Protein Dimerization Induced by Supramolecular Interactions with Cucurbit[8]uril.

The application of supramolecular chemistry to proteins provides a means of reversibly controlling protein properties. For example, proteins have been decorated with synthetic guest ligands for immobilization on host-molecule-modified surfaces, and vesicles decorated with b-cyclodextrin have been used as a platform to immobilize adamantane-modified enzymes. The supramolecular dimerization of two peptides enables sequence-specific recognition of DNA, and a bcyclodextrin-based host–guest system can be used to control the functional reassembly of two fragments of green fluorescent protein (GFP). The supramolecular interaction of bcyclodextrin with lithocholic acid, both of which were attached to protein C termini, was exploited to induce selective protein heterodimerization in solution and in cells. Control over protein dimerization with synthetic molecules is highly desirable, and approaches in which small molecules bind to specific, additionally incorporated protein domains have been very successful. Control over protein dimerization by means of a very small, genetically encoded peptide motif is an attractive goal in this respect as it would require only very minor protein modifications. Synthetic supramolecular host molecules such as the cyclodextrins and cucurbituril have been shown to selectively recognize amino acids, and synthetic receptors have been developed to recognize protein elements, some of which with a high degree of recognition affinity and selectivity. However, synthetic supramolecular host molecules have not been applied thus far as inducers of protein dimerization. Herein we show that the supramolecular host molecule cucurbit[8]uril can be used to induce and reversibly control the dimerization of proteins having a genetically incorporated Nterminal phenyalanine–glycine–glycine (FGG) peptide motif (Figure 1). The FGG peptide tag is easily incorporated through molecular biology techniques. The induction of protein dimerization is stable and can be reversed by addition of a synthetic supramolecular guest molecule. The FGG tripeptide has been shown to bind as a dimer in the cavity of a cucurbit[8]uril molecule; a key interaction is that between the N-terminal amine functionality of the peptide and the carbonyl rim of cucurbit[8]uril. We therefore incorporated this motif at the N terminus of the proteins tested as a recognition element to induce protein dimerization mediated by cucurbit[8]uril. Proteins are typically expressed with an N-terminal methionine. Therefore intein-based protein expression was used to generate proteins with an Nterminal phenylalanine. Two monomeric fluorescent proteins incorporating the FGG motif, FGG-mCFP (monomeric cyan fluorescent protein) and FGG-mYFP (monomeric yellow fluorescent protein), were designed and generated. The monomeric character of the proteins ensures the absence of unspecific protein aggregation. Apart from an Nterminal intein domain, for postexpression autocleavage, the proteins featured an N-terminal chitin-binding domain (CBD) for purification. The FGG proteins were expressed at 15 8C, and subsequently the N-terminal intein tag was released from the target proteins by overnight incubation at room temperature and pH 7.0. Two normal fluorescent reference proteins featuring an N-terminal methionine were also generated (Met-CFP and Met-YFP). All proteins were isolated in pure form as evidenced by SDS-PAGE and LCESI-MS (see the Supporting Information). We first studied the cucurbit[8]uril-induced homodimerization of FGG-mYFP (Figure 1) with F rster resonance energy transfer (homo-FRET) studies (Figure 2a). The homodimerization of two fluorescent proteins typically results in a decrease of the fluorescence anisotropy as a result of intermolecular energy transfer. Indeed, upon the addition of cucurbit[8]uril to a solution of FGG-mYFP, the anisotropy of the YFP fluorescence decreased. The anisoFigure 1. Schematic representation of two monomeric yellow fluorescent proteins having an N-terminal FGG peptide motif and their dimerization which is induced by supramolecular interactions with cucurbit[8]uril.