonal immunoglobulins is a well-known but rare complication of B-cell neoplasia. We have designed an in vivo model of cryocrystalglobulinemia by grafting to mice hybridoma clones producing a pathogenic monoclonal immunogloblulin (Ig) G3kappa. One clone, 8A4, secreted a singular IgG3 that formed crystals both in the proliferating plasma cells and as mesangial and subendothelial deposits in the kidney glomeruli. Morphologic analysis of kidneys revealed neutrophil infiltration and endocapillary hyperplasia, while the morphology of deposits was reminiscent of those in cryocrystalglobulinemia patients. A variant clone that only differed from 8A4 by a 3-amino acid deletion in the V(kappa) CDR1 increased its secretion level by 7-fold and produced an abundant bona fide serum monoclonal cryoglobulin in mice, without crystal formation within tumoral cells; it yielded no subendothelial deposits but only amorphous precipitates in capillary lumens of kidney glomeruli, reminiscent of those seen in the human hyperviscosity syndrome, without other glomerular lesions. A limited variation in the V(kappa) domain thus proved able to increase secretion, to abrogate crystallization, and to modify patterns of glomerular lesions and deposits. Both the crystallizing and noncrystallizing IgG3kappa sequences were related to previously reported murine cryoglobulins, all including a gamma3 chain and canonical VH sequences. Two additional variants of 8A4 with identical VH and VL domains but having switched to IgG1 also lost crystal formation, further showing this feature of 8A4 to result from a unique 3-dimensional conformation of the complete immunoglobulin, relying on V and C domain primary structures of both chains.

Amino Acid Sequence↗
PubMed2000

Single-electron reduction of the oxidized state is coupled to proton uptake via the K pathway in Paracoccus denitrificans cytochrome c oxidase.

The reductive part of the catalytic cycle of cytochrome c oxidase from Paracoccus denitrificans was examined by using time-resolved potential measurements on black lipid membranes. Proteoliposomes were adsorbed to the black lipid membranes and Ru(II)(2, 2'-bipyridyl)(3)(2+) was used as photoreductant to measure flash-induced membrane potential generation. Single-electron reduction of the oxidized wild-type cytochrome c oxidase reveals two phases of membrane potential generation (tau(1) approximately 20 micros and tau(2) approximately 175 micros) at pH 7.4. The fast phase is not sensitive to cyanide and is assigned to electron transfer from Cu(A) to heme a. The slower phase is inhibited completely by cyanide and shows a kinetic deuterium isotope effect by a factor of 2-3. Although two enzyme variants mutated in the so-called D pathway of proton transfer (D124N and E278Q) show the same time constants and relative amplitudes as the wild-type enzyme, in the K pathway variant K354M, tau(2) is increased to 900 micros. This result suggests uptake of a proton through the K pathway during the transition from the oxidized to the one-electron reduced state. After the second laser flash under anaerobic conditions, a third electrogenic phase with a time constant of approximately 1 ms appears. The amplitude of this phase grows with increasing flash number. We explain this growth by injection of a second electron into the single-electron reduced enzyme. On multiple flashes, both D pathway mutants behave differently compared with the wild type and two additional slow phases of tau(3) approximately 2 ms and tau(4) approximately 15 ms are observed. These results suggest that the D pathway is involved in proton transfer coupled to the uptake of the second electron.

Amino Acid Substitution↗
PubMed2000

Uphill electron transfer in the tetraheme cytochrome subunit of the Rhodopseudomonas viridis photosynthetic reaction center: evidence from site-directed mutagenesis.

The cytochrome (cyt) subunit of the photosynthetic reaction center from Rhodopseudomonas viridis contains four heme groups in a linear arrangement in the spatial order heme1, heme2, heme4, and heme3. Heme3 is the direct electron donor to the photooxidized primary electron donor (special pair, P(+)). This heme has the highest redox potential (E(m)) among the hemes in the cyt subunit. The E(m) of heme3 has been specifically lowered by site-directed mutagenesis in which the Arg residue at the position of 264 of the cyt was replaced by Lys. The mutation decreases the E(m) of heme3 from +380 to +270 mV, i.e., below that of heme2 (+320 mV). In addition, a blue shift of the alpha-band was found to accompany the mutation. The assignment of the lowered E(m) and the shifted alpha-band to heme3 was confirmed by spectroscopic measurements on RC crystals. The structure of the mutant RC has been determined by X-ray crystallography. No remarkable differences were found in the structure apart from the mutated residue itself. The velocity of the electron transfer (ET) from the tetraheme cyt to P(+) was measured under several redox conditions by following the rereduction of P(+) at 1283 nm after a laser flash. Heme3 donates an electron to P(+) with t(1/2) = 105 ns, i.e., faster than in the wild-type reaction center (t(1/2) = 190 ns), as expected from the larger driving force. The main feature is that a phase with t(1/2) approximately 2 micros dominates when heme3 is oxidized but heme2 is reduced. We conclude that the ET from heme2 to heme3 has a t(1/2) of approximately 2 micros, i.e., the same as in the WT, despite the fact that the reaction is endergonic by 50 meV instead of exergonic by 60 meV. We propose that the reaction kinetics is limited by the very uphill ET from heme2 to heme4, the DeltaG degrees of which is about the same (+230 meV) in both cases. The interpretation is further supported by measurements of the activation energy (216 meV in the wild-type, 236 meV in the mutant) and by approximate calculations of ET rates. Altogether these results demonstrate that the ET from heme2 to heme3 is stepwise, starting with a first very endergonic step from heme2 to heme4.

