Biomedical subjects

Hartmut Michel

Publications and source records attributed to Hartmut Michel.

34 records · Page 2Linked to original sources

PubMed2004

Expression, purification, crystallization and preliminary X-ray analysis of strictosidine glucosidase, an enzyme initiating biosynthetic pathways to a unique diversity of indole alkaloid skeletons.

Strictosidine beta-D-glucosidase, a plant enzyme initiating biosynthetic pathways to about 2000 monoterpenoid indole alkaloids with an extremely large number of various carbon skeletons, has been functionally expressed in Escherichia coli and purified to homogeneity in mg scale. Crystals suitable for X-ray analysis were found by robot-mediated screening. Using the hanging-drop technique, optimum conditions were 0.3 M ammonium sulfate, 0.1 M sodium acetate, pH 4.6 and PEG 4000 (10%) as precipitant buffer. The crystals of strictosidine glucosidase belong to the space group P42(1)2 with unit cell dimensions of a=157.63, c=103.59 A and diffract X-rays to 2.48-A resolution.

Catharanthus↗

PubMed2004

Crystallization and preliminary X-ray crystallographic analysis of strictosidine synthase from Rauvolfia: the first member of a novel enzyme family.

Strictosidine synthase is a central enzyme involved in the biosynthesis of almost all plant monoterpenoid indole alkaloids. Strictosidine synthase from Rauvolfia serpentina was heterologously expressed in Escherichia coli. Crystals of the purified recombinant enzyme have been obtained by the hanging-drop technique at 303 K with potassium sodium tartrate tetrahydrate as precipitant. The crystals belong to the space group R3 with cell dimensions of a=b=150.3 A and c=122.4 A. Under cryoconditions (120 K), the crystals diffract to about 2.95 A.

Base Sequence↗

PubMed2004

Tyrosine 167: the origin of the radical species observed in the reaction of cytochrome c oxidase with hydrogen peroxide in Paracoccus denitrificans.

Determination of the three-dimensional structure of cytochrome c oxidase, the terminal enzyme of the respiratory chain, from Paracoccus denitrificans offers the possibility of site-directed mutagenesis studies to investigate the relationship between the structure and the catalytic function of the enzyme. The mechanism of electron-coupled proton transfer is still, however, poorly understood. The P(M) intermediate of the catalytic cycle is an oxoferryl state the generation of which requires one additional electron, which cannot be provided by the two metal centers. It is suggested that the missing electron is donated to this binuclear site by a tyrosine residue that forms a radical species, which can then be detected in both the P(M) and F(*) intermediates of the catalytic cycle. One possibility to produce P(M) and F(*) intermediates artificially in cytochrome c oxidase is the addition of hydrogen peroxide to the fully oxidized enzyme. Using electron paramagnetic resonance (EPR) spectroscopy, we assign a radical species detected in this reaction to a tyrosine residue. To address the question, which tyrosine residue is the origin of the radical species, several tyrosine variants of subunit I are investigated. These variants are characterized by their turnover rates, as well as using EPR and optical spectroscopy. From these experiments, it is concluded that the origin of the radical species appearing in P(M) and F(*) intermediates produced with hydrogen peroxide is tyrosine 167. The significance of this finding for the catalytic function of the enzyme is discussed.

Bacterial Proteins↗

PubMed2004

Vinorine synthase from Rauvolfia: the first example of crystallization and preliminary X-ray diffraction analysis of an enzyme of the BAHD superfamily.

Crystals of vinorine synthase (VS) from medicinal plant Rauvolfia serpentina expressed in Escherichia coli have been obtained by the hanging-drop technique at 305 K with ammonium sulfate and PEG 400 as precipitants. The enzyme is involved in the biosynthesis of the antiarrhythmic drug ajmaline and is a member of the BAHD superfamily of acyltransferases. So far, no three-dimensional structure of a member of this enzyme family is known. The crystals belong to the space group P2(1)2(1)2(1) with cell dimensions of a=82.3 A, b=89.6 A and c=136.2 A. Under cryoconditions (120 K), a complete data set up to 2.8 A was collected at a synchrotron source.

Alkaloids↗

PubMed2004

The MAM (meprin/A5-protein/PTPmu) domain is a homophilic binding site promoting the lateral dimerization of receptor-like protein-tyrosine phosphatase mu.

