Biomedical subjects

Harald Seitz

Publications and source records attributed to Harald Seitz.

11 recordsLinked to original sources

PubMed2006

Differential binding studies applying functional protein microarrays and surface plasmon resonance.

A variety of different in vivo and in vitro technologies provide comprehensive insights in protein-protein interaction networks. Here we demonstrate a novel approach to analyze, verify and quantify putative interactions between two members of the S100 protein family and 80 recombinant proteins derived from a proteome-wide protein expression library. Surface plasmon resonance (SPR) using Biacore technology and functional protein microarrays were used as two independent methods to study protein-protein interactions. With this combined approach we were able to detect nine calcium-dependent interactions between Arg-Gly-Ser-(RGS)-His6 tagged proteins derived from the library and GST-tagged S100B and S100A6, respectively. For the protein microarray affinity-purified proteins from the expression library were spotted onto modified glass slides and probed with the S100 proteins. SPR experiments were performed in the same setup and in a vice-versa approach reversing analytes and ligands to determine distinct association and dissociation patterns of each positive interaction. Besides already known interaction partners, several novel binders were found independently with both detection methods, albeit analogous immobilization strategies had to be applied in both assays.

Biosensing Techniques↗

PubMed2006

Bacteriophage replication modules.

Bacteriophages (prokaryotic viruses) are favourite model systems to study DNA replication in prokaryotes, and provide examples for every theoretically possible replication mechanism. In addition, the elucidation of the intricate interplay of phage-encoded replication factors with 'host' factors has always advanced the understanding of DNA replication in general. Here we review bacteriophage replication based on the long-standing observation that in most known phage genomes the replication genes are arranged as modules. This allows us to discuss established model systems--f1/fd, phiX174, P2, P4, lambda, SPP1, N15, phi29, T7 and T4--along with those numerous phages that have been sequenced but not studied experimentally. The review of bacteriophage replication mechanisms and modules is accompanied by a compendium of replication origins and replication/recombination proteins (available as supplementary material online).

Bacteriophages↗

PubMed2005

Recent advances of protein microarrays.

Technological innovations and novel applications have greatly advanced the field of protein microarrays. Over the past two years, different types of protein microarrays have been used for serum profiling, protein abundance determinations, and identification of proteins that bind DNA or small compounds. However, considerable development is still required to ensure common quality standards and to establish large content repertoires. Here, we summarize applications available to date and discuss recent technological achievements and efforts on standardization.

Animals↗

PubMed2005

High throughput identification of potential Arabidopsis mitogen-activated protein kinases substrates.

Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance.

Arabidopsis↗

PubMed2005

Miniaturization in functional genomics and proteomics.

Proteins are the key components of the cellular machinery responsible for processing changes that are ordered by genomic information. Analysis of most human proteins and nucleic acids is important in order to decode the complex networks that are likely to underlie many common diseases. Significant improvements in current technology are also required to dissect the regulatory processes in high-throughtput and with low cost. Miniaturization of biological assays is an important prerequisite to achieve these goals in the near future.

Biological Assay↗

PubMed2004

A catalog of human cDNA expression clones and its application to structural genomics.

We describe here a systematic approach to the identification of human proteins and protein fragments that can be expressed as soluble proteins in Escherichia coli. A cDNA expression library of 10,825 clones was screened by small-scale expression and purification and 2,746 clones were identified. Sequence and protein-expression data were entered into a public database. A set of 163 clones was selected for structural analysis and 17 proteins were prepared for crystallization, leading to three new structures.

Catalogs as Topic↗

PubMed2004

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein-DNA interactions.

