BiFC Assay: A Powerful Tool for Studying Protein-Protein Interactions

Protein-protein interactions, the cornerstone of many biological processes, from signal transduction to gene regulation and immune response, hold paramount importance in unraveling the intricacies of cellular pathways and developing targeted therapeutics. However, comprehending the underlying mechanisms that govern these interactions remains an elusive goal.

Enter the bimolecular fluorescence complementation (BiFC) assay, a cutting-edge method that has emerged as a powerful tool for investigating protein-protein interactions in living cells. By leveraging the properties of fluorescent proteins, this technique enables the detection of protein-protein interactions in real-time, providing researchers with valuable insights into the dynamics and regulation of these interactions.

Despite its promise, the BiFC assay is not without its limitations. One key challenge is the need for precise experimental conditions to ensure accurate results. Nonetheless, the potential of this method in shedding light on the complex and dynamic world of protein-protein interactions makes it a compelling avenue for future research.

How Does BiFC Work?

The BiFC assay perates on the basis of a principle which involves the fusion of two non-fluorescent protein fragments to two interacting proteins. When these proteins interact, the protein fragments come in close proximity and unite to create a fully functional fluorescent protein, generating a fluorescence signal that acts as a readout of the protein-protein interaction taking place in vivo.

Yellow fluorescent protein (YFP) is the most commonly used protein fragment for BiFC, where the N-terminal and C-terminal fragments of YFP, known as YFPn and YFPc, respectively, are non-fluorescent when expressed individually. However, upon their fusion with two interacting proteins, the co-expression of YFPn and YFPc in a cell can result in the formation of a functional fluorescent YFP molecule, provided the two proteins interact. The fluorescence signal produced can be visualized using fluorescence microscopy or quantified through fluorescence spectrometry.

The adaptability of the BiFC assay allows for the exploration of protein-protein interactions between full-length proteins, domains, or peptides across diverse systems ranging from mammalian cells, yeast, plants, and bacteria.

Principle of multicolour bimolecular fluorescence complementation (Kerppola et al., 2006)

Advantages of BiFC Assay

The BiFC assay offers several advantages over other methods for studying protein-protein interactions:

• Allows protein-protein interactions to be studied in living cells, providing a more physiologically consistent environment than in vitro methods.
• Study protein-protein interactions with high spatial and temporal resolution. Fluorescent signals can be observed at specific subcellular compartments or at specific time points in the cellular process.
• Can be used to measure protein-protein interactions quantitatively by fluorescence spectroscopy and thus compare protein-protein interactions under different conditions or between different proteins.
• Suitable for multiplexing, allowing simultaneous detection of multiple protein-protein interactions in the same cell.

Quantitative BiFC

Quantitative BioFC (qBiFC) is a modified bioFC protocol. In qBiFC, the fluorescence signal is normalized to the expression level of the interacting proteins, allowing the calculation of the binding affinity constant (Kd) for protein-protein interactions. This makes it possible to determine the strength of the interaction between two proteins.

qBiFC has been used to study the interactions of various proteins, including transcription factors, enzymes, and signaling molecules. The method is highly reproducible and sensitive.

Differences between FRET and BiFC

Fluorescence resonance energy transfer (FRET) is a widely embraced technique for the study of protein-protein interactions. FRET methodology is based on the concept that when two fluorophores exist in close proximity, energy can be transmitted from an excited donor fluorophore to an acceptor fluorophore, leading to a fluorescence signal. This signal, in turn, enables scientists to investigate protein-protein interactions.

One of the primary distinctions between FRET and BiFC lies in the readout. FRET generates the fluorescence signal by transferring energy between two fluorophores, whereas BiFC is characterized by the fluorescence signal arising from the reconstitution of a fluorescent protein. This variance results in BiFC being more sensitive to protein-protein interactions that bring about a conformational change or stimulate the association of two proteins.

BiFC and FRET are two cell-based techniques to monitor protein–protein interactions (Geiss et al., 2009)

Another difference between FRET and BiFC involves the specificity of the interaction detected. FRET has the capability of detecting any protein-protein interaction that leads to a change in distance between the two fluorophores, irrespective of whether the interaction is direct or indirect. Conversely, BiFC only identifies direct protein-protein interactions between the two proteins that are fused to the YFP fragments.

Finally, it is imperative to note that FRET and BiFC have varied restrictions concerning the size and location of the proteins being studied. While FRET is adaptable to the study of protein-protein interactions between small molecules or domains, BiFC necessitates larger proteins capable of accommodating the fused YFP fragments. Moreover, the location of the interaction can affect the detection of the fluorescence signal in both FRET and BiFC, making them challenging to use in certain cases.