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The MAM (meprin/A5-protein/PTPmu) domain is present in numerous proteins with diverse functions. PTPμ 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 PTPμ. We demonstrate that the MAM domain forms oligomers in solution and binds to the PTPμ 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 PTPμ 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 PTPμ at the cell surface. This finding contributes to the understanding of the signal transduction mechanism in MAM-containing PTPs.
Supersilylated tetrachlorodigermane (tBu3Si)Cl2GeGeCl2(SitBu3) and trigermoxetane (tBu3Si)3Ge3Cl3O
(2004)
In contrast to the tetrachlorodigermane (tBu3Si)Cl2Ge-GeCl2(SitBu3), the cis,transcyclotrigermane (tBu3SiGeCl)3 is sensitive to oxygen. Its treatment with O2 at ambient temperature leads to the trigermoxetane (tBu3Si)3Ge3Cl3O. According to an X-ray structure analysis of single crystals consisting of cocrystallized (tBu3Si)3Ge3Cl3O and (tBu3Si)Cl2Ge-GeCl2(SitBu3) the trigermaoxetane contains an almost planar Ge3O-ring while the tetrachlorodigermane (tBu3Si)Cl2Ge- GeCl2(SitBu3) possesses a Si-Ge-Ge-Si chain which is exactly all trans,
The major light-harvesting complex (LHC-II) of higher plants plays a crucial role in capturing light energy for photosynthesis and in regulating the flow of energy within the photosynthetic apparatus. Native LHC-II isolated from plant tissue consists of three isoforms, Lhcb1, Lhcb2, and Lhcb3, which form homo- and heterotrimers. All three isoforms are highly conserved among different species, suggesting distinct functional roles. We produced the three LHC-II isoforms by heterologous expression of the polypeptide in Escherichia coli and in vitro refolding with purified pigments. Although Lhcb1 and Lhcb2 are very similar in polypeptide sequence and pigment content, Lhcb3 is clearly different because it lacks an N-terminal phosphorylation site and has a higher chlorophyll a/b ratio, suggesting the absence of one chlorophyll b. Low temperature absorption and fluorescence emission spectra of the pure isoforms revealed small but significant differences in pigment organization. The oligomeric state of the pure isoforms and of their permutations was investigated by native gel electrophoresis, sucrose density gradient centrifugation, and SDS-PAGE. Lhcb1 and Lhcb2 formed trimeric complexes by themselves and with one another, but Lhcb3 was able to do so only in combination with one or both of the other isoforms. We conclude that the main role of Lhcb1 and Lhcb2 is in the adaptation of photosynthesis to different light regimes. The most likely role of Lhcb3 is as an intermediary in light energy transfer from the main Lhcb1/Lhcb2 antenna to the photosystem II core.
The transporter associated with antigen processing (TAP) is a key component of the cellular immune system. As a member of the ATP-binding cassette (ABC) superfamily, TAP hydrolyzes ATP to energize the transport of peptides from the cytosol into the lumen of the endoplasmic reticulum. TAP is composed of TAP1 and TAP2, each containing a transmembrane domain and a nucleotide-binding domain (NBD). Here we investigated the role of the ABC signature motif (C-loop) on the functional non-equivalence of the NBDs, which contain a canonical C-loop (LSGGQ) for TAP1 and a degenerate C-loop (LAAGQ) for TAP2. Mutation of the leucine or glycine (LSGGQ) in TAP1 fully abolished peptide transport. However, TAP complexes with equivalent mutations in TAP2 still showed residual peptide transport activity. To elucidate the origin of the asymmetry of the NBDs of TAP, we further examined TAP complexes with exchanged C-loops. Strikingly, the chimera with two canonical C-loops showed the highest transport rate whereas the chimera with two degenerate C-loops had the lowest transport rate, demonstrating that the ABC signature motifs control peptide transport efficiency. All single site mutants and chimeras showed similar activities in peptide or ATP binding, implying that these mutations affect the ATPase activity of TAP. In addition, these results prove that the serine of the C-loop is not essential for TAP function but rather coordinates, together with other residues of the C-loop, the ATP hydrolysis in both nucleotide-binding sites.
