Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry
| Autor: | Bernhard Spengler, Luis Giron-Monzon, Jan Kosinski, Robert Ahrends, Dieter Kirsch, Peter Friedhoff, Oliver Schulz, Lars Hummerich, Laura Manelyte |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2006 |
| Předmět: |
Streptavidin
Light Stereochemistry macromolecular substances Biology Mass spectrometry Article Chromatography Affinity Evolution Molecular Maleimides chemistry.chemical_compound Benzophenones Affinity chromatography Biotin Genetics Moiety Amino Acid Sequence Cysteine Sulfhydryl Compounds Binding site Peptide sequence Adenosine Triphosphatases Binding Sites Endodeoxyribonucleases C-terminus Escherichia coli Proteins technology industry and agriculture Protein Structure Tertiary DNA-Binding Proteins Cross-Linking Reagents DNA Repair Enzymes MutL Proteins Biochemistry chemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Mutagenesis Site-Directed Peptides Dimerization Sequence Alignment Peptide Hydrolases |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | To investigate protein-protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH-MutL-DNA complex. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|