Supramolecular control of split-GFP reassembly by conjugation of β-cyclodextrin and coumarin units

Seiji Sakamoto, Kazuaki Kudo

Research output: Contribution to journalArticlepeer-review

32 Citations (Scopus)

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, β-cyclodextrin (βCDx) and coumarin units were introduced into a C-terminal fragment (residues 214-230) of split GFP. The C-terminal peptide with βCDx and coumarin, DC-M2-βCDx, 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 βCDx 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.

Original languageEnglish
Pages (from-to)9574-9582
Number of pages9
JournalJournal of the American Chemical Society
Volume130
Issue number29
DOIs
Publication statusPublished - 2008 Jul 23

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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