“L201 and L207” - Nakagawa et al. also reported the identification of two residues (L201 and L207) in the 10th β-strand that, when mutated, could increase the S/N ratio in Venus-based BiFC (44) (Figure 3; Table 2). Based on the three-dimensional structure of Venus, it was predicted that L201 in the C-terminal fragment of Venus interacts with Y74, F84, V150, and I152 in the N-terminal fragment of Venus, and that L207 in the C-terminal fragment of Venus interacts with V61, Y143, and Y145 in the N-terminal fragment of Venus (44). The two leucines appear to form a hydrophobic core between the N-terminal and C-terminal fragments. When the L201V and L207V mutations were introduced, the S/N ratios increased by 4.2- and 3.0-fold, respectively (44). Because L201 interacts with V150 and I152, the underlying mechanism of the L201V mutation on the S/N ratio might be similar to that of the V150L, V150A, and I152L mutations.
In summary, the search for mutations to reduce self-assembly has demonstrated that the V150L, V150A, I152L, L201V, and L207V mutations can increase S/N ratios in the Venus-based BiFC system. Because different animal model systems were used in these studies, it is difficult to conclude which one is better. Nevertheless, results from these three independent studies provide evidence that the S/N ratio can be improved by a single substitution (Table 2). Since these studies focused on the interactions between the 7th and 10th β-strands, it would be interesting to test whether introduction of mutations into other sites could show increased improvement. Alternatively, introduction of multiple mutations might be necessary to achieve higher S/N ratios. It is interesting to note that the V152L mutation had little effect on the S/N ratio in the Cerulean-based BiFC assay (43), indicating other fluorescent protein-based BiFC systems should be individually tested for improvement by other mutations.Irreversibility of BiFC
The irreversibility of BiFC complexes has been well documented (10), and it would appear that most, if not all, fluorescent protein-based BiFC systems are irreversible (8, 33, 43, 48-51). Although this irreversibility offers a significant advantage for analysis of transient or weak protein-protein interactions (48) and when performing BiFC-based PPI screening (49, 50), the irreversibility does limit the use of the BiFC assay for dynamic interactions. Given that PPIs are tightly regulated, development of reversible BiFC systems would provide opportunities to use BiFC for many biological studies. It should be noted that several groups have reported that BiFC complexes are reversible (52-54). These claims are largely based on observations that EYFP-based BiFC fluorescence was slightly decreased after chemical treatments (52-54). However, we and others only observed slight decreases in fluorescence intensity when using a rapamycin-inducible PPI system (43, 51). It is also interesting to note that the mutations that were identified to increase the S/N ratio had little effect on irreversibility (43). Therefore, it will be necessary to take a systematic approach (e.g., screening or directed evolution) to the development of a reversible BiFC system. If successful, it would not be surprising to see a full blooming of the BiFC assay.Controls for BiFC assay
As noted previously, BiFC complementation occurs when two complementary non-fluorescent fragments of an FP are brought together by the pair of interacting proteins they are fused to, thus reconstituting a functional FP (Figure 1C and 4A). Alternatively, two FP fragments also can come together by random collision when co-expressed in the same subcellular compartment even though the two proteins may not interact. Fluorescence signals generated through random collisions are considered non-specific signal, but in these cases, both the interaction-driven fluorescence complementation and the interaction-independent non-specific assembly contribute to the overall fluorescence in the BiFC assay. In order to reveal specific protein interactions, the contribution of non-specific fluorescence in BiFC experiments must be determined. Because the expression level and subcellular localization of the non-fluorescent fragments that can self-assemble may differ from the fusion proteins, co-expression of the two non-fluorescent fragments may not provide such information (Figure 4). In many cases, the fluorescent signal resulting from co-expression of the two non-fluorescent fragments not fused to interacting proteins is even stronger than that resulting from co-expression of the two fusion proteins. Because of these concerns, we and others have recommended the use of negative controls, in which a mutation or small deletion is introduced into the interaction interface in one of the two proteins (55-61). However, a review of published BiFC experiments has led to the disappointing finding that some BiFC experiments do not include appropriate negative controls (Figure 4, B and C). Examples of inappropriate negative controls include the use of one fragment only, of a third protein that has a different structure or localization from the two proteins under study (Figure 4, D-L), or of mutants with different localization patterns than the wild-type protein or that have lower expression levels (e.g., because of shorter half-life or decreased stability). Several BiFC protocols have provided detailed discussions on the design and use of negative controls (55-58). It may be possible to forgo mutant controls in BiFC experiments if the same two fusion constructs are used under different experimental conditions and the change in BiFC signal is the end result. For example, the increase in BiFC signal for two proteins under study with and without the addition of a chemical (e.g., in the rapamycin-inducible PPI system) (43, 51) can be compared, provided that the chemical does not increase the expression level of the two fusion proteins.