Бимолекулярная флуоресцентная комплементация: метод визуализации белковых взаимодействий

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. 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. 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.

Biochemical complementation was first reported in subtilisin cleaved bovine pancreatic ribonuclease, then expanded using β galactosidase mutants that allowed cells to grow on lactose. 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. 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. 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.

Fluorophore activation occurs through an autocatalytic cyclization reaction that occurs after the protein has been folded correctly. 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. 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. 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.

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. 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. If fluorescence is detected when the fusion proteins are expressed, but is lacking or significantly reduced after the expression of the mutated negative control, it is likely that a specific interaction occurs between the two target proteins of interest. However, if the fluorescence intensity is not significantly different between the mutated negative control fusion protein and its wild type counterpart, then the fluorescence is likely caused by non specific protein interactions, so a different combination of fusion protein conformations should be tested. If no fluorescence is detected, an interaction may still exist between the proteins of interest, as the creation of the fusion protein may alter the structure or interaction face of the target protein or the fluorescence fragments may be physically unable to associate. To ensure that this result is not a false negative, that there is no interaction, the protein interaction must be tested in a situation where fluorescence complementation and activation requires an external signal. In this case, if the external signal fails to cause fluorescence fragment association, it is likely that the proteins do not interact or there is a physical impediment to fluorescence complementation.

Unlike many in vivo protein interaction assays, BiFC does not require protein complexes to be formed by a large proportion of the proteins or at stoichiometric proportions. Instead, BiFC can detect interactions among protein subpopulations, weak interactions, and low expression proteins due to the stable complementation of the fluorescent reporter protein.

BiFC does not require specialised equipment, as visualisation is possible with an inverted fluorescence microscope that can detect fluorescence in cells.

The fluorescent signal only is produced after the proteins have interacted, which is generally in the order of hours. Hence BiFC is unable to provide real time detection of protein interactions. The delay for chemical reactions to generate fluorophore may also have an effect on the dynamics of complex dissociation and partner exchange.

BiFC complex formation is only reversible during the initial step of fluorescent reporter protein re assembly, typically in the order of milliseconds. Once the fluorochrome has been reconstituted, it is essentially irreversible in vitro. This prevents proteins from interacting with others and may disrupt the association/disassociation of protein complexes in dynamic equilibrium. However certain organisms, especially apicomplexa, have a higher autofluorescence that make it harder to apply BiFC in them. Certain fungi, such as Candida albicans, also have a high autofluorescent background, but BiFC can often still be performed when the proper controls and strains are used.

Because endogenous wild type proteins cannot be visualised in vivo, fusion proteins must be created and their plasmids transfected into the cells studied. These fusion proteins may not recapitulate the functions, localisation, and interactions common to their wild type counterparts, providing an inaccurate picture of the proteins in question. This problem can be alleviated by using structural information and the location of interaction sites to rationally identify fusion sites on the proteins of interest, using appropriate controls, and comparing the expression levels and functions of the fusion and wild type proteins through Western Blots and functional assays.

Because fluorophore reconstitution can occur at a distance of 7 nm or more, fluorescence complementation may indicate either a direct or indirect (i. e. within the same complex) interaction between the fluorescent fragments' fused proteins. The process of ribosomes assembly involves nucleation of ribosomal proteins in proper order and orientation. Perturbations in assembly can lead to structural defects in ribosomal subunits which as a result cannot join in the correct orientation to form fully functional ribosomes. Thus, the events of subunit joining signaled by the appearance of BiFC is an easy way to monitor ribosome biogenesis in contrast to laborious polysome profiling methods.

The fluorescent protein fragments used in BiFC have been expanded to include the colours blue, cyan, green, yellow, red, cherry, and Venus. This range in colours has made the development of multicolour fluorescence complementation analysis possible. Thus, BiFC is an important tool in understanding transcription factor localisation in subcellular compartments.

BiFC has been coupled with flow cytometry (BiFC FC). This allows protein–protein interaction surfaces to be mapped through the introduction of site directed or random mutations that affect complex formation. Finally, by studying proteins in vitro, one is unable to determine the influence of specific protein–protein interactions in the cell on the functional or physiological consequences. Other in vivo assays most commonly used to study protein–protein interactions include fluorescence resonance energy transfer (FRET) and yeast two hybrid (Y2H) assay. Each of these assays has their advantages and disadvantages in comparison to BiFC:

Fluorescence resonance energy transfer (FRET), also known as förster resonance energy transfer, resonance energy transfer (RET) or electronic energy transfer (EET), is based on the transfer of energy from an excited (donor) chromophore or fluorophore (if the chromophores are fluorescent) to a nearby acceptor. In this method, fluorophores are chemically linked or genetically fused to two proteins hypothesised to interact. If the proteins interact, this will bring the fluorophores into close spatial proximity. If the fluorophores are oriented in a manner that exposes the fluorophores to one another, usually ensured when designing and constructing the fluorophore protein linkage/fusion, then the energy transfer from the excited donor fluorophore will result in a change in the fluorescent intensities or lifetimes of the fluorophores.