The adaptive response of Sorghum bicolor landraces from Egypt to drought stress and following recovery was analyzed using two-dimensional difference gel electrophoresis, 2D-DIGE. Physiological measurements and proteome alterations of accession number 11434, drought tolerant, and accession number 11431, drought sensitive, were compared to their relative control values after drought stress and following recovery. Differentially expressed proteins were analysed by Matrix assisted laser desorption ionisation time-of-flight mass spectrometry, MALDI-TOF-MS. Alterations in protein contents related to the energy balance, metabolism (sensu Mewes et al. 1997), and chaperons were the most apparent features to elucidate the differences between the drought tolerant and sensitive accessions. Further alterations in the levels of proteins related to transcription and protein synthesis are discussed.
Two tetrahydroisoquinoline alkaloids were extracted from the alkaloid fraction of a methanol extract of the seeds of Calycotome Villosa Subsp. intermedia. Their structures were established as (R)-1-hydroxymethyl-7-8-dimethoxy-1,2,3,4-tetrahydro- isoquinoline (1) and (S)-7-hydroxymethyl-2-3-dimethoxy-7,8,9,10-tetrahydroisoquinoline chloride (2) by spectroscopic techniques and X-ray diffraction analysis.
Die bei der Photosynthese verwendete Lichtenergie wird zu einem großen Anteil von Lichtsammlersystemen bereitgestellt. In der pflanzlichen Photosynthese wird unterschieden zwischen Lichtsammlersytem I (light harvesting complex I, LHC-I), assoziiert mit Photosystem I (PS-I) und Lichtsammlersystem II (light harvesting complex II, LHC-II), assoziiert mit Photosystem II (PS-II). LHC-II ist der häufigste Protein-Pigment Komplex der Chloroplasten und bindet bis zu 50% aller Chlorophylle in der Thylakoidmembran. Der Protein-Pigment Komplex LHC-II hat vier, teils miteinander verwandte Funktionen in der Photosynthese. I) Die Sammlung und Weiterleitung von Lichtenergie, II) Stabilisierung der Granastapel, III) Ausgleich der Anregungsenergie von PS-I und PS-II, IV) Schutz der Photosynthese vor Überanregung mittels nichtphotochemischer Eliminierung von Anregungsenergie (NPQ). In der Pflanze bildet LHC-II Trimere in verschiedenen Kombinationen dreier Isoformen (Lhcb1, Lhcb2 und Lhcb3), wobei Lhcb1 mit 70-90% den Hauptteil des LHC-II stellt. Jedes Monomer bindet 8 verschiedene Co-Faktoren in unterschiedlichen Mengen, die ca. 30% seiner Masse ausmachen. Die drei Isoformen des LHC-II sind in allen Pflanzen stark konserviert. Die funktionelle Bedeutung der Isoformen ist jedoch weitestgehend unklar. Dies liegt vor allem an der schwierigen Isolierung reiner Isoformen aus Pflanzenmaterial. Im ersten Teil dieser Arbeit wurden deshalb alle drei Isoformen rekombinant hergestellt und mit getrennt isolierten Lipiden und photosynthetischen Pigmenten in ihre native Form gefaltet. Die anschließende biochemische und spektroskopische Charakterisierung zeigte einen hohen Grad an Homologie zwischen den drei Isoformen, wobei Lhcb3 die größten Unterschiede aufwies (Standfuss und Kühlbrandt 2004). Die wahrscheinlichsten Funktionen für Lhcb1 und Lhcb2 ist die Anpassung der Photosynthese an variierende Lichtbedingungen. LHC-II Heterotrimere mit Lhcb3 Anteil könnten an der Weiterleitung von Lichtenergie von der Haupt Lhcb1/Lhcb2 Antenne zum PS-II Reaktionszentrum beteiligt sein. Für die Erforschung des LHC-II war das mittels Cryo-Elektronenmikroskopie an 2D Kristallen erstellte atomare Modell des Komplexes von enormer Bedeutung. Ein tiefes Verständnis der Funktionen des LHC-II benötigt jedoch eine Struktur von höherer Auflösung, welche mit 2D Kristallen nur schwer zu erreichen ist. Im Verlauf der Arbeit wurden deshalb mehr als 100000 3D Kristallisationsexperimente durchgeführt, wodurch die Kristallisation von aus Erbsenblättern