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Borries Demeler

Publications and source records attributed to Borries Demeler.

27 records · Page 2Linked to original sources
PubMed2004

Kinetic mechanism and quaternary structure of Aminobacter aminovorans NADH:flavin oxidoreductase: an unusual flavin reductase with bound flavin.

The homodimeric NADH:flavin oxidoreductase from Aminobacter aminovorans is an NADH-specific flavin reductase herein designated FRD(Aa). FRD(Aa) was characterized with respect to purification yields, thermal stability, isoelectric point, molar absorption coefficient, and effects of phosphate buffer strength and pH on activity. Evidence from this work favors the classification of FRD(Aa) as a flavin cofactor-utilizing class I flavin reductase. The isolated native FRD(Aa) contained about 0.5 bound riboflavin-5'-phosphate (FMN) per enzyme monomer, but one bound flavin cofactor per monomer was obtainable in the presence of excess FMN or riboflavin. In addition, FRD(Aa) holoenzyme also utilized FMN, riboflavin, or FAD as a substrate. Steady-state kinetic results of substrate titrations, dead-end inhibition by AMP and lumichrome, and product inhibition by NAD(+) indicated an ordered sequential mechanism with NADH as the first binding substrate and reduced FMN as the first leaving product. This is contrary to the ping-pong mechanism shown by other class I flavin reductases. The FMN bound to the native FRD(Aa) can be fully reduced by NADH and subsequently reoxidized by oxygen. No NADH binding was detected using 90 microM FRD(Aa) apoenzyme and 300 microM NADH. All results favor the interpretation that the bound FMN was a cofactor rather than a substrate. It is highly unusual that a flavin reductase using a sequential mechanism would require a flavin cofactor to facilitate redox exchange between NADH and a flavin substrate. FRD(Aa) exhibited a monomer-dimer equilibrium with a K(d) of 2.7 microM. Similarities and differences between FRD(Aa) and certain flavin reductases are discussed.

Alphaproteobacteria↗
PubMed2004

Chaperone activity of cytosolic small heat shock proteins from wheat.

Small Hsps (sHsps) and the structurally related eye lens alpha-crystallins are ubiquitous stress proteins that exhibit ATP-independent molecular chaperone activity. We studied the chaperone activity of dodecameric wheat TaHsp16.9C-I, a class I cytosolic sHsp from plants and the only eukaryotic sHsp for which a high resolution structure is available, along with the related wheat protein TaHsp17.8C-II, which represents the evolutionarily distinct class II plant cytosolic sHsps. Despite the available structural information on TaHsp16.9C-I, there is minimal data on its chaperone activity, and likewise, data on activity of the class II proteins is very limited. We prepared purified, recombinant TaHsp16.9C-I and TaHsp17.8C-II and find that the class II protein comprises a smaller oligomer than the dodecameric TaHsp16.9C-I, suggesting class II proteins have a distinct mode of oligomer assembly as compared to the class I proteins. Using malate dehydrogenase as a substrate, TaHsp16.9C-I was shown to be a more effective chaperone than TaHsp17.8C-II in preventing heat-induced malate dehydrogenase aggregation. As observed by EM, morphology of sHsp/substrate complexes depended on the sHsp used and on the ratio of sHsp to substrate. Surprisingly, heat-denaturing firefly luciferase did not interact significantly with TaHsp16.9C-I, although it was fully protected by TaHsp17.8C-II. In total the data indicate sHsps show substrate specificity and suggest that N-terminal residues contribute to substrate interactions.

Amino Acid Sequence↗
PubMed2003

Functional requirement for symmetric assembly of archaeal box C/D small ribonucleoprotein particles.

Box C/D small ribonucleoprotein particles (sRNPs) are archaeal homologs of small nucleolar ribonucleoprotein particles (snoRNPs) in eukaryotes that are responsible for site specific 2'-O-methylation of ribosomal and transfer RNAs. The function of box C/D sRNPs is characterized by step-wise assembly of three core proteins around a box C/D RNA that include fibrillarin, Nop5p, and L7Ae. The most distinct structural feature in all box C/D RNAs is the presence of two conserved box C/D motifs accompanied by often a single, and sometimes two, antisense elements located immediately upstream of either the D or D' box. Despite this asymmetric distribution of antisense elements, the bipartite feature of the box C/D motifs appears to be in pleasing agreement with a recently reported three-dimensional structure of the core protein complex between fibrillarin and Nop5p. This investigates functional implications of the symmetric features both in box C/D RNAs and in the fibrillarin-Nop5p complex. Site-directed mutagenesis was employed to generate box C/D RNAs lacking one of the two box C/D motifs and a mutant fibrillarin-Nop5p complex deficient in self-association. The ability of the mutated components to assemble and to direct methyl transfer reactions was assessed by gel mobility-shift, analytical ultracentrifugation, and in vitro catalysis studies. The results presented here suggest that, while a box C/D sRNP is capable of asymmetrical assembly, the symmetries in both the box C/D RNA and in the fibrillarin-Nop5p complex are required for efficient catalysis. These findings underscore the importance of functional assembly in methyl transfer reactions.

