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Michael Hippler

Publications and source records attributed to Michael Hippler.

At least 19 recordsLinked to original sources
PubMed2006

Infrared spectroscopy of hydrogen-bonded CHCl3-SO2 in the gas phase.

A molecular association between chloroform and sulfur dioxide in the gas phase at room temperature was studied by Fourier transform infrared spectroscopy. Since the intensity of the CH-stretching fundamental vibration of monomer chloroform is very weak but much stronger upon complexation, a simple subtraction procedure isolated the CH-stretching vibration spectrum of the complex. The presence of a 1:1 complex was confirmed by two dilution series, where the monomer concentrations were varied. The molecular association manifested itself as a shift of the peak absorbance of the CH-stretching vibration of CHCl3-SO2 by +7 cm(-1) and of the CD-stretching vibration of CDCl3-SO2 by +5 cm(-1) to higher wave numbers compared to monomer chloroform, accompanied by a considerable broadening of the band contour. In agreement with previous ab initio calculations, this indicates a "blueshifting" or more appropriately, a "C-H contracting" hydrogen bond between chloroform and sulfur dioxide. An estimate of the complex concentration was made based on ab initio calculations for the integrated band strength and the measured spectrum. With this estimate, the equilibrium constant Kp (295 K)=0.014 (po=10(5) Pa) for the dimerization was calculated, providing one of the very few cases where the formation of a hydrogen-bonded gas phase complex at room temperature could be quantitatively studied by infrared spectroscopy.

Chemistry, Physical↗
PubMed2006

Modifications of the lipoamide-containing mitochondrial subproteome in a yeast mutant defective in cysteine desulfurase.

Comparison and identification of mitochondrial matrix proteins from wild-type and cysteine desulfurase-defective (nfs1-14, carrying a hypomorphic allele of NFS1) yeast strains, using two-dimensional gel electrophoresis coupled to mass spectrometry analyses, revealed large changes in the amounts of various proteins. Protein spots that were specifically increased in the nfs1-14 mutant included subunits of lipoamide-containing enzyme complexes: Kgd2, Lat1, and Gcv3, subunits of the mitochondrial alpha-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and glycine cleavage system complexes, respectively. Moreover the increased protein spots corresponded to lipoamide-deficient forms in the nfs1-14 mutant. The increased proteins migrated as separate, cathode-shifted spots, consistent with gain of a lysine charge due to lack of lipoamide addition. Lack of lipoylation of these proteins was further validated using an antibody specific for lipoamide-containing proteins. In addition, this antibody revealed a fourth lipoamide-containing protein, probably corresponding to the E2 component of the branched-chain keto acid dehydrogenase complex. Like the lipoamide-containing forms of Kgd2, Lat1, and Gcv3, this protein also showed decreased lipoic acid reactivity in the nfs1-14 mutant. Cysteine desulfurases, such as yeast NFS1, are required for sulfur addition to iron-sulfur clusters and other sulfur-requiring processes. The results demonstrate that Nfs1 protein is required for the proper post-translational modification of the lipoamide-containing mitochondrial subproteome in yeast and pave the road toward a thorough understanding of its precise role in lipoic acid synthesis.

Carbon-Sulfur Lyases↗
PubMed2005

Quantum-chemical study of CHCl3-SO2 association.

CHCl(3)-SO(2) association is studied by high-level quantum-chemical calculations of stationary points of the dimer electronic potential-energy hypersurface, including correlated second-order Moller-Plesset and CCSD(T) calculations with basis sets up to 6-311++G(d,p). During geometry optimization, frequency, and energy calculations, a self-written computer code embedding the GAMESS ab initio program suite applies counterpoise correction of the basis set superposition error. A CH...O hydrogen-bonded complex (DeltaE(0)=-8.73 kJmol) with a 2.4 A intermolecular H...O distance and two very weak van der Waals complexes (DeltaE(0)=-3.78 and -2.94 kJmol) are located on the counterpoise-corrected potential-energy surface. The intermolecular interactions are characterized by Kitaura-Morokuma interaction energy decompositions and Mulliken electron population analyses. The unusual hydrogen bond is distinguished by a CH-bond contraction, a pronounced enhancement of the IR intensity and a shift to higher frequency ("blueshift") of the CH-stretching vibration compared to the CHCl(3) monomer. Spectroscopy and association in liquid solution is also discussed; our results provide an alternative explanation for features in the CH-stretching vibration spectrum of chloroform dissolved in liquid sulfur dioxide which have been attributed previously to an intermolecular Fermi resonance.

Algorithms↗
PubMed2005

NAB1 is an RNA binding protein involved in the light-regulated differential expression of the light-harvesting antenna of Chlamydomonas reinhardtii.

