title: Supramolecular control of split-GFP reassembly by conjugation of beta-cyclodextrin and coumarin units.
abstract: The design of proteins whose structure and function can be manipulated by binding with specific ligands has been of great interest in the field of protein engineering. Some successful examples of small-molecule-dependent proteins have been reported, but their ligand-binding domains have mainly been limited to those derived from natural proteins. The introduction of synthetic components for ligand responsiveness may expand the versatility of small-molecule-dependent proteins. In this study, we designed and constructed a fragmented green fluorescent protein (split GFP) whose reassembly could be modulated by the non-natural supramolecular interaction. In the design of split GFP, beta-cyclodextrin (betaCDx) and coumarin units were introduced into a C-terminal fragment (residues 214-230) of split GFP. The C-terminal peptide with betaCDx and coumarin, DC-M2-betaCDx, which contains both host and guest moieties in the same peptide chain, formed an intramolecular inclusion complex in the absence of exogenous guest molecules. This interaction strongly inhibited reconstitution with the GFP N-terminal fragment (residues 2-214) (GFP 1-10 OPT). However, the addition of suitable guest molecules for betaCDx terminated the intramolecular host-guest interaction in the C-terminal peptide, leading to reassembly of the protein fragments and concomitant fluorescence recovery due to the formation of mature GFP. These results successfully demonstrated direct control of protein structure and function by application of synthetic supramolecular interaction to a fragmented protein. The combined system of fragmented protein and synthetic supramolecular elements is expected to be a useful and flexible strategy for regulation of protein structure and function via binding to synthetic ligands.
authors.affiliation: Institute of Industrial Science, University of Tokyo, Komaba 4-6-1, Meguro-ku, Tokyo 153-8505, Japan. sakamoto@tagen.tohoku.ac.jp
authors.forename: Seiji
authors.initials: S
authors.lastname: Sakamoto
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Kudo
mesh_terms: D002942:Circular Dichroism; D003374:Coumarins / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D047392:beta-Cyclodextrins / Q000737:chemistry*
chemical_list: D003374:Coumarins; D010446:Peptide Fragments; D047392:beta-Cyclodextrins; D049452:Green Fluorescent Proteins; C031215:betadex