Amino Acid Substitution↗
PubMed2000

The monoclonal antibody 1F6 identifies a pH-dependent conformational change in the hydrophilic NH(2) terminus of NhaA Na(+)/H(+) antiporter of Escherichia coli.

One of the most interesting properties of the NhaA Na(+)/H(+) antiporter of Escherichia coli is the strong regulation of its activity by pH. This regulation is accompanied by a conformational change that can be probed by digestion with trypsin and involves the hydrophilic loop connecting the transmembrane helices VIII-IX. In the present work we show that a monoclonal antibody (mAb), 1F6, recognizes yet another domain of NhaA in a pH-dependent manner. This antibody binds NhaA at pH 8.5 but not at pH 4.5, whereas two other mAbs bind to NhaA independently of pH. The epitope of mAb 1F6 was located at the NH(2) terminus of NhaA by probing proteolytic fragments in Western blot analysis and amino acid sequencing. The antibody bound to the peptide HLHRFFSS, starting at the third amino acid of NhaA. A synthetic peptide with this sequence was shown to bind mAb 1F6 both at acidic and alkaline pH suggesting that this peptide is accessible to mAb 1F6 in the native protein only at alkaline pH. Although slightly shifted to acidic pH, the pH profile of the binding of mAb 1F6 to the antiporter is similar to that of both the Na(+)/H(+) antiporter activity as well as to its sensitivity to trypsin. We thus suggest that these pH profiles reflect a pH-dependent conformational change, which leads to activation of the antiporter. Indeed, a replacement of Gly-338 by Ser (G338S), which alleviates the pH dependence of both the NhaA activity as well as its sensitivity to trypsin, affects in a similar pattern the binding of mAb 1F6 to NhaA. Furthermore, the binding site of mAb 1F6 is involved in the functioning of the antiporter as follows: a double Cys replacement H3C/H5C causes an acidic shift by half a pH unit in the pH dependence of the antiporter; N-ethylmaleimide, which does not inhibit the wild-type protein, inhibits H3C/H5C antiporter to an extent similar to that exerted by mAb 1F6.

Amino Acid Sequence↗
PubMed2000

Engineering of a proteolytically stable human beta 2-adrenergic receptor/maltose-binding protein fusion and production of the chimeric protein in Escherichia coli and baculovirus-infected insect cells.

The hydrophobic human beta 2 adrenergic receptor was produced in fusion to the hydrophilic maltose-binding protein (MalE) in Escherichia coli. Photoaffinity labeling with the adrenergic ligand [125I]cyanopindolole-diazirine indicated that the majority of the protein was proteolyzed in the intergenic region between the fusion partners after production in E. coli. The simple and fast genetics of the bacterium enabled us to engineer a linker with an increased proteolytic stability. The fusion protein produced in E. coli was fully functional with respect to binding of adrenergic ligands and coupling to stimulatory GTP-binding protein. The production level with 3 pmol receptor fusion protein per mg membrane protein in a crude membrane preparation was significantly higher than those reported for other beta 2 adrenergic receptor constructs in E. coli. After solubilization with dodecanoyl sucrose, the fusion protein was purified to near homogeneity by affinity chromatography on immobilized Ni2+ ions (binding to a C-terminal His6-tag) and on crosslinked amylose (binding to the MalE). In order to achieve higher production levels, the fusion protein preceded by an insect signal peptide was produced in baculovirus-infected insect cells. As expected, the production level with about 17 pmol receptor per mg membrane protein was higher in the insect cells than in E. coli. The receptor fusion protein produced in the insect cells bound adrenergic ligands and activated heterotrimeric GTP-binding proteins with biochemical properties comparable to that of the unfused receptor.

ATP-Binding Cassette Transporters↗
PubMed2000

Functional properties of the heme propionates in cytochrome c oxidase from Paracoccus denitrificans. Evidence from FTIR difference spectroscopy and site-directed mutagenesis.

By specific (13)C labeling of the heme propionates, four bands in the reduced-minus-oxidized FTIR difference spectrum of cytochrome c oxidase from Paracoccus denitrificans have been assigned to the heme propionates [Behr, J., Hellwig, P., Mäntele, W., and Michel, H. (1998) Biochemistry 37, 7400-7406]. To attribute these signals to the individual propionates, we have constructed seven cytochrome coxidase variants using site-directed mutagenesis of subunit I. The mutant enzymes W87Y, W87F, W164F, H403A, Y406F, R473K, and R474K were characterized by measurement of enzymatic turnover, proton pumping activity, and Vis and FTIR spectroscopy. Whereas the mutant enzymes W164F and Y406F were found to be structurally altered, the other cytochrome c oxidase variants were suitable for band assignment in the infrared. Reduced-minus-oxidized FTIR difference spectra of the mutant enzymes were used to identify the ring D propionate of heme a as a likely proton acceptor upon reduction of cytochromic oxidase. The ring D propionate of heme a(3) might undergo conformational changes or, less likely, act as a proton donor.

Amino Acid Substitution↗