Biomedical subjects

Hartmut Michel

Publications and source records attributed to Hartmut Michel.

At least 19 recordsLinked to original sources

PubMed2006

Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems.

Production of recombinant receptors has been one of the major bottlenecks in structural biology on G protein-coupled receptors (GPCRs). The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus (SFV) vectors. Evaluation by immunodetection demonstrated that 50% of a total of 103 GPCRs were expressed in bacterial inclusion bodies, 94% in yeast cell membranes and 95% in SFV-infected mammalian cells. The expression levels varied from low to high and the various GPCR families and subtypes were analyzed for their expressability in each expression system. More than 60% of the GPCRs were expressed at milligram levels or higher in one or several systems, compatible to structural biology applications. Functional activity was determined by binding assays in yeast and mammalian cells and the correlation between immunodetection and binding activity was analyzed.

Animals↗

PubMed2006

Functional overexpression and characterization of human bradykinin subtype 2 receptor in insect cells using the baculovirus system.

Bradykinin exerts its actions via binding to B1 and B2 receptors (B1R and B2R), which are members of G protein-coupled receptor superfamily. B2R is constitutively expressed in a variety of cells such as endothelial cells, vascular smooth muscle cells, and cardiomyocytes and it is an important drug target for the treatment of cardiovascular disorders. During this study, the human B2R was functionally overexpressed in insect cells using the baculovirus expression system. The maximum expression level in Sf9 cells under optimized condition was 10 pmol/mg. This corresponds to approximately 0.25 mg active receptor per liter culture. The recombinant receptor showed high affinity for its endogenous ligand bradykinin, similar to the B2R expressed in native tissues. Functional coupling of the recombinant receptor to the endogenous G alpha(s) protein was demonstrated via cAMP release assay upon agonist stimulation. Confocal laser scanning microscopy and immunogold-labeling experiment revealed that the recombinant B2R was mainly localized intracellularly and only a minor fraction of the recombinant receptor reached the plasma membrane. To our knowledge, this is the first report of high level expression of recombinant B2R in insect cells and provides a way for large scale production and structural characterization of this receptor.

Animals↗

PubMed2006

Comparative analysis of the human angiotensin II type 1a receptor heterologously produced in insect cells and mammalian cells.

Angiotensin II type 1a receptor (AT1aR) is a member of GPCR superfamily and it plays crucial roles in the regulation of blood pressure, hormone secretion and renal functions. Here, we report functional overexpression and characterization of the human AT1aR in insect cells using the baculovirus system and in mammalian cells using the Semliki Forest virus system. The recombinant receptor was expressed at a level of 29-32 pmol/mg and it binds to angiontensin II with high affinity (Kd=0.98-1.1 nM). Angiotensin II stimulated accumulation of inositol phosphate and phosphorylation of MAP kinase was also observed, which indicated that the recombinant AT1aR could couple to endogenous Galphaq protein. Confocal laser scanning microscopy revealed that the recombinant receptor was predominantly localized in the plasma membrane and agonist induced internalization of the recombinant AT1aR was also observed. The recombinant AT1aR was expressed in glycosylated form and in vivo inhibition of glycosylation suppressed its surface expression.

Animals↗

PubMed2006

Biochemical and electron microscopic characterization of the F1F0 ATP synthase from the hyperthermophilic eubacterium Aquifex aeolicus.

The F(1)F(0) ATP synthase has been purified from the hyperthermophilic eubacterium Aquifex aeolicus and characterized. Its subunits have been identified by MALDI-mass spectrometry through peptide mass fingerprinting and MS/MS. It contains the canonical subunits alpha, beta, gamma, delta and epsilon of F(1) and subunits a and c of F(0). Two versions of the b subunit were found, which show a low sequence homology to each other. Most likely they form a heterodimer. An electron microscopic single particle analysis revealed clear structural details, including two stalks connecting F(1) and F(0). In several orientations the central stalk appears to be tilted and/or kinked. It is unclear whether there is a direct connection between the peripheral stalk and the delta subunit.

Amino Acid Sequence↗

PubMed2006

Crucial steps in the structure determination of the Na+/H+ antiporter NhaA in its native conformation.

Sodium proton antiporters are ubiquitous membrane proteins. Their importance for cell viability is the result of their role in homeostasis of intracellular pH, cellular Na+ content and cell volume. Recently, the first structure of this family of secondary transporters, namely of NhaA from Escherichia coli, revealed a novel fold and elucidated the molecular basis for the mechanism of transport and its regulation by pH. Here, we describe the key steps for the structure determination of NhaA, an iterative process of improving protein quality as well as crystallization conditions. Protein quality was optimized by shortening the purification to a single step and by changing the expression host. The major steps for crystal improvement were the exchange of the detergent during protein purification from the beta- to the alpha-anomer of DDM, the addition of OG to the crystallization set ups, and the growth of the crystals under conditions suitable for cryo-temperatures. Unexpectedly, the dimeric association of the transporter in the 3D crystal lattice is non-physiological. A comparison of the X-ray structure with the electron density map from cryo-electron microscopy of 2D crystals demonstrates that the NhaA helix packing in the 3D crystal is identical with the one in the lipid environment. Thus, the antiporter is in a native conformation in the 3D crystals.