The MAM (meprin/A5-protein/PTPmu) domain is present in numerous proteins with diverse functions. PTPmu belongs to the MAM-containing subclass of protein-tyrosine phosphatases (PTP) able to promote cell-to-cell adhesion. Here we provide experimental evidence that the MAM domain is a homophilic binding site of PTPmu. We demonstrate that the MAM domain forms oligomers in solution and binds to the PTPmu ectodomain at the cell surface. The presence of two disulfide bridges in the MAM molecule was evidenced and their integrity was found to be essential for MAM homophilic interaction. Our data also indicate that PTPmu ectodomain forms oligomers and mediates the cellular adhesion, even in the absence of MAM domain homophilic binding. Reciprocally, MAM is able to interact homophilically in the absence of ectodomain trans binding. The MAM domain therefore contains independent cis and trans interaction sites and we predict that its main role is to promote lateral dimerization of PTPmu at the cell surface. This finding contributes to the understanding of the signal transduction mechanism in MAM-containing PTPs.

Amino Acid Sequence↗

PubMed2004

Dynamic water networks in cytochrome C oxidase from Paracoccus denitrificans investigated by molecular dynamics simulations.

We present a molecular dynamics study of cytochrome c oxidase from Paracoccus denitrificans in the fully oxidized state, embedded in a fully hydrated dimyristoylphosphatidylcholine lipid bilayer membrane. Parallel simulations with different levels of protein hydration, 1.125 ns each in length, were carried out under conditions of constant temperature and pressure using three-dimensional periodic boundary conditions and full electrostatics to investigate the distribution and dynamics of water molecules and their corresponding hydrogen-bonded networks inside cytochrome c oxidase. The majority of the water molecules had residence times shorter than 100 ps, but a few water molecules are fixed inside the protein for up to 1.125 ns. The hydrogen-bonded network in cytochrome c oxidase is not uniformly distributed, and the degree of water arrangement is variable. The average number of solvent sites in the proton-conducting K- and D-pathways was determined. In contrast to single water files in narrow geometries we observe significant diffusion of individual water molecules along these pathways. The highly fluctuating hydrogen-bonded networks, combined with the significant diffusion of individual water molecules, provide a basis for the transfer of protons in cytochrome c oxidase, therefore leading to a better understanding of the mechanism of proton pumping.

Computer Simulation↗

PubMed2004

Comparative analysis of high-affinity ligand binding and G protein coupling of the human CXCR1 chemokine receptor and of a CXCR1-Galpha fusion protein after heterologous production in baculovirus-infected insect cells.

In order to perform biochemical and pharmacological characterization of CXCR1, we designed several CXCR1 constructs. All constructs, including a CXCR1-G(i2)alpha fusion protein, were produced in insect cells after infection with recombinant baculovirus. The recombinant receptors exhibited specific high-affinity binding of (125)I-labelled interleukin-8, and Scatchard transformation of the binding data indicated the presence of a population of single homogenous binding sites. Furthermore, the pharmacological profiles for the different CXCR1 constructs produced in the baculovirus-infected insect cells were almost identical to those reported for CXCR1 on human neutrophils. Interestingly, when the CXCR1 constructs were coproduced with G(i2) protein as a result of coinfection with baculoviruses encoding the G(i2)alpha-, the beta- and the gamma- subunits, the B(max) values were significantly increased. Hence, the level of FlagCXCR1Bio, after coproduction with G(i2) protein, was found to be almost 10 times higher than that of the FlagCXCR1Bio alone. However, no differences in the K(i) values were observed of the receptor constructs produced either after single infection or coinfection of insect cells. The addition of guanyl-5'-yl imidodiphosphate resulted in a dramatic reduction of the number of binding sites; however, the K(i) values remained unchanged, indicating coupling of the receptor to the guanine nucleotide-binding protein.

Animals↗

PubMed2004

Production of the human D2S receptor in the methylotrophic yeast P. pastoris.

In order to evaluate the methylotrophic yeast Pichia pastoris as means for high-yield production of homogenous D(2S) receptor protein, we have expressed the unmodified D(2S) receptor and various D(2S) receptor fusion constructs under the transcriptional control of the highly inducible promotor of the P. pastoris alcoholoxidase 1 gene in strain SMD1163. Fusion of the D(2S) receptor gene to the alpha-factor preprosequence proved to be essential for receptor production. For the receptor fusion constructs a gene dosage of more than two copies per cell increased production levels three- to sixfold. Adding various dopaminergic ligands to the induction medium increased yields up to tenfold, reaching 51,500 +/- 5700 receptors/cell. Immunoblot analysis of the effect of tunicamycin on D(2S) receptor fusion proteins and immunoprecipitation of metabolically labeled wild-type and glycosylation-deficient D(2S) receptor fusion proteins revealed that the high-mannose-type glycosylation of the D(2S) receptor prevents cleavage of the alpha-factor prosequence by the Kex2 endopeptidase. Abolishing glycosylation restored correct processing. Immunogold electron microscopy showed that recombinant yeast cells overproducing the D(2S) receptor developed membrane stacks harboring the receptor protein. The pharmacological profile of the recombinant D(2S) receptor was similar to that reported for neuronal D(2) receptors independent of glycosylation and processing. In conclusion, the D(2S) receptor can readily be produced in P. pastoris with high yield suitable for receptor purification and future structural studies.