isoliertem und in vitro gefaltetem LHC-II gelang. Die 3D Kristalle aus nativem Material zeigten einen für die röntgenkristallographische Strukturaufklärung ausreichenden Ordnungsgrad und führten zu einer Struktur des LHC-II bei 2.5 Å Auflösung (Standfuss et al., eingereicht). Die Struktur zeigt 223 der 232 Aminosäuren und die Position und Orientierung von 4 Carotinoiden (2 Luteine, 1 Neoxanthin und 1 Violaxanthin), 14 Chlorophyllen (8 Chl a und 6 Chl b) und zwei Lipiden (PG und DGDG) pro Monomer. Diese Informationen sind essentiell für das Verständnis des Energietransfers innerhalb des LHC-II und zu den Photoreaktionszentren und sollten zusammen mit der großen Anzahl von spektroskopischen Untersuchungen eine zukünftige detaillierte Modellierung dieser ultraschnellen und extrem effizienten Energietransfer Prozesse ermöglichen. Auf Basis der Ladungsverteilung der stromalen Seite des Komplexes konnte ein Modell für die Beteiligung des LHC-II an der Stapelung von Grana in Chloroplasten erstellt werden. Dieses liefert außerdem eine plausible Erklärung für den mittels Phosphorylierung des N-Terminus gesteuerten Ausgleich von Anregungsenergie zwischen PS-I und PS-II. Die 2.5 Å Struktur des LHC-II zeigt schließlich einen einfachen aber effektiven Mechanismus zur Optimierung und Schutz des Photosyntheseapparates mittels NPQ. Dieser benötigt keine Strukturänderungen des LHC-II oder der restlichen Lichtsammelantenne und beruht auf der reversiblen Bindung der Xanthophylle Violaxanthin und Zeaxanthin an LHC-II. Diese Arbeit liefert damit Beiträge zu allen Funktionen des LHC-II Komplexes und hilft damit grundlegende Regulationsmechanismen und die Bereitstellung von solarer Energie für die pflanzliche Photosynthese zu verstehen.
The cytochrome bc1 complex is a cornerstone in bioenergetic electron transfer chains, where it carries out tasks as diverse as respiration, photosynthesis, and nitrogen fixation. This homodimeric multisubunit membrane protein has been studied extensively for several decades and the enzyme mechanism is described with the modified protonmotive Q cycle. Still, the molecular and kinetic description of the catalytic cycle is not complete and questions remain regarding the bifurcation of electron transfer at the quinol oxidation (Qo) site, substrate occupancy, pathways of proton conduction, and the nature of the Rieske protein domain movement. We used competitive inhibitors to study the molecular architecture at the Qo site with X-ray crystallography. The structure of the enzyme with the substrate analog 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole (HHDBT) bound at the Qo site was determined at 2.5 Å resolution. Spectroscopic studies showed that HHDBT is negatively charged when bound at the active site. Mechanistic interpretations from inhibitor binding are in line with single occupancy model for quinol oxidation and structural analysis supports the proposed proton transfer pathway. For functional insight into the enzyme mechanism, redox-sensitive protonation changes were studied by Fourier transform infrared spectroscopy. The protein purification procedure was optimized for less delipidation and the isolated enzyme was more active. Furthermore, two new phospholipids were identified in the X-ray structures, including a cardiolipin. Strikingly, conserved lipid binding cavities were observed in structural comparison with homologous enzymes. The functional role of tightly bound phospholipids will be discussed. Finally, the Qo site is a target for various compounds of agricultural and pharmaceutical importance. Importantly, the X-ray structures permit detailed analysis of the molecular reasons for acquired resistance to and treatment failure of Qo site inhibitors, such as atovaquone, that is used to treat malaria and pneumonia, as discussed herein.
