Bimolecular fluorescence complementation (also known as BiFC) is a technology typically used to validate protein interactions. It is based on the association of fluorescent protein fragments that are attached to components of the same macromolecular complex. Proteins that are postulated to interact are fused to unfolded complementary fragments of a fluorescent reporter protein and expressed in live cells. Interaction of these proteins will bring the fluorescent fragments within proximity, allowing the reporter protein to reform in its native three-dimensional structure and emit its fluorescent signal.Kerppola, T. K. Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat. Protoc. 1, 1278–1286 (2006). This fluorescent signal can be detected and located within the cell using an inverted fluorescence microscope that allows imaging of fluorescence in cells. In addition, the intensity of the fluorescence emitted is proportional to the strength of the interaction, with stronger levels of fluorescence indicating close or direct interactions and lower fluorescence levels suggesting interaction within a complex.Morell, M., Espargaro, A., Aviles, F. X. & Ventura, S. Study and selection of in vivo protein interactions by coupling bimolecular fluorescence complementation and flow cytometry. Nat. Protoc. 3, 22–33 (2008). Therefore, through the visualisation and analysis of the intensity and distribution of fluorescence in these cells, one can identify both the location and interaction partners of proteins of interest.
History Biochemical complementation was first reported in subtilisin-cleaved bovine pancreatic ribonuclease, then expanded using β-galactosidase mutants that allowed cells to grow on lactose.Richards, F. M. On the Enzymic Activity of Subtilisin-Modified Ribonuclease. Proc. Natl. Acad. Sci. U.S.A. 44, 162–166 (1958).Ullmann, A., Jacob, F. & Monod, J. Characterization by in vitro complementation of a peptide corresponding to an operator-proximal segment of the beta-galactosidase structural gene of Escherichia coli. J. Mol. Biol. 24, 339–343 (1967).Ullmann, A., Jacob, F. & Monod, J. On the subunit structure of wild-type versus complemented beta-galactosidase of Escherichia coli. J. Mol. Biol. 32, 1–13 (1968).
Recognition of many proteins' ability to spontaneously assemble into functional complexes as well as the ability of protein fragments to assemble as a consequence of the spontaneous functional complex assembly of interaction partners to which they are fused was later reported for ubiquitin fragments in yeast protein interactions.Johnsson, N. & Varshavsky, A. Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl. Acad. Sci. U.S.A. 91, 10340-10344 (1994).
In 2000, Ghosh et al developed a system that allowed a green fluorescent protein (GFP) to be reassembled using an anti-parallel leucine zipper in E. coli cells.Ghosh, I., Hamilton, A. D. & Regan, L. Antiparallel Leucine Zipper-Directed Protein Reassembly: Application to the Green Fluorescent Protein. Journal of the American Chemical Society 122, 5658 (2000). This was achieved by dissecting GFP into C- and N-terminal GFP fragments. As the GFP fragment was attached to each leucine zipper by a linker, the heterodimerisation of the anti-parallel leucine zipper resulted in a reconstituted, or re-formed, GFP protein that could be visualised. The successful fluorescent signal indicated that the separate GFP peptide fragments were able to correctly reassemble and achieve tertiary folding. It was, therefore, postulated that using this technique, fragmented GFP could be used to study interaction of protein–protein pairs that have their N–C termini in close proximity.
After the demonstration of successful fluorescent protein fragment reconstitution in mammalian cells, Hu et al. described the use of fragmented yellow fluorescent protein (YFP) in the investigation of bZIP and Rel family transcription factor interactions. This was the first report bZIP protein interaction regulation by regions outside of the bZIP domain, regulation of subnuclear localization of the bZIP domains Fos and Jun by their different interacting partners, and modulation of transcriptional activation of bZIP and Rel proteins through mutual interactions. In addition, this study was the first report of an in vivo technique, now known as the bimolecular fluorescence complementation (BiFC) assay, to provide insight into the structural basis of protein complex formation through detection of fluorescence caused by the assembly of fluorescent reporter protein fragments tethered to interacting proteins.Hu, C. D., Chinenov, Y. & Kerppola, T. K. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol. Cell 9, 789–798 (2002).
Fluorescent labeling Fluorophore activation occurs through an autocatalytic cyclization reaction that occurs after the protein has been folded correctly.Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998). This was advanced with the successful reconstitution of the YFP fluorophore from protein fragments that had been fused to interacting proteins within 8 hours of transfection, reported in 2002.
