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Bimolecular fluorescence complementation (BiFC): A 5-year update and future perspectives
Yutaka Kodama1 and Chang-Deng Hu2
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Recently, Dronpa, a photoswitchable GFP-like protein isolated from Pectiniidae, was also demonstrated to support BiFC analysis (34) (Table 1). Dronpa exhibits green fluorescence, with an emission maximum of 518 nm when excited at 503 nm (35). It can be photoswitched to a non-fluorescent state by strong light irradiation at around 490 nm and then switched back to a fluorescent state by minimal irradiation at 405 nm. It was reported that a Dronpa-based BiFC system possesses the same reversible photoswitching characteristic and could be used in repeated photo-switching experiments (34). Since Dronpa was successfully used for visualization of nucleocytoplasmic shuttling proteins (35), and many transcriptional regulatory proteins function as dimers and shuttle between the cytoplasm and nucleus, Dronpa-based BiFC analysis should help uncover how transcriptional regulatory proteins shuttle between the cytoplasm and nucleus. Likewise, Dronpa-based BiFC could also facilitate the study of protein complex translocation between various cellular compartments.

Given the strong evidence that most, if not all, fluorescent proteins are BiFC-competent, it is plausible to predict that other photoactivatable and photoconvertible fluorescent proteins, such as PA-GFP, EosFP, and Kaede, could be good candidates for BiFC analysis (36-38). If true, such fluorescent protein-based BiFC systems could enable new applications, including multicolor photoregulatable BiFC as well as super resolution imaging of BiFC.

Split sites

Circular permutation of proteins is a technique to change the order of amino acids in a protein sequence (39). The original N terminus of a protein is ligated with its original C terminus, and new N and C termini are generated by splitting the circularized protein in other structural regions. One of the applications of circular permutation is to determine the position where an insertion can be introduced without affecting the overall structure and protein folding. Several studies provided evidence that circularly permutated GFP could be split at positions in the loop between the 6th and 7th β-strands, in the 7th β-strand, in the loop between the 7th and 8th β-strands, in the 8th β-strand, and in the loop between the 8th and 9th β-strands (40). These positions were later employed to split EYFP for the development of BiFC (8). The identified split sites have been successfully used for BiFC analysis with several fluorescent proteins. For example, there are YN173/YC173 from EYFP, CN173/CC173 from ECFP, and RN169/RC169 from mRFP (Q66T), which are all split at a position between the 8th and 9th β-strands. Another split site is the loop between 7th and 8th β-strands. Examples are YN155/YC155 from EYFP and CN155/CC155 from ECFP (Figure 1, A and B). This split site is likely available for the GFP variants from jellyfish Aequorea victoria, but not for RFP variants from Discosoma sp. (31) (Figure 1, A and B). Although the above two split sites are widely used as canonical split sites of BiFC assay, other split sites have been reported for BiFC assays. For example, superfolder GFP can be split at the loop between the 10th and 11th β-strands (23) (Figure 1B). Because of high background fluorescence resulting from fluorescence complementation using this split site, several point mutations were introduced into these fragments. More recently, an extensive screen was performed by splitting Venus at 13 different sites in the hopes of developing a Venus-based BiFC assay with low background fluorescence (41). Interestingly, this screen identified the same loop between the 10th and 11th β-strands as the best split site for BiFC analysis (41). Contrary to the sfGFP-based BiFC, the Venus-based BiFC assay had the lowest background fluorescence among all combinations tested (41). Given the overall structural homology between sfGFP and Venus, it remains to be determined whether the differential effect of the same split site on these two proteins might be related to the proteins of interest used or the design of their BiFC plasmids (23, 41). Nevertheless, the fact that the C-terminal fragment from the 11th β-strand, which consists of 15 or 29 amino acids (sfGFP: a.a. 215–229, Venus: a.a. 210–238) (23, 41) (Figure 1B), should have less effect on the structure and folding of target proteins, and should be useful for BiFC analysis of proteins that are more sensitive to structural alterations.

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