Photosynthetic organisms respond to changes in ambient light by modulating the size and composition of their light-harvesting complexes, which in the case of the green alga Chlamydomonas reinhardtii consists of >15 members of a large extended family of chlorophyll binding subunits. How their expression is coordinated is unclear. Here, we describe the analysis of an insertion mutant, state transitions mutant3 (stm3), which we show has increased levels of LHCBM subunits associated with the light-harvesting antenna of photosystem II. The mutated nuclear gene in stm3 encodes the RNA binding protein NAB1 (for putative nucleic acid binding protein). In vitro and in vivo RNA binding and protein expression studies have confirmed that NAB1 differentially binds to LHCBM mRNA in a subpolysomal high molecular weight RNA-protein complex. Binding of NAB1 stabilizes LHCBM mRNA at the preinitiation level via sequestration and thereby represses translation. The specificity and affinity of binding are determined by an RNA sequence motif similar to that used by the Xenopus laevis translation repressor FRGY2, which is conserved to varying degrees in the LHCBM gene family. We conclude from our results that NAB1 plays an important role in controlling the expression of the light-harvesting antenna of photosystem II at the posttranscriptional level. The similarity of NAB1 and FRGY2 of Xenopus implies the existence of similar RNA-masking systems in animals and plants.

Amino Acid Sequence↗
PubMed2005

Release of oxidized plastocyanin from photosystem I limits electron transfer between photosystem I and cytochrome b6f complex in vivo.

We used fast absorbance spectroscopy to investigate in vivo binding dynamics and electron transfer between plastocyanin (pc) and photosystem I (PSI), and cytochrome (cyt) f oxidation kinetics in Chlamydomonas reinhardtii mutants in which either the binding or the release of pc from PSI was diminished. Under single flash-excitation conditions, electron flow between PSI and the cyt complex was not affected by a 5-fold lowering of the binding affinity of pc to PSI, as induced by a mutation replacing the tryptophan-651 of the PsaA subunit by a serine residue (PsaA-W651S). On the other hand, electron flow from PSI to the cyt b(6)f complex was very sensitive to a 2- to 3-fold decrease in the rate of pc release from PSI, obtained by replacing the glutamic acid residue 613 of the PsaB subunit with glutamine (PsaB-E613N). Thus, our data indicate that under these experimental conditions the release of oxidized pc limits electron transfer between cyt b(6)f complex and PSI in vivo.

Animals↗
PubMed2005

N-terminal processing of Lhca3 Is a key step in remodeling of the photosystem I-light-harvesting complex under iron deficiency in Chlamydomonas reinhardtii.

Iron deficiency induces a remodeling of the photosynthetic apparatus in Chlamydomonas reinhardtii. In this study we showed that a key mechanistic event in the remodeling process of photosystem I (PSI) and its associated light-harvesting proteins (LHCI) is the N-terminal processing of Lhca3. N-terminal processing of Lhca3 is documented independently by two-dimensional gel electrophoresis and tandem mass spectrometric (MS/MS) analysis as well as by quantitative comparative MS/MS peptide profiling using isotopic labeling of proteins. Dynamic remodeling of the LHCI complex under iron deficiency is further exemplified by depletion of Lhca5 and up-regulation of Lhca4 and Lhca9 polypeptides in respect to photosystem I. Most importantly, the induction of N-terminal processing of Lhca3 by progression of iron deficiency correlates with the functional drop in excitation energy transfer efficiency between LHCI and PSI as assessed by low temperature fluorescence emission spectroscopy. Using an RNA interference (RNAi) strategy, we showed that the truncated form of Lhca3 is essential for the structural stability of LHCI. Depletion of Lhca3 by RNAi strongly impacted the efficiency of excitation energy transfer between PSI and LHCI, as is the case for iron deficiency. However, in contrast to iron deficiency, comparative MS/MS peptide profiling using isotopic labeling of proteins demonstrated that RNAi depletion of Lhca3 caused strong reduction of almost all Lhca proteins in isolated PSI particles.

Amino Acid Sequence↗
PubMed2005

Chlamydomonas reinhardtii proteomics.

Proteomics, based on the expanding genomic resources, has begun to reveal new details of Chlamydomonas reinhardtii biology. In particular, analyses focusing on subproteomes have already provided new insight into the dynamics and composition of the photosynthetic apparatus, the chloroplast ribosome, the oxidative phosphorylation machinery of the mitochondria, and the flagellum. It assisted to discovered putative new components of the circadian clockwork as well as shed a light on thioredoxin protein-protein interactions. In the future, quantitative techniques may allow large scale comparison of protein expression levels. Advances in software algorithms will likely improve the use of genomic databases for mass spectrometry (MS) based protein identification and validation of gene models that have been predicted from the genomic DNA sequences. Although proteomics has only been recently applied for exploring C. reinhardtii biology, it will likely be utilized extensively in the near future due to the already existing genetic, genomic, and biochemical tools.