Workflow
Selection of fusion protein production system There are different production systems that can be used for the fusion protein generated. Transient gene expression is used to identify protein–protein interactions in vivo as well as in subcellular localisation of the BiFC complex. However, one must be cautious against protein over-expression, as this may skew both preferential localisation and the predominant protein complexes formed. Instead, weak promoters, the use of low levels of plasmid DNA in the transfection, and plasmid vectors that do not replicate in mammalian cells should be used to express proteins at or near their endogenous levels to mimic the physiological cellular environment. Also, careful selection of the fluorescent protein is important, as different fluorescent proteins require different cellular environments. For example, GFP can be used in E. coli cells, while YFP is used in mammalian cells.Kerppola, T. K. Complementary methods for studies of protein interactions in living cells. Nat. Methods 3, 969–971 (2006).
Stable cell lines with the expression vector integrated into its genome allows more stable gene expression in the cell population, resulting in more consistent results.
Determination of fusion sites When deciding the linker fusion site on the protein surface, there are three main considerations. First, the fluorescent protein fragments must be able to associate with one another when their tethered proteins interact. Structural information and the location of the interaction surface may be useful when determining the fusion site to the linker, although the information is not necessary, as multiple combinations and permutations can be screened. Secondly, the creation of the fusion protein must not significantly alter the localisation, stability, or expression of the proteins to which the fragments are linked as compared to the endogenous wild-type proteins. Finally, the addition of the fluorescent fragment fusion must not affect the biological function of the protein, preferably verified using assays that evaluate all of the proteins' known functions.
Designing linkers A linker is a short amino acid sequence that tethers the fluorescent reporter protein fragment to the protein of interest, forming the fusion protein. When designing a linker sequence, one must ensure that the linker is sufficiently soluble and long to provide the fluorescent protein fragments with flexibility and freedom of movement so that the fragment and its partner fragment will collide frequently enough to reconstitute during the interaction of their respective fused proteins. Although it is not documented, it is possible that the length or the sequence of the linker may influence complementation of some proteins. Reported linker sequences RSIAT and RPACKIPNDLKQKVMNH (single amino acid code) and AAANSSIDLISVPVDSR (Sigma) have been successfully used in BiFC experiments.Hu, C. D. & Kerppola, T. K. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat. Biotechnol. 21, 539–545 (2003).
Creating proper plasmid expression vectors When designing plasmid vectors to express the proteins of interest, the construct must be able to express proteins that are able to form fusion proteins with fluorescent protein fragments without disrupting the protein's function. In addition, the expected protein complex must be able to accept stabilisation of the fluorescent protein fragment interaction without affecting the protein complex function or the cell being studied. Many fluorescent protein fragments that combine in several ways can be used in BiFC. Generally, YFP is recommended to serve as the reporter protein, cleaved at residue 155 (N-terminal consisting of residues 1–154 and C-terminal consisting of residues 155–238) or residue 173 in particular, as these sets of fragments are highly efficient in their complementation when fused to many interacting proteins and they produce low levels fluorescence when fused to non-interacting proteins. It is suggested that each target protein is fused to both the N- and C-terminal fragments of the fluorescent reporter protein in turn, and that the fragments are fused at each of the N- and C-terminal ends of the target proteins. This will allow a total of eight different permutations, with interactions being tested:
N-terminal fragment fused at the N-terminal protein 1 + C-terminal fragment fused at the N-terminal protein 2 N-terminal fragment fused at the N-terminal protein 1 + C-terminal fragment fused at the C-terminal protein 2 N-terminal fragment fused at the C-terminal protein 1 + C-terminal fragment fused at the N-terminal protein 2 N-terminal fragment fused at the C-terminal protein 1 + C-terminal fragment fused at the C-terminal protein 2 C-terminal fragment fused at the N-terminal protein 1 + N-terminal fragment fused at the N-terminal protein 2 C-terminal fragment fused at the N-terminal protein 1 + N-terminal fragment fused at the C-terminal protein 2 C-terminal fragment fused at the C-terminal protein 1 + N-terminal fragment fused at the N-terminal protein 2 C-terminal fragment fused at the C-terminal protein 1 + N-terminal fragment fused at the C-terminal protein 2
Selection of appropriate cell culture system As previously stated, it is important to ensure that the fluorescent reporter protein being used in BiFC is appropriate and can be expressed in the cell culture system of choice, as not all reporter proteins can fluoresce or be visualised in all model systems.
Selection of appropriate controls Fluorescent protein fragments can associate and fluoresce at low efficiency in the absence of a specific interaction. Therefore, it is important to include controls to ensure that the fluorescence from fluorescent reporter protein reconstitution is not due to unspecific contact.Morell, M. et al. Monitoring the interference of protein–protein interactions in vivo by bimolecular fluorescence complementation: the DnaK case. Proteomics 8, 3433–3442 (2008).
Some controls include fluorophore fragments linked to non-interacting proteins, as the presence of these fusions tend to decrease non-specific complementation and false positive results.