Biomedical subjects

Susanne Schiffmann

Publications and source records attributed to Susanne Schiffmann.

6 recordsLinked to original sources

PubMed2004

Metal sites in 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with the substrate ribulose 5-phosphate.

The crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal centre has recently been determined at 1.7 A resolution. The enzyme converts ribulose 5-phosphate into 3,4-dihydroxy-2-butanone 4-phosphate, while its C4 atom is released as formate. The resulting four-carbon body supplies all eight C atoms for the xylene moiety of riboflavin. Three of the four hydroxyl groups of ribulose 5-phosphate were coordinated by the metal ions. Based on crystallographic refinement, the metals were assigned as zinc and calcium, which were present in the crystallization buffer. Neither metal supports the enzymatic reaction. In the present study, the correctness of this assignment is assessed using anomalous diffraction data collected at the high-energy side of the zinc absorption edge (lambda = 1.2823 A). Only the three tentative zinc ions give strong peaks in an anomalous difference Fourier map (>20sigma), whereas the four tentative calcium ions do not show anomalous signals above the noise level. These results confirm the initial assignment. In addition, the resolution was improved to 1.55 A.

Binding Sites↗

PubMed2004

Double-chip protein arrays: force-based multiplex sandwich immunoassays with increased specificity.

Protein assays provide direct access to biologically and pharmacologically relevant information. To obtain a maximum of information from the very smallest amounts of complex biological samples, highly multiplexed protein assays are needed. However, at present, cross-reactions of binding reagents restrict the use of such assays to selected cases and severely limit the potential for up-scaling the technology. Here we describe a double-chip format, which can effectively overcome this specificity problem for sandwich immunoassays. This format consists of a capture array and a reference array with fluorescent labeled detection antibodies coupled to the reference array via DNA duplexes. This format allows for the local application of the labeled detection antibodies onto their corresponding specific spots on the capture array. Here we show that this double-chip format allows for the use of cross-reactive antibodies without generating false positive signals, and an assay for the parallel detection of seven different cytokines was set up. Even without further optimization, the dynamic range and the limit of detection for interleukin 8 were found to be comparable to those obtained with other types of multiplexed sandwich immunoassays.

Animals↗

PubMed2004

Double chip protein arrays using recombinant single-chain Fv antibody fragments.

Protein arrays permit the parallel analysis of many different markers in a small sample volume. However, the problem of cross-reactivity limits the degree of multiplexing in parallel sandwich immunoassays (using monoclonal antibodies (mAbs)), meaning antibodies must be prescreened in order to reduce false positives. In contrast, we use a second chip surface for the local application of detection antibodies, thereby efficiently eliminating antibody cross-reactions. Here, we illustrate the potential advantages of using single-chain Fv fragments rather than mAbs as capture and detection molecules with this double chip technology.

Biotinylation↗

PubMed2003

Structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with divalent metal ions and the substrate ribulose 5-phosphate: implications for the catalytic mechanism.

Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.7-A resolution. The carbonyl group (O2) and two out of three free hydroxyl groups (OH3 and OH4) of the substrate are metal-coordinated. We correlate previous mutational studies on this enzyme with the present structural results. Residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity. Only Glu-185 of the second coordination sphere cannot be replaced without complete loss of activity. It contacts the C3 hydrogen atom directly and probably initiates enediol formation in concert with both metal ions to start the reaction sequence. Mechanistic similarities to Rubisco acting on the similar substrate ribulose 1,5-diphosphate in carbon dioxide fixation as well as other carbohydrate (reducto-) isomerases are discussed.

Binding Sites↗

PubMed2003

DNA: a programmable force sensor.

Direct quantification of biomolecular interaction by single-molecule force spectroscopy has evolved into a powerful tool for materials and life sciences. We introduce an approach in which the unbinding forces required to break intermolecular bonds are measured in a differential format by comparison with a known reference bond (here, a short DNA duplex). In addition to a marked increase in sensitivity and force resolution, which enabled us to resolve single-base pair mismatches, this concept allows for highly specific parallel assays. This option was exploited to overcome cross-reactions of antibodies in a protein biochip application.

Animals↗

PubMed2002

Biosynthesis of riboflavin in archaea studies on the mechanism of 3,4-dihydroxy-2-butanone-4-phosphate synthase of Methanococcus jannaschii.

The hypothetical protein predicted by the open reading frame MJ0055 of Methanococcus jannaschii was expressed in a recombinant Escherichia coli strain under the control of a synthetic gene optimized for translation in an eubacterial host. The recombinant protein catalyzes the formation of the riboflavin precursor 3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 174 nmol mg(-1) min(-1) at 37 degrees C. The homodimeric 51.6-kDa protein requires divalent metal ions, preferentially magnesium, for activity. The reaction involves an intramolecular skeletal rearrangement as shown by (13)C NMR spectroscopy using [U-(13)C(5)]ribulose 5-phosphate as substrate. A cluster of charged amino acid residues comprising arginine 25, glutamates 26 and 28, and aspartates 21 and 30 is essential for catalytic activity. Histidine 164 and glutamate 185 were also shown to be essential for catalytic activity.

Amino Acid Sequence↗