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Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins.

Publication ,  Journal Article
Moore, DA; Whatley, ZN; Joshi, CP; Osawa, M; Erickson, HP
Published in: J Bacteriol
January 1, 2017

UNLABELLED: FtsZ, a bacterial tubulin homologue, is a cytoskeletal protein that assembles into protofilaments that are one subunit thick. These protofilaments assemble further to form a "Z ring" at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane and also serves as a scaffold to recruit cell wall remodeling proteins for complete cell division in vivo One model of the Z ring proposes that protofilaments associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of Escherichia coli FtsZ by inserting either small peptides or whole fluorescent proteins (FPs). Among the four lateral surfaces on FtsZ protofilaments, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174, located on the left and right surfaces, completely blocked function, and these sites were identified as possible sites for essential lateral interactions. However, the insert at R174 did not interfere with association of protofilaments into sheets and bundles in vitro Another goal was to find a location within FtsZ that supported insertion of FP reporter proteins while allowing the FtsZ-FPs to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by superresolution techniques. IMPORTANCE: One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the involvement of the front and back faces of the protofilament in essential interactions in vivo but pointed to two potential lateral bond sites, on the right and left sides. We also identified an FtsZ loop where various fluorescent proteins could be inserted without blocking function; these FtsZ-FPs functioned as the sole source of FtsZ. This advance provides improved tools for all fluorescence imaging of the Z ring and may be especially important for superresolution imaging.

Duke Scholars

Published In

J Bacteriol

DOI

EISSN

1098-5530

Publication Date

January 1, 2017

Volume

199

Issue

1

Location

United States

Related Subject Headings

  • Protein Conformation
  • Protein Binding
  • Mutation
  • Mutagenesis, Site-Directed
  • Mutagenesis, Insertional
  • Models, Molecular
  • Microbiology
  • Gene Expression Regulation, Bacterial
  • Fluorescence
  • Escherichia coli
 

Citation

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Moore, D. A., Whatley, Z. N., Joshi, C. P., Osawa, M., & Erickson, H. P. (2017). Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins. J Bacteriol, 199(1). https://doi.org/10.1128/JB.00553-16
Moore, Desmond A., Zakiya N. Whatley, Chandra P. Joshi, Masaki Osawa, and Harold P. Erickson. “Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins.J Bacteriol 199, no. 1 (January 1, 2017). https://doi.org/10.1128/JB.00553-16.
Moore, Desmond A., et al. “Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins.J Bacteriol, vol. 199, no. 1, Jan. 2017. Pubmed, doi:10.1128/JB.00553-16.

Published In

J Bacteriol

DOI

EISSN

1098-5530

Publication Date

January 1, 2017

Volume

199

Issue

1

Location

United States

Related Subject Headings

  • Protein Conformation
  • Protein Binding
  • Mutation
  • Mutagenesis, Site-Directed
  • Mutagenesis, Insertional
  • Models, Molecular
  • Microbiology
  • Gene Expression Regulation, Bacterial
  • Fluorescence
  • Escherichia coli