Amino Acid Sequence↗

PubMed2006

Dimethylsulphoxide as a tool to increase functional expression of heterologously produced GPCRs in mammalian cells.

High-level overexpression of G protein-coupled receptors GPCRs in mammalian cells remains a difficult task inspite of newly developed virus based expression systems. Here, we show that the functional expression level of the recombinant bradykinin receptor (B(2)R) in mammalian cells can be increased up to sixfold just by the addition of dimethylsulphoxide in the culture medium. Total expression level, cellular localization and binding affinity of the recombinant receptor for its endogenous ligand remains unaltered. The strategy presented here, with recombinant B(2)R as a case example, is applicable to other GPCRs and provides a generic tool to improve the functional expression level of recombinant GPCRs in mammalian cells.

Animals↗

PubMed2006

Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen.

We have optimized the expression level of 20 mammalian G protein-coupled receptors (GPCRs) in the methylotrophic yeast Pichia pastoris. We found that altering expression parameters, including growth temperature, and supplementation of the culture medium with specific GPCR ligands, histidine, and DMSO increased the amount of functional receptor, as assessed by ligand binding, by more than eightfold over standard expression conditions. Unexpectedly, we found that the overall amount of GPCR proteins expressed, in most cases, varied only marginally between standard and optimized expression conditions. Accordingly, the optimized expression conditions resulted in a marked fractional increase in the ratio of ligand binding-competent receptor to total expressed receptor. The results of this study suggest a general approach for increasing yields of functional mammalian GPCRs severalfold over standard expression conditions by using a set of optimized expression condition parameters that we have characterized for the Pichia expression system. Overall, we have more than doubled the number of GPCR targets that can be produced in our laboratories in sufficient amounts for structural studies.

Amino Acid Sequence↗

PubMed2006

Multiconformation continuum electrostatics analysis of the NhaA Na+/H+ antiporter of Escherichia coli with functional implications.

Sodium proton antiporters are essential enzymes that catalyze the exchange of sodium ions for protons across biological membranes. Protonations and deprotonations of individual amino acid residues and of clusters formed by these residues play an important role in activating these enzymes and in the mechanism of transport. We have used multiconformation continuum electrostatics method to investigate the protonation states of residues in the sodium proton exchanger NhaA from Escherichia coli, the structure of which has been determined recently by x-ray crystallography. Our calculations identify four clusters of electrostatically tightly interacting residues as well as long-range interactions between residues required for activation. The importance of many of these residues has been demonstrated by the characterization of site-directed mutants. A number of residues with extreme pKa values, including several of the "pH sensor," can only undergo protonation/deprotonation reactions subsequent to conformational changes. The results of the calculations provide valuable information on the activation of the antiporter and the role of individual amino acid residues, and provide a solid framework for further experiments.

Amino Acid Sequence↗

PubMed2006

The role of tryptophan 272 in the Paracoccus denitrificans cytochrome c oxidase.

The mechanism of electron coupled proton transfer in cytochrome c oxidase (CcO) is still poorly understood. The P(M)-intermediate of the catalytic cycle is an oxoferryl state whose generation requires one additional electron, which cannot be provided by the two metal centres. The missing electron has been suggested to be donated to this binuclear site by a tyrosine residue. A tyrosine radical species has been detected in the P(M) and F* intermediates (formed by addition of H2O2) of the Paraccocus denitrificans CcO using electron paramagnetic resonance (EPR) spectroscopy. From the study of conserved variants its origin was determined to be Y167 which is surprising as this residue is not part of the active site. Upon inspection of the active site it becomes evident that W272 could be the actual donor of the missing electron, which can then be replenished from Y167 or from the Y280-H276 cross link in the natural cycle. To address the question, whether such a direct electron transfer pathway to the binuclear centre exists two tryptophan 272 variants in subunit I have been generated. These variants are characterised by their turnover rates as well as using EPR and optical spectroscopy. From these experiments it is concluded, that W272 is an important intermediate in the formation of the radical species appearing in P(M) and F* intermediates produced with hydrogen peroxide. The significance of this finding for the catalytic function of the enzyme is discussed.