The cytochrome bc1 complex or ubiquinol:cytochrome c oxidoreductase (QCR) catalyses electron transfer from ubiquinol to cytochrome c in respiration and photosynthesis coupled to a vectorial proton transport across the membrane, in which the enzyme resides. In both bacteria and eukaryotic organisms, QCR participates in supramolecular assembly of membrane proteins that comprise the respiratory or photosynthetic chain. In the present work, proton transfer pathways, substrate binding and the supramolecular assembly of the respiratory chain in yeast were probed by structure-based site-directed mutagenesis and characterization of the variants. Both active sites centre P, the place of quinol oxidation, and centre N, where quinone reduction takes place, lack direct access to the bulk solvent necessary for proton release and uptake. Based on the X-ray structure, proton transfer pathways were postulated. Analysis at centre P showed, that E272 and Y132 of cytochrome b are important for QCR catalysis as indicated by increased superoxide production and lowered Cyc1p reductase activity in these variants. Pre-steady state heme reduction kinetics in combination with stigmatellin resistance indicated that charge and length of the side chain at position 272 are crucial for efficient docking of the ISP to form the enzyme substrate complex and for electron bifurcation at centre P. Variants of Y312 and F129, both residues of cytochrome b, showed an increased Km indicating participation of these residues in coordination of ubiquinol or the possible intermediate semiquinone anion radical. F129 proved to be crucial for a functional Q-cycle as indicated by respiratory negative growth phenotype and a lowered H+/e- stoichiometry of F129 variants. At centre N, the postulated CL/K and E/R proton transfer pathways are located at opposite sites of the bound ubiquinone. Variants in the surface residues R218 (cytochrome b) and E52 (Qcr7) of the E/R pathway and E82 (Qcr7) of the CL/K pathway showed instability upon purification indicating an important role of these residues for QCR integrity. The slowed down centre N reduction kinetics in H85 (CL/K), R218 and N208 (both E/R) variant was attributed to a destabilised semiquinone anion consistent with the observed decreased sensitivity towards the site-specific inhibitor antimycin and an increased Km. Variants of residues of both pathway, E82Q and R218M, exhibited a decreased H+/e- stoichiometry indicating a crucial role of both residue for maintaining a working Q-cycle and supporting the proposed protonation of the substrate via the Cl/K and the E/R pathway. Long-range interaction between centre N and centre P were observed by altered reduction kinetics of the high potential chain and increased superoxide production in the centre N variants. The role of the cation-pi-interaction between F230 of Cyt1p and R19 of cytochrome c in binding of the redox carrier to QCR was analysed. In F230L hydrophobic interaction were partially lost as was deduced from the ionic strength dependence of Cyc1p reductase activity and Cycp1 binding, as detected by ionic strength sensitive Kd and Km for Cyc1p. The decreased enzymatic rate of F230W could be explained by a disturbed binding of Cyc1p to the variant enzyme. F230 may influence the heme mid point potential and thereby the electron transfer rate to Cyc1p. Reduction of Cobp via both centre P and centre N was disturbed suggesting an interaction between high and low potential chain. Supramolecular association between QCR and cytochrome c oxidase (COX) in yeast mitochondria was probed by affinity chromatography of a his-tagged QCR in the presence of the mild detergent digitonin. In comparison to purification with laurylmaltoside, the presence of both QCR and COX subunits was detected in the elution fractions by SDS-PAGE, Cyc1p reductase and TMPD oxidase activity assays and immunoblot analysis. The CL-dependent formation of the supercomplex between QCR and COX was analysed by replacement variants in the CL-binding site of QCR in CL containing and CL free environment. With an increasing number of replacements of the three lysines the CL-binding pocket supercomplex formation was not abolished, when CL is present as shown by BN-PAGE analysis. This was supported by the synergetic decrease in enzyme activity for both enzymes upon increased number of replacements. In the CL-free environment, no supracomplex formation was observed for a wildtype CL binding site. By replacements of two lysines in the CL-binding pocket, supercomplex formation could be recovered as revealed by BN-PAGE. This indicates, that CL may serve as a charge neutralizer for the lysines near the presumed interaction domain between complex III and complex IV. The obtained results for centre P provide new information of residues critical for stabilisation of ubiquinol and controlling electron short circuit reactions. The observations for centre N variants clearly support the proposed two proton transfer pathways and the role of the bound phospholipids in centre N kinetics. Variants in the Cyc1p binding site suggest a role for F230 both in Cyc1p binding and electron transfer. Clear interaction between the high and low potential chain in both Cyt1p and centre N variants strongly support long-range interactions in the complex. Studies on the supramolecular association of complex III and complex IV indicate a new role of Cl in stabilising a supracomplex.