Archaeal Proteins↗
PubMed2003

Insights into dimerization and four-helix bundle formation found by dissection of the dimer interface of the GrpE protein from Escherichia coli.

The GrpE heat shock protein from Escherichia coli has a homodimeric structure. The dimer interface encompasses two long alpha-helices at the NH(2)-terminal end from each monomer (forming a "tail"), which lead into a small four-helix bundle from which each monomer contributes two short sequential alpha-helices in an antiparallel topological arrangement. We have created a number of different deletion mutants of GrpE that have portions of the dimer interface to investigate requirements for dimerization and to study four-helix bundle formation. Using chemical crosslinking and analytical ultracentrifugation techniques to probe for multimeric states, we find that a mutant containing only the long alpha-helical tail portion (GrpE1-88) is unable to form a dimer, most likely due to a decrease in alpha-helical content as determined by circular dichroism spectroscopy, thus one reason for a dimeric structure for the GrpE protein is to support the tail region. Mutants containing both of the short alpha-helices (GrpE1-138 and GrpE88-197) are able to form a dimer and presumably the four-helix bundle at the dimer interface. These two mutants have equilibrium constants for the monomer-dimer equilibrium that are very similar to the full-length protein suggesting that the tail region does not contribute significantly to the stability of the dimer. Interestingly, one mutant that contains just one of the short alpha-helices (GrpE1-112) exists as a tetrameric species, which presumably is forming a four-helix bundle structure. A proposed model is discussed for this mutant and its relevance for factors influencing four-helix bundle formation.

Bacterial Proteins↗
PubMed2002

Cytotoxic cell granule-mediated apoptosis. Characterization of the macromolecular complex of granzyme B with serglycin.

We have recently shown that the physiological mediator of granule-mediated apoptosis is a macromolecular complex of granzymes and perforin complexed with the chondroitin-sulfate proteoglycan, serglycin (Metkar, S. S., Wang, B., Aguilar-Santelises, M., Raja, S. M., Uhlin-Hansen, L., Podack, E., Trapani, J. A., and Froelich, C. J. (2002) Immunity 16, 417-428). We now report our biophysical studies establishing the nature of granzyme B-serglycin (GrB.SG) complex. Dynamic laser light scattering studies establish that SG has a hydrodynamic radius of approximately 140 +/- 23 nm, comparable to some viral particles. Agarose mobility shift gels and surface plasmon resonance (SPR), show that SG binds tightly to GrB and has the capacity to hold 30-60 GrB molecules. SPR studies also indicate equivalent binding affinities (K(d) approximately 0.8 microm), under acidic (granule pH) and neutral isotonic conditions (extra-cytoplasmic pH), for GrB.SG interaction. Finally, characterization of GrB.SG interactions within granules revealed complexes of two distinct molecular sizes, one held approximately 4-8 molecules of GrB, whereas the other contained as many as 32 molecules of GrB or other granule proteins. These studies provide a firm biophysical basis for our earlier reported observations that the proapoptotic granzyme is exocytosed predominantly as a macromolecular complex with SG.

Apoptosis↗
PubMed2002

The quaternary structure of the HisZ-HisG N-1-(5'-phosphoribosyl)-ATP transferase from Lactococcus lactis.