To gain insights into complex biological processes, such as transcription and replication, the analysis of protein-DNA interactions and the determination of their sequence requirements are of central importance. In this study, we probed protein microarray technology and ultraviolet crosslinking combined with mass spectrometry (MS) for their practicability to study protein-DNA interactions. We chose as a model system the well-characterized interaction of bacterial replication initiator DnaA with its cognate binding site, the DnaA box. Interactions of DnaA domain 4 with a high-affinity DnaA box (R4) and with a low-affinity DnaA box (R3) were compared. A mutant DnaA domain 4, A440V, was included in the study. DnaA domain 4, wt, spotted onto FAST slides, revealed a strong signal only with a Cy5-labeled, double-stranded, 21-mer oligonucleotide containing DnaA box R4. No signals were obtained when applying the mutant protein. Ultraviolet crosslinking combined with nanoLC/MALDI-TOF MS located the site of interaction to a peptide spanning amino acids 433- 442 of Escherichia coli DnaA. This fragment contains six residues that were identified as being involved in DNA binding by recently published crystal structure and nuclear magnetic resonance (NMR) analysis. In the future, the technologies applied in this study will become important tools for studying protein-DNA interactions.

Amino Acid Sequence↗

PubMed2002

Protein identification by MALDI-TOF-MS peptide mapping: a new strategy.

A new strategy for identifying proteins by MALDI-TOF-MS peptide mapping is reported. In contrast to current approaches, the strategy does not rely on a good relative or absolute mass accuracy as the criterion that discriminates false positive results. The protein sequence database is first searched for all proteins that match a minimum five of the submitted masses within the maximum expected relative errors when the default or externally determined calibration constants are used, for instance, +/-500 ppm. Typically, this search retrieves many thousand candidate sequences. Assuming initially that each of these is the correct protein, the relative errors of the matching peptide masses are calculated for each candidate sequence. Linear regression analysis is then performed of the calculated relative errors as a function of m/z for each candidate sequence, and the standard deviation to the regression is used to distinguish the correct sequence among the candidates. We show that this parameter is independent of whether the mass spectrometric data were internally or externally calibrated. The result is a search engine that renders internal spectrum calibration unnecessary and adapts to the quality of the raw data without user interference. This is made possible by a dynamic scoring algorithm, which takes into account the number of matching peptide masses, the percentage of the protein's sequence covered by these peptides and, as new parameter, the determined standard deviation. The lower the standard deviation, the less cleavage peptides are required for identification and vice versa. Performance of the new strategy is demonstrated and discussed. All necessary computing has been implemented in a computer program, free access to which is provided in the Internet.

Algorithms↗

PubMed2002

Generation of minimal protein identifiers of proteins from two-dimensional gels and recombinant proteins.

We describe the technical feasibility and methodology to characterize a protein by a minimal set of structural information generated by matrix assisted laser desorption/ionization (MALDI)-mass spectrometry, termed a "minimal protein Identifier" (MPI). MPIs can be determined for proteins from two-dimensional gels and recombinant proteins and can be used to compare and identify proteins from these sources.

Brain↗

PubMed2002

Strand-specific loading of DnaB helicase by DnaA to a substrate mimicking unwound oriC.

We analysed the enzymatic activity (strand dis-placement) of the Escherichia coli DnaB helicase on a mirror-image pair of oligonucleotide-based substrates mimicking the unwound replication origin oriC. Loading of the helicase complex occurred exclusively to the single-stranded 'lower strand' part of the substrates. Full helicase activity required DnaA bound to the double-stranded part of the substrates (oriC DnaA box R1) and to their single-stranded 'upper strand' part. We assume that in vivo DnaA also loads the first of two helicase complexes - required for the assembly of two replication forks - to the lower strand of oriC during initiation of bidirectional chromosome replication in E. coli.

Bacterial Proteins↗

PubMed2001

The double mechanism of incompatibility between lambda plasmids and Escherichia coli dnaA(ts) host cells.

For plasmids derived from bacteriophage lambda, the initiation of bidirectional DNA replication from orilambda depends on the stimulation of transcription from the p(R) promoter by the host replication initiator protein DnaA. Certain Escherichia coli dnaA(ts) mutants cannot be transformed by wild-type lambda plasmids even at the temperature permissive to cell growth. This plasmid-host incompatibility appeared to be due to inefficient stimulation of transcription from the p(R) promoter by the mutant DnaA protein. This paper shows that there is a second mechanism for the incompatibility between lambda plasmids and dnaA(ts) hosts, exemplified in this study by the dnaA46 mutant. This is based on the competition between the lambda P protein and the host DnaA and DnaC proteins for DnaB helicase. Both mechanisms must be operative for the incompatibility.

Bacterial Proteins↗