Cloning, Molecular↗

PubMed2003

Direct observation of tiers in the energy landscape of a chromoprotein: a single-molecule study.

Single-molecule spectroscopic techniques were applied to individual pigments embedded in a chromoprotein. A sensitive tool to monitor structural fluctuations of the protein backbone in the local environment of the chromophore is provided by recording the changes of the spectral positions of the pigment absorptions as a function of time. The data provide information about the organization of the energy landscape of the protein in tiers that can be characterized by an average barrier height. Additionally, a correlation between the average barrier height within a distinct tier and the time scale of the structural fluctuations is observed.

Bacterial Proteins↗

PubMed2003

1.3 A X-ray structure of an antibody Fv fragment used for induced membrane-protein crystallization.

The antibody Fv fragment 7E2 has previously been employed in the induced crystallization of the integral membrane protein cytochrome c oxidase from Paracoccus denitrificans. The 1.3 A X-ray structure of the uncomplexed antibody fragment reveals conserved water networks on the surfaces of the framework regions. A novel consensus motif for water coordination, XX(S/T), is found along the edges of the beta-sandwich, where a water molecule forms hydrogen bonds to the carbonyl O atom of a residue at position N and the OG hydroxyl groups of conserved serines or threonines at position N + 2. Multiple conformations were found in the hydrophobic core for residues IleL21, LeuL33 and the disulfide bridges. An internal water molecule that is compatible with only one of the three packing states of the V(L) core suggests local 'breathing' of the variable domain. TrpH47, a conserved key residue of the V(H)/V(L) interface, is crucially involved in the formation of the antigen-binding site by adopting a novel conformation that specifically stabilizes the non-canonical CDR-L3 loop. Finally, a comparison with 7E2-cytochrome c oxidase complexes demonstrates that binding of this membrane-bound antigen proceeds without major conformational changes of the 7E2 antibody fragment.

Antibodies↗

PubMed2003

Isolation, characterization and electron microscopic single particle analysis of the NADH:ubiquinone oxidoreductase (complex I) from the hyperthermophilic eubacterium Aquifex aeolicus.

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence. The purified detergent solubilized enzyme is highly active above 50 degrees C. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 degrees C. The A. aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine. SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits. N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G. The isolated complex is highly stable and active in a temperature range from 50 to 90 degrees C, with a half-life of about 10 h at 80 degrees C. The activity shows a linear Arrhenius plot at 50-85 degrees C with an activation energy at 31.92 J/mol K. Single particle electron microscopy shows that the A. aeolicus complex I has the typical L-shape. However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources. In addition, the angle (90 degrees ) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant.

Bacteria↗

PubMed2003

Single-molecule study of the electronic couplings in a circular array of molecules: light-harvesting-2 complex from Rhodospirillum molischianum.

Applying single-molecule spectroscopic techniques allowed us to determine the mutual angles between the transition-dipole moments associated with optical transitions of the eight bacteriochlorophyll a molecules which form the so-called B800 ring of the light-harvesting-2 complex from Rhodospirillum molischianum. The orientation of the transition-dipole moment is a sensitive probe for the strength of the local intermolecular interactions because of the well-defined arrangement of the individual molecules within the B800 ring. Our data reveal that the strength of the electronic coupling between individual molecules in the ring is subjected to spatial as well as temporal variations.

Bacteriochlorophyll A↗

PubMed2003

Low-intensity pump-probe measurements on the B800 band of Rhodospirillum molischianum.