Animals↗
PubMed2004

Proteomic analysis of the photosystem I light-harvesting antenna in tomato (Lycopersicon esculentum).

Until now, more genes of the light-harvesting antenna of higher-plant photosystem I (PSI) than proteins have been described. To improve our understanding of the composition of light-harvesting complex I (LHCI) of tomato (Lycopersicon esculentum), we combined one- and two-dimensional (1-D and 2-D, respectively) gel electrophoresis with immunoblotting and tandem mass spectrometry (MS/MS). Separation of PSI with high-resolution 1-D gels allowed separation of five bands attributed to proteins of LHCI. Immunoblotting with monospecific antibodies and MS/MS analysis enabled the correct assignment of the four prominent bands to light-harvesting proteins Lhca1-4. The fifth band was recognized by only the Lhca1 antibody. Immunodetection as well as mass spectrometric analysis revealed that these protein bands contain not only the eponymous protein but also other Lhca proteins, indicating a heterogeneous protein composition of Lhca bands. Additionally, highly sensitive MS/MS allowed detection of a second Lhca4 isoform and of Lhca5. These proteins had not been described before on the protein level in higher plants. Two-dimensional gel electrophoresis revealed an even more diverse composition of individual Lhca proteins than was apparent from 1-D gels. For each of the four prominent Lhca proteins, four to five isoforms with different isoelectric points could be identified. In the case of Lhca1, Lhca4, and Lhca3, additional isoforms with slightly differing molecular masses were identified. Thus, we were able to detect four to ten isoforms of each individual Lhca protein in PSI. Reasons for the origin of Lhca heterogeneity are discussed. The observed variety of Lhca proteins and their isoforms is of particular interest in the context of the recently published crystal structure of photosystem I from pea, which showed the presence of only four Lhca proteins per photosystem I. These findings indicate that several populations of photosystem I that differ in their Lhca composition may exist.

Amino Acid Sequence↗
PubMed2004

Comparison of the subunit compositions of the PSI-LHCI supercomplex and the LHCI in the green alga Chlamydomonas reinhardtii.

Although the light-harvesting chlorophyll protein complex I (LHCI) of photosystem I (PSI) is intimately associated with the PSI core complex and forms the PSI-LHCI supercomplex, the LHCI is normally synthesized in PSI-deficient mutants. In this paper, we compared the subunit compositions of the PSI-LHCI supercomplex and the LHCI by immunoblot analysis and two-dimensional gel electrophoresis combined with mass spectrometry. The PSI-LHCI supercomplex and the LHCI were purified by sucrose density gradient centrifugation and (diethylamino)ethyl column chromatography from n-dodecyl-beta-D-maltoside-solubilized thylakoids of the wild-type and DeltapsaB mutant of the green alga Chlamydomonas reinhardtii. The PSI-LHCI supercomplex contained all of the nine Lhca polypeptides (Lhca1-9) that are detected in wild-type thylakoids. In contrast, the LHCI retained only six Lhca polypeptides, whereas Lhca3 and two minor polypeptides, Lhca2 and Lhca9, were lost during the purification procedure. Sucrose density gradient centrifugation showed that the purified LHCI retains an oligomeric structure with an apparent molecular mass of 300-400 kDa. We therefore concluded that Lhca2, Lhca3, and Lhca9 are not required for the stable oligomeric structure of the LHCI and that the association of these polypeptides in the LHCI is stabilized by the presence of the PSI core complex. Finally, we discuss the possible localization and function of Lhca polypeptides in the LHCI.

Amino Acid Sequence↗
PubMed2004

Light induces phosphorylation of glucan water dikinase, which precedes starch degradation in turions of the duckweed Spirodela polyrhiza.