The N-1-(5'-phosphoribosyl)-ATP transferase (ATP-PRTase) encoded by the hisG locus catalyzes the condensation of ATP with PRPP, the first reaction in the biosynthesis of histidine. Unlike the homohexameric forms of the enzyme found in Escherichia coli and Salmonella typhimurium, the ATP-PRTase from Lactococcus lactis and a number of other bacterial species consists of two different polypeptides, both of which are required for catalytic activity (Sissler et al. (1999) Proc. Natl. Acad. Sci. 96, 8985-8990). The first of these is a truncated version of HisG that is approximately 100 amino acids shorter than the canonical versions. The second, HisZ, is a 328-residue version of a class II aminoacyl-tRNA synthetase catalytic domain that possesses no aminoacylation function. Here, the molecular mass and subunit composition of the L. lactis HisZ-HisG heteromeric ATP-PRTase is investigated using liquid chromatography, analytical ultracentrifugation, and quantitative protein sequencing. Individually, HisZ and HisG form inactive but stable dimers with association constants in the range of 2.5-3.3 x 10(5) M(-1). When both types of subunits are present, a quaternary octamer complex is formed with a sedimentation coefficient of 10.1 S. Incubation of this complex with ATP promotes a shift to 10.7 S. By contrast, incubation with the allosteric modulators AMP and histidine destabilizes the complex, resulting in a shift to multiple species in equilibrium with an average of 9.3 S. While this octameric structure is unique to both the phosphoribosyl transferases and the aminoacyl-tRNA synthetases, the change in sedimentation behavior elicited by substrates and inhibitors suggests the presence of allosteric regulatory mechanisms reminiscent of other multisubunit enzymes of metabolic importance.

Amino Acyl-tRNA Synthetases↗
PubMed2002

Crystal structure and dimerization equilibria of PcoC, a methionine-rich copper resistance protein from Escherichia coli.

PcoC is a soluble periplasmic protein encoded by the plasmid-born pco copper resistance operon of Escherichia coli. Like PcoA, a multicopper oxidase encoded in the same locus and its chromosomal homolog CueO, PcoC contains unusual methionine rich sequences. Although essential for copper resistance, the functions of PcoC, PcoA, and their conserved methionine-rich sequences are not known. Similar methionine motifs observed in eukaryotic copper transporters have been proposed to bind copper, but there are no precedents for such metal binding sites in structurally characterized proteins. The high-resolution structures of apo PcoC, determined for both the native and selenomethionine-containing proteins, reveal a seven-stranded beta barrel with the methionines unexpectedly housed on a solvent-exposed loop. Several potential metal-binding sites can be discerned by comparing the structures to spectroscopic data reported for copper-loaded PcoC. In the native structure, the methionine loop interacts with the same loop on a second molecule in the asymmetric unit. In the selenomethionine structure, the methionine loops are more exposed, forming hydrophobic patches on the protein surface. These two arrangements suggest that the methionine motifs might function in protein-protein interactions between PcoC molecules or with other methionine-rich proteins such as PcoA. Analytical ultracentrifugation data indicate that a weak monomer-dimer equilibrium exists in solution for the apo protein. Dimerization is significantly enhanced upon binding Cu(I) with a measured delta(deltaG degrees )<or=-8.0 kJ/mole, suggesting that copper might bind at the dimer interface.

Amino Acid Motifs↗
PubMed2001

Structure-function analysis of the heat shock factor-binding protein reveals a protein composed solely of a highly conserved and dynamic coiled-coil trimerization domain.

Heat shock factor-binding protein (HSBP) 1 is a small, evolutionarily conserved protein originally identified in a yeast two-hybrid screen using the trimerization domain of heat shock factor (HSF) 1 as the bait. Similar in size to HSF1 trimerization domain, human HSBP1 contains two arrays of hydrophobic heptad repeats (designated HR-N and HR-C) characteristic of coiled-coil proteins. Proteins of the HSBP family are relatively small (<100 residues), comprising solely a putative coiled-coil oligomerization domain without any other readily recognizable structural or functional motif. Our biophysical and biochemical characterization of human HSBP1 reveals a cooperatively folded protein with high alpha-helical content and moderate stability. NMR analyses reveal a single continuous helix encompassing both HR-N and HR-C in the highly conserved central region, whereas the less conserved carboxyl terminus is unstructured and accessible to proteases. Unlike previously characterized coiled-coils, backbone 15N relaxation measurements implicate motional processes on the millisecond time scale in the coiled-coil region. Analytical ultracentrifugation and native PAGE studies indicate that HSBP1 is predominantly trimeric over a wide concentration range. NMR analyses suggest a rotationally symmetric trimer. Because the highly conserved hydrophobic heptad repeats extend over 60% of HSBP1, we propose that HSBP most likely regulates the function of other proteins through coiled-coil interactions.

Amino Acid Sequence↗