1 CHARACTERIZATION OF CAULOBACTER CRESCENTUS FTSZ USING DYNAMIC LIGHT SCATTERING Sen Hou, 1 Stefan A. Wieczorek, 1 Tomasz S. Kaminski, 1 Natalia Ziebacz, 1 Marcin Tabaka, 1 Nohemy A. Sorto, 2 Marie H. Foss, 3 Jared T. Shaw, 2 Martin Thanbichler, 4 Douglas B. Weibel, 3 Krzysztof Nieznanski, 5 Robert Holyst, 1* Piotr Garstecki 1* 1 Institute of Physical Chemistry PAS, Kasprzaka 44/52, 01-224 Warsaw, Poland 2 Department of Chemistry, University of California at Davis, 1 Shields Ave, Davis, CA 95616 USA 3 Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA 4 Max Planck Institute for Terrestrial Microbiology, LOEWE Center for Synthetic Microbiology and Faculty of Biology, Philipps University, D-35043 Marburg, Germany 5 Department of Biochemistry, Nencki Institute of Experimental Biology, Pasteur 3, 02-093 Warsaw, Poland Address correspondence to: Piotr Garstecki, Robert Holyst, Institute of Physical Chemistry PAS, Kasprzaka 44/52, 01-224 Warsaw, Poland; E-mail: holyst@ptys.ichf.edu.pl (RH), garst@ichf.edu.pl (PG) Keywords: bacteria; cytoskeleton; dynamic light scattering; electron microscopy (EM); protein self-assembly; C. crescentus; FtsZ; PC190723 Background: Self-assembly of the tubulin-homologue FtsZ is critical in bacterial cell division. Results: Dynamic light scattering (DLS) measurements provide insight into the kinetics and stable length of Caulobacter crescentus FtsZ in vitro. Conclusion: Caulobacter crescentus FtsZ forms short linear polymers in solution with the assembly rate depending on the concentrations of GTP and GDP. Significance: DLS is a valuable technique for studying the polymerization of cytoskeletal proteins. SUMMARY The self-assembly of the tubulin homologue FtsZ at the mid-cell is a critical step in bacterial cell division. We introduce dynamic light scattering spectroscopy (DLS) as a new method to study the polymerization kinetics of FtsZ in solution. Analysis of the DLS data indicates that the FtsZ polymers are remarkably monodisperse in length, independent of the concentrations of GTP, GDP and FtsZ monomers. Measurements of the diffusion coefficient of the polymers demonstrate that their length is remarkably stable until the free GTP is consumed. We estimated the mean size of the FtsZ polymers within this interval of stable length to be between 9 and 18 monomers. The rate of FtsZ polymerization and depolymerization are likely influenced by the concentration of GDP, as the repeated addition of GTP to FtsZ increased the rate of polymerization and slowed down depolymerization. Increasing the FtsZ concentration did not change the size of FtsZ polymers; however, it increased the rate of the depolymerization reaction by depleting free GTP. Using transmission electron microscopy we observed that FtsZ forms linear polymers in solutions that rapidly convert to large bundles upon contact http://www.jbc.org/cgi/doi/10.1074/jbc.M111.309492 The latest version is at JBC Papers in Press. Published on May 9, 2012 as Manuscript M111.309492 Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc. by guest, on June 19, 2012 www.jbc.org Downloaded from