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Rapid in vitro assembly dynamics and subunit turnover of FtsZ demonstrated by fluorescence resonance energy transfer.

Publication ,  Journal Article
Chen, Y; Erickson, HP
Published in: J Biol Chem
June 10, 2005

We have developed an assay for the assembly of FtsZ based on fluorescence resonance energy transfer (FRET). We mutated an innocuous surface residue to cysteine and labeled separate pools with fluorescein (donor) and tetramethylrhodamine (acceptor). When the pools were mixed and GTP was added, assembly produced a FRET signal that was linearly proportional to FtsZ concentration from 0.7 microm (the critical concentration (C(c))) to 3 microm. At concentrations greater than 3 microm, an enhanced FRET signal was observed with both GTP and GDP, indicating additional assembly above this second C(c). This second C(c) varied with Mg(2+) concentration, whereas the 0.7 microm C(c) did not. We used the FRET assay to measure the kinetics of initial assembly by stopped flow. The data were fit by the simple kinetic model used previously: monomer activation, a weak dimer nucleus, and elongation, although with some differences in kinetic parameters from the L68W mutant. We then studied the rate of turnover at steady state by pre-assembling separate pools of donor and acceptor protofilaments. When the pools were mixed, a FRET signal developed with a half-time of 7 s, demonstrating a rapid and continuous disassembly and reassembly of protofilaments at steady state. This is comparable with the 9-s half-time for FtsZ turnover in vivo and the 8-s turnover time of GTP hydrolysis in vitro. Finally, we found that an excess of GDP caused disassembly of protofilaments with a half-time of 5 s. Our new data suggest that GDP does not exchange into intact protofilaments. Rather, our interpretation is that subunits are released following GTP hydrolysis, and then they exchange GDP for GTP and reassemble into new protofilaments, all on a time scale of 7 s. The mechanism may be related to the dynamic instability of microtubules.

Duke Scholars

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

June 10, 2005

Volume

280

Issue

23

Start / End Page

22549 / 22554

Location

United States

Related Subject Headings

  • Time Factors
  • Rhodamines
  • Protein Binding
  • Polymers
  • Mutation
  • Models, Chemical
  • Microtubules
  • Microscopy, Electron
  • Magnesium
  • Kinetics
 

Citation

APA
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MLA
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Chen, Y., & Erickson, H. P. (2005). Rapid in vitro assembly dynamics and subunit turnover of FtsZ demonstrated by fluorescence resonance energy transfer. J Biol Chem, 280(23), 22549–22554. https://doi.org/10.1074/jbc.M500895200
Chen, Yaodong, and Harold P. Erickson. “Rapid in vitro assembly dynamics and subunit turnover of FtsZ demonstrated by fluorescence resonance energy transfer.J Biol Chem 280, no. 23 (June 10, 2005): 22549–54. https://doi.org/10.1074/jbc.M500895200.
Chen, Yaodong, and Harold P. Erickson. “Rapid in vitro assembly dynamics and subunit turnover of FtsZ demonstrated by fluorescence resonance energy transfer.J Biol Chem, vol. 280, no. 23, June 2005, pp. 22549–54. Pubmed, doi:10.1074/jbc.M500895200.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

June 10, 2005

Volume

280

Issue

23

Start / End Page

22549 / 22554

Location

United States

Related Subject Headings

  • Time Factors
  • Rhodamines
  • Protein Binding
  • Polymers
  • Mutation
  • Models, Chemical
  • Microtubules
  • Microscopy, Electron
  • Magnesium
  • Kinetics