title: DNA-Directed Assembly of Capture Tools for Constitutional Studies of Large Protein Complexes.
abstract: Large supramolecular protein complexes, such as the molecular machinery involved in gene regulation, cell signaling, or cell division, are key in all fundamental processes of life. Detailed elucidation of structure and dynamics of such complexes can be achieved by reverse-engineering parts of the complexes in order to probe their interactions with distinctive binding partners in vitro. The exploitation of DNA nanostructures to mimic partially assembled supramolecular protein complexes in which the presence and state of two or more proteins are decisive for binding of additional building blocks is reported here. To this end, four-way DNA Holliday junction motifs bearing a fluorescein and a biotin tag, for tracking and affinity capture, respectively, are site-specifically functionalized with centromeric protein (CENP) C and CENP-T. The latter serves as baits for binding of the so-called KMN component, thereby mimicking early stages of the assembly of kinetochores, structures that mediate and control the attachment of microtubules to chromosomes in the spindle apparatus. Results from pull-down experiments are consistent with the hypothesis that CENP-C and CENP-T may bind cooperatively to the KMN network.
authors: Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG 1), Hermann-von-Helmholtz-Platz 1, D-76344, Eggenstein-Leopoldshafen, Germany.
authors: Rebecca
authors: R
authors: Meyer
authors: Max-Planck Institut für Molekulare Physiologie, Otto-Hahn-Straße 11, 44227, Dortmund, Germany.
authors: Alex
authors: A
authors: Faesen
authors: TU Dortmund, Fakultät für Chemie und Chemische Biologie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Str. 6, 44227, Dortmund, Germany.
authors: Katrin
authors: K
authors: Vogel
authors: Max-Planck Institut für Molekulare Physiologie, Otto-Hahn-Straße 11, 44227, Dortmund, Germany.
authors: Sadasivam
authors: S
authors: Jeganathan
authors: Max-Planck Institut für Molekulare Physiologie, Otto-Hahn-Straße 11, 44227, Dortmund, Germany.|Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitätsstrasse 5, 45141, Essen, Germany.
authors: Andrea
authors: A
authors: Musacchio
authors: Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG 1), Hermann-von-Helmholtz-Platz 1, D-76344, Eggenstein-Leopoldshafen, Germany.
authors: Christof M
authors: CM
authors: Niemeyer
mesh_terms: D001665:Binding Sites; D040761:Biomimetic Materials / Q000138:chemical synthesis*; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000648:ultrastructure; D018386:Kinetochores / Q000737:chemistry* / Q000648:ultrastructure; D008422:Materials Testing; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000648:ultrastructure; D011485:Protein Binding
chemical_list: C534001:CENPT protein, human; D002868:Chromosomal Proteins, Non-Histone; D046912:Multiprotein Complexes; C060905:centromere protein C
keywords: DNA; biomolecules; kinetochores; nanostructures; self-assembly