Published online 13 June 2006
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry
1 Institut für Biochemie (FB 08), Justus-Liebig-Universität D-35392 Giessen, Germany 2 Institut für Chemie, Humboldt-Universität zu Berlin Brook-Taylor-Strasse 2, 12489 Berlin 3 Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology 4 Ks. Trojdena, 02-109 Warsaw, Poland 4 Institut für Anorganische und Analytische Chemie (FB 08), Justus-Liebig-Universität D-35392 Giessen, Germany
*To whom correspondence should be addressed. Tel: +49 641 9935407; Fax: +49 641 9935409; Email: friedhoff{at}chemie.bio.uni-giessen.de
Received February 13, 2006. Revised May 10, 2006. Accepted May 16, 2006.
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.
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