title: Development of a homogeneous fluorescence anisotropy assay to monitor and measure FtsZ assembly in solution.
abstract: We present here a fluorescence anisotropy method for the quantification of the polymerization of FtsZ, an essential protein for cytokinesis in prokaryotes whose GTP-dependent assembly initiates the formation of the divisome complex. Using Alexa 488 labeled wild-type FtsZ as a tracer, the assay allows determination of the critical concentration of FtsZ polymerization from the dependence of the measured steady-state fluorescence anisotropy on the concentration of FtsZ. The incorporation of the labeled protein into FtsZ polymers and the lack of spectral changes on assembly were independently confirmed by time-resolved fluorescence and fluorescence correlation spectroscopy. Critical concentration values determined by this new assay are compatible with those reported previously under the same conditions by other well-established methods. As a proof of principle, data on the sensitivity of the assay to changes in FtsZ assembly in response to Mg(2+) concentration or to the presence of high concentrations of Ficoll 70 as crowding agent are shown. The proposed method is sensitive, low sample consuming, rapid, and reliable, and it can be extended to other cooperatively polymerizing systems. In addition, it can help to discover new antimicrobials that may interfere with FtsZ polymerization because it can be easily adapted to systematic screening assays.
authors.affiliation: Instituto de Química-Física Rocasolano, Consejo Superior de Investigaciones Científicas, E-28006 Madrid, Spain.
authors.forename: Belén
authors.initials: B
authors.lastname: Reija
authors.forename: Begoña
authors.initials: B
authors.lastname: Monterroso
authors.forename: Mercedes
authors.initials: M
authors.lastname: Jiménez
authors.forename: Miguel
authors.initials: M
authors.lastname: Vicente
authors.forename: Germán
authors.initials: G
authors.lastname: Rivas
authors.forename: Silvia
authors.initials: S
authors.lastname: Zorrilla
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002413:Cations, Divalent; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D055503:Protein Multimerization; D012996:Solutions
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D003598:Cytoskeletal Proteins; C071525:FtsZ protein, Bacteria; D012996:Solutions; D006160:Guanosine Triphosphate; D008274:Magnesium