A polymerizable GFP variant

Klein, Ágnes; Tóth, Balázs; Jankovics, Hajnalka; Muskotál, Adél; Vonderviszt, Ferenc

doi:10.1093/protein/gzs003

Cited by 22 publications

(20 citation statements)

References 26 publications

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“…Previous studies have exploited the functional potential of the flagellin hypervariable region in vitro by replacing it with adhesive peptides 34 , 35 , fluorescence domains 36 , and glycosyl hydrolases 37 . However, prior to our discovery, only structural and adhesive functions for flagellins have been observed in nature.…”

Section: Discussionmentioning

confidence: 99%

Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella

et al. 2017

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Bacterial flagella are cell locomotion and occasional adhesion organelles composed primarily of the polymeric protein flagellin, but to date have not been associated with any enzymatic function. Here, we report the bioinformatics-driven discovery of a class of enzymatic flagellins that assemble to form proteolytically active flagella. Originating by a metallopeptidase insertion into the central flagellin hypervariable region, this flagellin family has expanded to at least 74 bacterial species. In the pathogen, Clostridium haemolyticum, metallopeptidase-containing flagellin (which we termed flagellinolysin) is the second most abundant protein in the flagella and is localized to the extracellular flagellar surface. Purified flagellar filaments and recombinant flagellin exhibit proteolytic activity, cleaving nearly 1000 different peptides. With ~ 20,000 flagellin copies per ~ 10-μm flagella this assembles the largest proteolytic complex known. Flagellum-mediated extracellular proteolysis expands our understanding of the functional plasticity of bacterial flagella, revealing this family as enzymatic biopolymers that mediate interactions with diverse peptide substrates.

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Section: Discussionmentioning

confidence: 99%

Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella

et al. 2017

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“…High-resolution structural studies revealed that the conserved terminal regions of flagellin are involved in subunit interactions in the filament core while the hypervariable central portion of the polypeptide chain, comprising residues 190–284, forms the D3 domain exposed on the filament surface 14 , 15 . We demonstrated that D3 is a structurally independent part of flagellin which has no direct role in filament formation 16 and it can be removed or replaced by foreign proteins without influencing polymerization ability 17 , 18 .…”

Section: Introductionmentioning

confidence: 95%

“…The prototype of flagellin-based polymerizable proteins has been successfully created by replacing the D3 domain of flagellin with the amino acid sequence of the xylanase A enzyme 17 . A polymerizable GFP variant has been also fabricated which exhibited intensive fluorescence and was capable of efficient filament formation 18 . In these cases, it was relatively simple to construct these fusions because the terminal regions of the inserted proteins were close to each other similarly to the two internal ends of flagellin generated upon removal of D3.…”

Section: Introductionmentioning

confidence: 99%

Nanobody-Displaying Flagellar Nanotubes

Klein

Kovács

Muskotál

et al. 2018

Sci Rep

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In this work we addressed the problem how to fabricate self-assembling tubular nanostructures displaying target recognition functionalities. Bacterial flagellar filaments, composed of thousands of flagellin subunits, were used as scaffolds to display single-domain antibodies (nanobodies) on their surface. As a representative example, an anti-GFP nanobody was successfully inserted into the middle part of flagellin replacing the hypervariable surface-exposed D3 domain. A novel procedure was developed to select appropriate linkers required for functional internal insertion. Linkers of various lengths and conformational properties were chosen from a linker database and they were randomly attached to both ends of an anti-GFP nanobody to facilitate insertion. Functional fusion constructs capable of forming filaments on the surface of flagellin-deficient host cells were selected by magnetic microparticles covered by target GFP molecules and appropriate linkers were identified. TEM studies revealed that short filaments of 2–900 nm were formed on the cell surface. ITC and fluorescent measurements demonstrated that the fusion protein exhibited high binding affinity towards GFP. Our approach allows the development of functionalized flagellar nanotubes against a variety of important target molecules offering potential applications in biosensorics and bio-nanotechnology.

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“…Furthermore, the PEG side-chains of the polymer form a comblike structure on the surface, leaving very limited ability to control the surface orientation and local environment of the RGD motifs. [19,20].…”

Section: Introductionmentioning

confidence: 99%