Degradation of storage starch in turions, survival organs of Spirodela polyrhiza, is induced by light. Starch granules isolated from irradiated (24 h red light) or dark-stored turions were used as an in vitro test system to study initial events of starch degradation. The starch-associated pool of glucan water dikinase (GWD) was investigated by two-dimensional gel electrophoresis and by western blotting using antibodies raised against GWD. Application of this technique allowed us to detect spots of GWD, which are light induced and absent on immunoblots prepared from dark-adapted plants. These spots, showing increased signal intensity following incubation of the starch granules with ATP, became labeled by randomized [betagamma-33P]ATP but not by [gamma-33P]ATP and were removed by acid phosphatase treatment. This strongly suggests that they represent a phosphorylated form(s) of GWD. The same light signal that induces starch degradation was thus demonstrated for the first time to induce autophosphorylation of starch-associated GWD. The in vitro assay system has been used to study further effects of the light signal that induces autophosphorylation of GWD and starch degradation. In comparison with starch granules from dark-adapted plants, those from irradiated plants showed increase in (1) binding capacity of GWD by ATP treatment decreased after phosphatase treatment; (2) incorporation of the beta-phosphate group of ATP into starch granules; and (3) rate of degradation of isolated granules by starch-associated proteins, further enhanced by phosphorylation of starch. The presented results provide evidence that autophosphorylation of GWD precedes the initiation of starch degradation under physiological conditions.

Adenosine Triphosphate↗
PubMed2004

Subunit composition of NDH-1 complexes of Synechocystis sp. PCC 6803: identification of two new ndh gene products with nuclear-encoded homologues in the chloroplast Ndh complex.

Cyanobacteria contain several genes, annotated ndh, whose products show sequence similarities to subunits found in complex I (NADH:ubiquinone oxidoreductase) of eubacteria and mitochondria. However, it is still unclear whether the cyanobacterial ndh gene products actually form a single large protein complex or exist as smaller independent complexes. To address this, we have constructed a strain of Synechocystis sp. PCC 6803 in which the C terminus of the NdhJ subunit was fused to an His(6) tag to aid isolation. Three major NdhJ-containing complexes were resolved by blue native polyacrylamide gel electrophoresis, with approximate apparent molecular masses of 460, 330, and 110 kDa. N-terminal sequencing and mass spectrometry revealed that the 460-kDa complex contained ten annotated ndh gene products. Detergent-induced fragmentation experiments indicated that the 460-kDa complex was composed of hydrophobic (150 kDa) and hydrophilic (110-130 kDa) modules similar to that found in the minimal form of complex I found in Escherichia coli, except that the electron input module was not conserved. The difference in size between the 460- and 330-kDa complexes is attributed to differences in the stoichiometry of the hydrophilic and hydrophobic modules in the complex, either 2:1 or 1:1, respectively. We have also detected the presence of two new Ndh subunits (slr1623 and sll1262) that are unrelated to subunits in the eubacterial complex I but which have homologues in the closely related chloroplast Ndh complex of maize (Funk, E., Schäfer, E., and Steinmüller, K. (1999) J. Plant Physiol. 154, 16-23). The presence of these additional subunits might reflect the use by the NDH-1 and Ndh complexes of a different, so far unidentified, electron input module.

Amino Acid Sequence↗
PubMed2004

Successful herbivore attack due to metabolic diversion of a plant chemical defense.

Plants protect themselves against herbivory with a diverse array of repellent or toxic secondary metabolites. However, many herbivorous insects have developed counteradaptations that enable them to feed on chemically defended plants without apparent negative effects. Here, we present evidence that larvae of the specialist insect, Pieris rapae (cabbage white butterfly, Lepidoptera: Pieridae), are biochemically adapted to the glucosinolate-myrosinase system, the major chemical defense of their host plants. The defensive function of the glucosinolate-myrosinase system results from the toxic isothiocyanates that are released when glucosinolates are hydrolyzed by myrosinases on tissue disruption. We show that the hydrolysis reaction is redirected toward the formation of nitriles instead of isothiocyanates if plant material is ingested by P. rapae larvae, and that the nitriles are excreted with the feces. The ability to form nitriles is due to a larval gut protein, designated nitrile-specifier protein, that by itself has no hydrolytic activity on glucosinolates and that is unrelated to any functionally characterized protein. Nitrile-specifier protein appears to be the key biochemical counteradaptation that allows P. rapae to feed with impunity on plants containing glucosinolates and myrosinases. This finding sheds light on the ecology and evolution of plant-insect interactions and suggests novel highly selective pest management strategies.

Amino Acid Sequence↗
PubMed2004

A new approach that allows identification of intron-split peptides from mass spectrometric data in genomic databases.

We present a new approach that allows the identification of intron-split peptides from mass spectrometric data in genomic databases. Our algorithm uses small regions of peptide sequence information which are automatically deduced from de novo amino acid sequence predictions together with the molecular mass information of the precursor ion. The sequence predictions are based on selected collision-induced mass spectrometric fragmentation spectra. Fragments of the predicted amino acid sequence are aligned with each of the six frames of the translated genome and the precursor mass information is used to assemble the corresponding tryptic peptides using the sequence as a matrix. Hereby, intron-split peptides can be gathered and in turn verified by mass spectrometric data interpretation tools such as Sequest.