Catalysis↗

PubMed2006

Comparative analysis and "expression space" coverage of the production of prokaryotic membrane proteins for structural genomics.

Membrane proteins comprise up to one-third of prokaryotic and eukaryotic genomes, but only a very small number of membrane protein structures are known. Membrane proteins are challenging targets for structural biology, primarily due to the difficulty in producing and purifying milligram quantities of these proteins. We are evaluating different methods to produce and purify large numbers of prokaryotic membrane proteins for subsequent structural and functional analysis. Here, we present the comparative expression data for 37 target proteins, all of them secondary transporters, from the mesophilic organism Salmonella typhimurium and the two hyperthermophilic organisms Aquifex aeolicus and Pyrococcus furiosus in three different Escherichia coli expression vectors. In addition, we study the use of Lactococcus lactis as a host for integral membrane protein expression. Overall, 78% of the targets were successfully produced under at least one set of conditions. Analysis of these results allows us to assess the role of different variables in increasing "expression space" coverage for our set of targets. This analysis implies that to maximize the number of nonhomologous targets that are expressed, orthologous targets should be chosen and tested in two vectors with different types of promoters, using C-terminal tags. In addition, E. coli is shown to be a robust host for the expression of prokaryotic transporters, and is superior to L. lactis. These results therefore suggest appropriate strategies for high-throughput heterologous overproduction of membrane proteins.

Bacterial Proteins↗

PubMed2006

Biochemical and pharmacological characterization of the human bradykinin subtype 2 receptor produced in mammalian cells using the Semliki Forest virus system.

Bradykinin, a vasoactive peptide, plays a crucial role in many cardiovascular processes via activation of the bradykinin subtype 2 receptor (B2R). B2R, a member of the G protein-coupled receptor (GPCR) superfamily, is a potential drug target in the treatment of cardiovascular disorders, pain and inflammation. In this study, human B2R was expressed at high levels in baby hamster kidney (BHK) cells using Semliki Forest virus-based vectors. The recombinant receptor was produced as a fusion protein with affinity tags and an expression level of 11 pmol/mg (i.e., approx. 0.2 mg of active receptor per liter of culture) was obtained. Radioligand binding analysis revealed that the recombinant receptor binds to its endogenous ligand bradykinin with high affinity (Kd = 0.12 nM) and its pharmacological profile was similar to that of B2R in native tissues. Bradykinin-stimulated accumulation of inositol phosphate was observed in BHK cells expressing the recombinant receptor, which indicated the activation of endogenous G alpha(q) protein by the recombinant B2R. Confocal laser scanning microscopy and immunogold staining revealed that the recombinant receptor was predominantly localized intracellularly. To the best of our knowledge, this is the first report of an affinity-tagged recombinant B2R been expressed at high levels in BHK cells and extensively characterized.

Amino Acid Sequence↗

PubMed2005

Rates and Equilibrium of CuA to heme a electron transfer in Paracoccus denitrificans cytochrome c oxidase.

Intramolecular electron transfer between CuA and heme a in solubilized bacterial (Paracoccus denitrificans) cytochrome c oxidase was investigated by pulse radiolysis. CuA, the initial electron acceptor, was reduced by 1-methylnicotinamide radicals in a diffusion-controlled reaction, as monitored by absorption changes at 825 nm, followed by partial restoration of the absorption and paralleled by an increase in the heme a absorption at 605 nm. The latter observations indicate partial reoxidation of the CuA center and the concomitant reduction of heme a. The rate constants for heme a reduction and CuA reoxidation were identical within experimental error and independent of the enzyme concentration and its degree of reduction, demonstrating that a fast intramolecular electron equilibration is taking place between CuA and heme a. The rate constants for CuA --> heme a ET and the reverse heme a --> CuA process were found to be 20,400 s(-1) and 10,030 s(-1), respectively, at 25 degrees C and pH 7.5, which corresponds to an equilibrium constant of 2.0. Thermodynamic and activation parameters of these intramolecular ET reactions were determined. The significance of the results, particularly the low activation barriers, is discussed within the framework of the enzyme's known three-dimensional structure, potential ET pathways, and the calculated reorganization energies.

Computer Simulation↗

PubMed2005

Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH.

The control by Na+/H+ antiporters of sodium/proton concentration and cell volume is crucial for the viability of all cells. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of Na+/H+ antiporters. Their activity is tightly controlled by pH. Here we present the crystal structure of pH-downregulated NhaA, the main antiporter of Escherichia coli and many enterobacteria. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of the membrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. We propose that the binding of charged substrates causes an electric imbalance, inducing movements, that permit a rapid alternating-access mechanism. This ion-exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the entry to the cytoplasmic funnel.

