Several methods enable the visualization of protein interactions in living cells. Most of these methods require either elaborate instrumentation and complex data processing, or staining with exogenous fluorophores or dyes. bimolecular fluorescence complementation (BiFC) assay enables simple and direct visualization of protein interactions in living cells (45
). The BiFC approach is based on the formation of a fluorescent complex when two proteins fused to non-fluorescent fragments of a fluorescent protein interact with each other (). The interaction between the fusion proteins facilitates the association between the fragments of the fluorescent protein. This approach enables visualization of the subcellular locations of specific protein complexes in the normal cellular environment. The approach can be used for the analysis of interactions between many types of proteins and does not require information about the structures of the interaction partners. It can be performed using a standard epifluorescence microscope, and does not require staining of the cells with exogenous fluorophores or dyes.
Figure 1 Schematic representation of the principle of the BiFC assay. Two non-fluorescent fragments (YN and YC) of the yellow fluorescent protein (YFP) are fused to putative interaction partners (A and B).The association of the interaction partners allows formation (more ...)
An Abbreviated History of Complementation Assays
Protein complementation has now been studied for about half a century. Fragments of many proteins have been shown to associate with each other to form a functional complex. Complementation between enzyme fragments was originally observed by Richards using subtilisin-cleaved bovine pancreatic ribonuclease (98
). Genetic complementation between different alleles of the same gene was characterized by Ullmann, Jacob and Monod using β-galactosidase mutants that conferred growth on lactose when co-expressed in the same cell (115
). Subsequently, fragments of many proteins have been shown to spontaneously associate to form a functional complex.
Of particular significance for the study of protein interactions was the demonstration that the association between some protein fragments could be facilitated by fusion of the fragments to specific interaction partners as first demonstrated for fragments of ubiquitin by Johnsson and Varshavsky, in yeast (52
). Subsequently, conditional complementation between fragments of β-galactosidase was visualized by Blau and coworkers in intact mammalian cells (99
). Conditional complementation by fragments of dihydrofolate reductase was reported by Michnick and collegues (89
Complementation between fragments of a green fluorescent protein (GFP) variant was first detected in E. coli
by Regan and coworkers using fusions to artificial, interacting peptides (34
). Fragments of the yellow fluorescent protein (YFP) were shown to produce fluorescent complexes in mammalian cells when fused to calmodulin and the M13 calmodulin binding peptide by the Miyawaki laboratory (76
). Conditional complementation between fragments of YFP in mammalian cells was demonstrated by Hu in my laboratory (45
). Fragments of several other proteins have been used in conditional complementation assays () (57
). Each complementation approach has specific advantages and limitations. This chapter will focus on complementation between fragments of fluorescent proteins.
Comparison of complementation methods using fragments of different proteins.
The structures of the complexes formed by complementation have not been determined. However, it is likely that the structures resemble those of the intact proteins since they reproduce many of their functions (). It is intriguing to note that proteins with a variety of different structures can be reconstituted from fragments. However, only a few of the peptide bonds in any particular protein can be broken to produce fragments that can associate to form a functional complex. This limitation may reflect the folding pathways of the respective proteins. Greater insight into the folding pathways of complexes formed by the protein fragments would be very valuable for understanding the factors that determine which protein fragments can associate to produce a functional complex.
Figure 2 Structures of proteins that have been used to study protein interactions using complementation approaches. The two fragments that have been used are shown in red and green based on the X-ray crystal structures of the intact proteins. In β-galactosidase, (more ...)
Comparison of the BiFC Approach and Other Complementation Assays
The advantage of the BiFC approach compared to other complementation methods is that the assembled complex has strong intrinsic fluorescence that allows direct visualization of the protein interaction. The interaction can therefore be detected without exogenous fluorogenic or chromogenic agents, avoiding potential perturbation of the cells by these agents. This also avoids potential problems caused by uneven distributions of the chromogenic or fluorogenic substrates or ligands. Using the BiFC approach, living cells can be observed over time and the possibility that experimental manipulations alter the result can be minimized. Moreover, as described below, multiple protein interactions can be visualized in parallel using spectrally distinct bimolecular fluorescent complexes.
One limitation of the BiFC approach is that there is a delay between the time when the fusion proteins interact with each other and the time when the complex becomes fluorescent (45
). This delay is due to the slow rate of the chemical reactions required to produce the fluorophore. The length of the delay depends on the sensitivity of the detection method since it is not necessary for all complexes to become fluorescent in order to observe the interaction. Nevertheless, the BiFC approach does not enable real-time detection of complex formation. In addition, formation of some bimolecular fluorescent complexes is irreversible at least in vitro
). These characteristics limit the BiFC assay to detection of the average efficiencies of complex formation over relatively long times (minutes to hours). Despite these limitations, the BiFC assay has been useful for investigation of interactions among a variety of structurally diverse proteins in many different cell types and organisms (58
). Thus, the BiFC assay is generally applicable for the visualization of a variety of protein complexes in living cells and organisms.
Comparison of BiFC Analysis with Alternative Visualization Methods
Several methods have been developed to study protein interactions in living cells. One of the most commonly employed methods is FRET analysis (43
). This assay is based on the use of two fluorophores, either chemically linked or genetically fused to two proteins whose interaction is to be examined. Compared to the BiFC assay, FRET analysis generally requires higher levels of protein expression to detect energy transfer. Also, structural information, or a great deal of luck in the case of proteins of moderate to large size, is required to place the two fluorophores within 100 Å of each other. This is the maximum distance over which significant energy transfer between fluorescent proteins can be detected. The fraction of proteins that form complexes must also be high enough to produce a sufficient change in the donor and acceptor fluorescence intensities. To exclude alternative interpretations of the results, numerous controls must be performed and the fluorescence intensities must be measured with high quantitative accuracy. Despite these limitations, FRET has been successfully used for the analysis of many protein interactions in living cells. A great advantage of FRET over BiFC analysis is that the complexes are in principle at equilibrium, allowing real-time detection of complex formation and dissociation.
Several characteristics of the BiFC assay make it valuable for many studies of protein interactions. First, it enables direct visualization of protein interactions and does not depend on detection of secondary effects. Second, the interactions can be visualized in living cells, eliminating potential artifacts associated with cell lysis or fixation. Third, the proteins are expressed in their normal cellular context, ideally at levels comparable to their endogenous counterparts. Thus, they are predicted to reflect the properties of the corresponding native proteins, including the effects of any post-translational modifications. Fourth, the BiFC assay does not require complex formation by a large fraction of the proteins but can detect interactions between subpopulations of each protein. Fifth, multicolor BiFC analysis allows simultaneous visualization of multiple protein complexes in the same cell and enables analysis of the competition between alternative interaction partners for complex formation with a shared subunit. Finally, BiFC analysis does not require specialized equipment, apart from an inverted fluorescence microscope equipped with objectives that allow imaging of fluorescence in cells. The direct detection of bimolecular complex fluorescence requires no post-acquisition image processing for interpretation of the data. In sum, BiFC is a powerful tool for cell biologists seeking to understand protein interactions in intact cells.