Algorithms↗
PubMed2004

The hydrophobic recognition site formed by residues PsaA-Trp651 and PsaB-Trp627 of photosystem I in Chlamydomonas reinhardtii confers distinct selectivity for binding of plastocyanin and cytochrome c6.

On the lumenal side of photosystem I (PSI), each of the two large core subunits, PsaA and PsaB, expose a conserved tryptophan residue to the surface. PsaB-Trp(627) is part of the hydrophobic recognition site that is essential for tight binding of the two electron donors plastocyanin and cytochrome c(6) to the donor side of PSI (Sommer, F., Drepper, F., and Hippler, M. (2002) J. Biol. Chem. 277, 6573-6581). To examine the function of PsaA-Trp(651) in binding and electron transfer of both donors to PSI, we generated the mutants PsaA-W651F and PsaA-W651S by site-directed mutagenesis and biolistic transformation of Chlamydomonas reinhardtii. The protein-protein interaction and the electron transfer between the donors and PSI isolated from the mutants were analyzed by flash absorption spectroscopy. The mutation PsaA-W651F completely abolished the formation of a first order electron transfer complex between plastocyanin (pc) and the altered PSI and increased the dissociation constant for binding of cytochrome (cyt) c(6) by more than a factor of 10 as compared with wild type. Mutation of PsaA-Trp(651) to Ser had an even larger impact on the dissociation constant. The K(D) value increased another 2-fold when the values obtained for the interaction and electron transfer between cyt c(6) and PSI from PsaA-W651S and PsaA-W651F are compared. In contrast, binding and electron transfer of pc to PSI from PsaA-W651S improved as compared with PSI from PsaA-W651F and admitted the formation of an inter-molecular electron transfer complex, resulting in a K(D) value of about 554 microm that is still five times higher than observed for wild type. These results demonstrate that PsaA-Trp(651) is, such as PsaB-Trp(627), crucial for high affinity binding of pc and cyt c(6) to PSI. Our results also indicate that the highly conserved structural recognition motif that is formed by PsaA-Trp(651) and PsaB-Trp(627) confers a differential selectivity in binding of both donors to PSI.

Animals↗
PubMed2004

Functional proteomics of circadian expressed proteins from Chlamydomonas reinhardtii.

In this study, functional proteomics was successfully applied for the characterization of circadian expressed, basic proteins. For this purpose, we have chosen the green model alga Chlamydomonas reinhardtii since its entire nuclear genome is available and it is ideally suited for biochemical enrichment procedures. Proteins from cells harvested during subjective day and night were heparin affinity purified. They were separated by two-dimensional gel electrophoresis suited for basic proteins and analyzed after tryptic digestion by electrospray ionization mass spectrometry. We can show for the first time that the expressions of a protein disulfide isomerase-like protein and a tetratricopeptide repeat protein change in a circadian manner. Interestingly, both proteins are known to be interaction partners in multiprotein complexes including RNA binding proteins.

Amino Acid Sequence↗
PubMed2004

Introduction.

Explore the source record for details and available documents.

Journal Article↗
PubMed2003

Proteomics of Chlamydomonas reinhardtii light-harvesting proteins.

With the recent development of techniques for analyzing transmembrane thylakoid proteins by two-dimensional gel electrophoresis, systematic approaches for proteomic analyses of membrane proteins became feasible. In this study, we established detailed two-dimensional protein maps of Chlamydomonas reinhardtii light-harvesting proteins (Lhca and Lhcb) by extensive tandem mass spectrometric analysis. We predicted eight distinct Lhcb proteins. Although the major Lhcb proteins were highly similar, we identified peptides which were unique for specific lhcbm gene products. Interestingly, lhcbm6 gene products were resolved as multiple spots with different masses and isoelectric points. Gene tagging experiments confirmed the presence of differentially N-terminally processed Lhcbm6 proteins. The mass spectrometric data also revealed differentially N-terminally processed forms of Lhcbm3 and phosphorylation of a threonine residue in the N terminus. The N-terminal processing of Lhcbm3 leads to the removal of the phosphorylation site, indicating a potential novel regulatory mechanism. At least nine different lhca-related gene products were predicted by comparison of the mass spectrometric data against Chlamydomonas expressed sequence tag and genomic databases, demonstrating the extensive variability of the C. reinhardtii Lhca antenna system. Out of these nine, three were identified for the first time at the protein level. This proteomic study demonstrates the complexity of the light-harvesting proteins at the protein level in C. reinhardtii and will be an important basis of future functional studies addressing this diversity.

Amino Acid Sequence↗