Introduction to Bimolecular Fluorescence Complementation

Bimolecular Fluorescence Complementation (BiFC) is a fairly recent method for the direct visualization of protein-protein interactions in a variety of living host (Walter et al., 2004). BiFC is based on the reconstitution of a fluorescent protein function when two non- fluorescent non complementary fragments of a fluorescent protein are brought together by a pair of interacting proteins (Figure 3.8A). Studies have shown that when the YFP fluorescent protein can be split at specific sites within a β-strand or be split at a loop; that the resulting two fragments of protein are (C. D. Hu, Chinenov, & Kerppola, 2002) capable of functioning as one functional fluorescing protein when brought into close enough proximity, similar to the two halves of the GAL4 domain in the Y2H system. The BiFC utilizes a two fusion protein system where in a protein of interest is fused to either the N- terminal or the C-terminal half of a fluorescent protein, and the second protein of interest is fused to the other half of the fluorescent protein(C.-D. Hu, Grinberg, & Kerppola, 2006). The reconstitution of the fluorescent protein function allows for the imaging of the fluorescence emitted. Fluorescent proteins cannot be seen under visible light, and require excitation by specific wavelengths of light to be visualized using confocal microscopy (Figure 3.8).

Functional fluorescing proteins are able to absorb a photon of higher energy and emit a photon with a lower energy longer wavelength that can be measured using a confocal microscope(Kerppola, 2006). A variety of fluorescence protein are compatible with BiFC, including the enhanced yellow fluorescence protein (YFP) is used, but other fluorescence proteins have also been used in studies, including the green fluorescent protein (GFP), and blue fluorescent protein (BFP)(Weinthal & Tzfira, 2009).

For the purposes of this study, Enhanced YFP (YFP) was used. The two nonfluorescent protein fragments, the C-terminal half of YFP (cYFP) and the N-terminal half of YFP (nYFP), are fused to two putative interacting partners. If interaction occurs between the two putative protein partners, the positive interaction can result in the proximal reconstitution of the YFP fluorescent protein and will emit an observable yellowish-green fluorescence (Figure 3.8A). If no interaction occurs between 2 proteins, no fluorescence should be observed as no reconstitution of the fluorescent signal can occur without a proximal interaction to bring the YFP halves together (Figure 3.8C).

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There are several locations where a fluorescent protein can be split. For YFP, there are two places that the YFP can be split according to previously published data. The first location is at amino acid 155. The second location is between amino acid 173 and 174(C.-D. Hu, et al., 2006; Kerppola, 2006). For this study, YFP was split between amino acids 155 and 156(Appendix 4).

BiFC is a relatively simple method of investigating protein-protein interactions and provides a platform that can be used to study protein-protein interactions in a more native host model. The system has been reported to be very sensitive in the detection of low expressing protein-protein interactions (C.-D. Hu, et al., 2006; Kerppola, 2006; Walter, et al., 2004). BiFC allows for the use of a plant host to test for protein-protein interactions, thereby providing a host system that is similar to the native Arabidopsis host. The use of a similar host organism greatly improves the chances of proper protein translation, folding, and subsequent interactions.

In order to provide a more accurate model for CPR5 interaction assays, a Nicotiana benthamiana plant model was chosen for transient infiltration of Agrobacterium and subsequent bimolecular fluorescence complementation.

If CPR5 interaction with the identified proteins orients the two YFP fragments at opposite ends of the protein complex, the YFP halves will not be in close enough proximity to reconstitute YFP function (Figure 3.8B). In order to ensure that the fusion proteins allow for the fragments of YFP to associate with each other when a protein of interest interacts with CPR5, several different constructs of CPR5 were produced to account for the orientation of potential protein interactions. The N-terminal YFP segment was tagged to the N-terminal of all genes of interest and the C-terminal YFP segment was tagged to both N-terminal and the C-terminal of CPR5 (cYFP-CPR5/ CPR5-cYFP). By fusing the cYFP segment of the YFP protein to both the C-terminal and the N-terminal of CPR5 respectively, we account for the possibility that the N-terminal region of the proteins of interest interaction will be in close proximity to either the C-terminal or N-terminal region of CPR5.

57 Figure 3.8: The Theory of BiFC assay.

Functional YFP fluorescent proteins are able to emit fluorescence when excited with 496nm wavelength light (argon). A) Proteins that are able to interact (X and Y) with the cYFP and nYFP tags fused to the proper protein terminals provide the close enough proximity for the YFP fragments to reconstitute function and emit a fluorescent signal. B) Interacting proteins with cYFP and nYFP fused to nonproximal terminals do not provide the proximity required for reconstitution of signal despite interaction between X and Y. C) Proteins that do not interaction (X and Z) are unable to provide the proximity required to reconstitute signal. The precise structures and flexibilities specific to each protein must be taken into account in order to determine the most viable terminal region for YFP fusion. (Figure was modified from Weinthal & Tzfira, 2009)