The assembly of two complementary non-fluorescent protein fragments when each is fused to one of a pair of interacting proteins is essential for the BiFC assay. However, self-assembly of the non-fluorescent protein fragments independent of the interacting proteins can contribute to false-positive fluorescence, reducing the signal-to-noise (S/N) ratio (10). Recently three groups have reported improvements of the S/N ratio in the Venus-based BiFC assay in mammalian cells (42-44). All three groups split the Venus fluorescent protein into two non-fluorescent fragments at a position between the 7th and 8th β-strands, focusing on the interaction between the 7th and 10th β-strands of Venus (Figure 3; Table 2). Four residues (V150 and I152 within the 7th β-strand, and L201V and L207V within the 10th β-strand) were reported to affect the S/N ratio in the Venus-based BiFC, with substitutions at these residues significantly improving the S/N ratio (Figure 3).
“V150” - Lin et al. identified the V150L mutation as having a higher S/N ratio based on the structure, protein stability and solvent accessibility of the 7th β-strand of Venus (42) (Figure 3; Table 2). To determine the effect of V150L on the S/N ratio, the interaction between Bcl-XL and Bak BH3, two peptides involved in apoptosis, was examined in mitochondria (45). Although V150L slightly reduced the BiFC signal (fluorescence intensity) with the peptides, it increased the S/N ratio (referred to as “BiFC specificity” in the paper) by 2-fold (42).
Based on the structure, the solvent accessibility of the 7th β-strand, and the properties of amino acid residues, we also identified the V150L mutation as a substitution that increased the S/N ratio by 4.5 fold (43) (Figure 3; Table 2). Using the interaction between the bZIP domains of Jun (bJun) and Fos (bFos) in the nucleus as a model system (43) (Table 2), we found the V150L mutation did increase the S/N ratio, although the signal was too low to be useful for BiFC analysis. Because the interaction between Bcl-XL and Bak BH3 peptides occurs in mitochondria, whereas the interaction between bJun and bFos takes place in the nucleus (Table 2), it is possible that the V150L mutation might only be useful for BiFC analysis of proteins localized in relatively small organelles such as mitochondria, peroxisomes, and plastids, where BiFC complexes can be locally concentrated.
Nakagawa et al. reported another mutation, V150A, for BiFC analysis, that was identified by predictions of hydrophobic amino acids and by screening of amino acids for substitutions (44) (Figure 3; Table 2). To determine the effect of V150A on BiFC analysis, the interaction between bJun and bFos was again used (Table 2). The results indicate that V150A decreases false-positive interactions more than V150L; thus the V150A mutation might be useful to improve the S/N ratio.
“I152” – Compared with V150L, we found the I152L mutation significantly improved the S/N ratio while preserving fluorescence brightness (43) (Figure 3; Table 2). To determine the effect of the mutation on the S/N ratio, an interaction between bJun and bFos was similarly used (Table 2). I152L decreased false-positive fluorescence and increased the S/N ratio by 4-fold (43). The I152L mutation not only increased the S/N ratio in the VN155/VC155-based BiFC, but also increased the S/N ratio in the VN173/VC173-based BiFC. The effect of this mutation has also been evaluated in other model organisms such as plant (Allium cepa) and worm (Caenorhabditis elegans), as different experimental systems using these model organisms could potentially affect the S/N ratio of BiFC (46, 47). For the purpose of comparison with the S/N ratio in mammalian cells (COS-1), bJun/bFos and bJun/ΔbFos were similarly used as positive and negative PPIs, respectively. Using the same VN155/VC155-based BiFC system, the S/N ratios in A. cepa and C. elegans were only 1 and 2, respectively, and there was almost no difference between positive and negative PPIs in A. cepa. Nevertheless, when the VN155-I152L mutation was introduced, S/N ratios increased by 2-fold, to 2 and 4 in A. cepa and C. elegans, respectively (Kodama, Duren, and Hu, unpublished observations). These results suggest that this improved Venus-based BiFC system may be applicable to multiple model organisms.