Binding Sites↗

PubMed2005

Crystallization and preliminary X-ray analysis of native and selenomethionyl vinorine synthase from Rauvolfia serpentina.

Vinorine synthase (VS) is a central enzyme of the biosynthesis of the antiarrhythmic drug ajmaline and is a member of the BAHD superfamily of acyltransferases. So far, no three-dimensional structure with significant sequence homology with VS is known. Crystals of VS and selenomethionyl-labelled VS from the medicinal plant Rauvolfia serpentina have been obtained by the hanging-drop technique at 305 K with ammonium sulfate and PEG 400 as precipitants. VS crystals diffract to 2.8 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 82.3, b = 89.6, c = 136.2 A. The selenomethionyl VS crystal was nearly isomorphous with the VS crystal.

Crystallization↗

PubMed2005

Crystallization and preliminary X-ray analysis of strictosidine synthase and its complex with the substrate tryptamine.

Strictosidine synthase (STR1) is a central enzyme that participates in the biosynthesis of almost all plant monoterpenoid indole alkaloids. After heterologous expression in Escherichia coli, crystals of STR1 and its substrate complex with tryptamine were obtained by the hanging-drop technique at 302-304 K with potassium sodium tartrate tetrahydrate as precipitant. All crystals belong to space group R3. The native STR1 crystals diffract to 2.95 A and have unit-cell parameters a = b = 150.3, c = 122.4 A. The tryptamine complex crystals diffract to 2.38 A, with unit-cell parameters a = b = 147.3, c = 122.3 A.

Carbon-Nitrogen Lyases↗

PubMed2005

Multivariate analysis of single-molecule spectra: surpassing spectral diffusion.

The full exploitation of single-molecule spectroscopy in disordered systems is often hampered by spectral diffusion processes of the optical transitions due to structural fluctuations in the local environment of the probe molecule which leads to temporal averaging of the signal. Multivariate statistical pattern recognition techniques, originally developed for single-molecule cryoelectron microscopy, allow us to retrieve detailed information from optical single-molecule spectra. As an example, we present the phonon side band of the B800 excitations of the light-harvesting 2 (LH2) complex from Rhodospirillum molischianum, revealing the electron-phonon coupling strength for these transitions. The measured Debye-Waller factors, ranging from 0.4 to 0.9, fall in the regime of weak electron-phonon coupling.

Journal Article↗

PubMed2005

Titration behavior of residues at the entrance of the D-pathway of cytochrome c oxidase from paracoccus denitrificans investigated by continuum electrostatic calculations.

Continuum electrostatic calculations were employed to investigate the titration curves of the fully oxidized state of wild type and several variants of cytochrome c oxidase from Paracoccus denitrificans (N131D, N131C, N131V, and D124N) for different values of the dielectric constant of the protein. The effects of the mutations at the entrance of the D-proton transfer pathway were found to be quite localized to their immediate surroundings. The results can be well interpreted in the light of the available biochemical and structural data and help understanding the effects of mutations on proton conductivity. The mutations of aspartic acid Asp-I-124 to a neutral residue resulted in a decreased pK(a) value of His-I-28 suggesting that the mutation of His-I-28 may have a significant influence on the coupling of electron and proton transfer in cytochrome c oxidase. We also investigated the effect of the mutations N131D, N131C, and N131V on the residue Glu-I-278 in terms of its pK(a) value and electrostatic interaction energies.

Amino Acid Substitution↗

PubMed2004

Structure at 1.3 A resolution of Rhodothermus marinus caa(3) cytochrome c domain.

The cytochrome c domain of subunit II from the Rhodothermus marinus caa(3) HiPIP:oxygen oxidoreductase, a member of the superfamily of heme-copper-containing terminal oxidases, was produced in Escherichia coli and characterised. The recombinant protein, which shows the same optical absorption and redox properties as the corresponding domain in the holo enzyme, was crystallized and its structure was determined to a resolution of 1.3 A by the multiwavelength anomalous dispersion (MAD) technique using the anomalous dispersion of the heme iron atom. The model was refined to final R(cryst) and R(free) values of 13.9% and 16.7%, respectively. The structure reveals the insertion of two short antiparallel beta-strands forming a small beta-sheet, an interesting variation of the classical all alpha-helical cytochrome c fold. This modification appears to be common to all known caa(3)-type terminal oxidases, as judged by comparative modelling and by analyses of the available amino acid sequences for these enzymes. This is the first high-resolution crystal structure reported for a cytochrome c domain of a caa(3)-type terminal oxidase. The R.marinus caa(3) uses HiPIP as the redox partner. The calculation of the electrostatic potential at the molecular surface of this extra C-terminal domain provides insights into the binding to its redox partner on one side and its interaction with the remaining subunit II on the other side.

Amino Acid Sequence↗