We have measured low-intensity, polarized one-color pump-probe traces in the B800 band of the light-harvesting complex LH2 of Rhodospirillum molischianum at 77 K. The excitation/detection wavelength was tuned through the B800 band. A single-wavelength and a global target analysis of the data were performed with a model that accounts for excitation energy transfer among the B800 molecules and from B800 to B850. By including the anisotropy of the signals into the fitting procedure, both transfer processes could be separated. It was estimated in the global target analysis that the intra-B800 energy transfer, i.e., the hopping of the excitation from one B800 to another B800 molecule, takes approximately 0.5 ps at 77 K. This transfer time increases with the excitation/detection wavelength from 0.3 ps on the blue side of the B800 band to approximately 0.8 ps on the red side. The residual B800 anisotropy shows a wavelength dependence as expected for energy transfer within an inhomogeneously broadened cluster of weakly coupled pigments. In the global target analysis, the transfer time from B800 to B850 was determined to be approximately 1.7 ps at 77 K. In the single-wavelength analysis, a speeding-up of the B800 --> B850 energy transfer rate toward the blue edge of the B800 band was found. This nicely correlates with the proposed position of the suggested high-exciton component of the B850 band acting as an additional decay channel for B800 excitations.

Anisotropy↗

PubMed2002

Vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans: FTIR and electrochemical studies on Tyr-D4-labeled and on Tyr280His and Tyr35Phe mutant enzymes.

A combined electrochemical and FTIR spectroscopic approach was used to identify the vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans which change upon electron transfer and coupled proton transfer. Electrochemically induced FTIR difference spectra of the Tyr-D4-labeled cytochrome c oxidase reveal that only small contributions arise from the tyrosines. Contributions between 1600 and 1560 cm(-1) are attributed to nu8a/8b(CC) ring modes. The nu19(CC) ring mode for the protonated form of tyrosines is proposed to absorb with an uncommonly small signal at 1525-1518 cm(-1) and for the deprotonated form at 1496-1486 cm(-1), accompanied by the increase of the nu19(CC) ring mode of the Tyr-D(4)-labeled oxidase at approximately 1434 cm(-1). A signal at 1270 cm(-1) can be tentatively attributed to the nu7'a(CO) and delta(COH) mode of a protonated tyrosine. Uncommon absorptions, like the mode at 1524 cm(-1), indicate the involvement of Tyr280 in the spectra. Tyr280 is a crucial residue close to the binuclear center and is covalently bonded to His276. The possible changes of the spectral properties are discussed together with the absorbance spectra of tyrosine bound to histidine. The vibrational modes of Tyr280 are further analyzed in combination with the mutation to histidine, which is assumed to abolish the covalent bonding. The electrochemically induced FTIR difference spectra of the Tyr280His mutant point to a change in protonation state in the environment of the binuclear center. Together with an observed decrease of a signal at 1736 cm(-1), previously assigned to Glu278, a possible functional coupling is reflected. In direct comparison to the FTIR difference spectra of the D4-labeled compound and comparing the spectra at pH 7 and 4.8, the protonation state of Tyr280 is discussed. Furthermore, a detailed analysis of the mutant is presented, the FTIR spectra of the CO adduct revealing a partial loss of Cu(B). Electrochemical redox titrations reflect a downshift of the heme a3 midpoint potential by 95 +/- 10 mV. Another tyrosine identified to show redox dependent changes upon electron transfer is Tyr35, a residue in the proposed D-pathway of the cytochrome c oxidase.

Electrochemistry↗

PubMed2002

Reduction of cytochrome c oxidase by a second electron leads to proton translocation.

Cytochrome c oxidase, the terminal enzyme of cellular respiration in mitochondria and many bacteria, reduces O(2) to water. This four-electron reduction process is coupled to translocation (pumping) of four protons across the mitochondrial or bacterial membrane; however, proton pumping is poorly understood. Proton pumping was thought to be linked exclusively to the oxidative phase, that is, to the transfer of the third and fourth electron. Upon re-evaluation of these data, however, this proposal has been questioned, and a transport mechanism including proton pumping in the reductive phase--that is, during the transfer of the first two electrons--was suggested. Subsequently, additional studies reported that proton pumping during the reductive phase can occur, but only when it is immediately preceded by an oxidative phase. To help clarify the issue we have measured the generation of the electric potential across the membrane, starting from a defined one-electron reduced state. Here we show that a second electron transfer into the enzyme leads to charge translocation corresponding to pumping of one proton without necessity for a preceding turnover.

Biological Transport↗

PubMed2002

Crystallisation of membrane proteins mediated by antibody fragments.

X-ray structures of three different membrane proteins in complex with antibody fragments have been published. The binding of Fv or Fab fragments enlarges the hydrophilic part of integral membrane proteins, thereby providing additional surface for crystal contacts and space for the detergent micelle. In all reported cases, antibody binding was either essential for the crystallisation of the membrane protein or it substantially improved the diffraction quality of the crystals. Antibody-fragment-mediated crystallisation appears to be a valuable tool in particular for membrane proteins with very small hydrophilic or flexible domains.

Antibodies, Monoclonal↗