Cucurbit[8]uril-mediated protein homotetramerization

Abstract

A supramolecular protein tetramerization approach has been devised which enables the controlled formation of a discrete protein tetramer. The supramolecular element cucurbit[8]uril has been used as an inducer of the protein tetramerization in combination with intrinsic affinities between the proteins, which preorganize the protein in dimerized form. The combination of a dimerizing interface on the fluorescent proteins under study (dYFP, dCFP), with a genetically encoded N-terminal phenylalanine-glycine-glycine (FGG) peptide motif allows cucurbit[8]uril to selectively induce FGG-dYFP or FGG-dCFP tetramerization. The concept of cucurbit[8]uril-induced protein tetramerization was elucidated by using a combination of fluorescence anisotropy, dynamic light scattering and size exclusion chromatography experiments. The cucurbit[8]uril-induced tetrameric protein complex is formed via a “dimers of dimers” pathway, is highly stable and can be separated by size exclusion chromatography. This supramolecular induced protein tetramerization approach opens up a novel entry in generating well-defined synthetic protein assemblies.