**3.1. Principle of FlimPIA**

In contrast to PCA, in which the structure of Fluc is divided into two domains as the probes, FlimPIA divides the reaction catalyzed by Fluc into two half-reactions. Fluc catalyzes the conversion of firefly d-luciferin (LH<sup>2</sup> ) to the excited state oxyluciferin (OxL) by a two-step catalysis, namely, an adenylation step and oxidative luminescence steps. In the adenylation step, LH<sup>2</sup> is converted to d-luciferyl adenylate (LH<sup>2</sup> -AMP), and in the oxidative luminescence steps, LH<sup>2</sup> -AMP is converted to OxL, and then excited OxL emits light. It was recently supposed that Fluc, which consists of a large N-terminal domain and a small C-terminal domain connected by a flexible hinge region, rotates its C-terminal domain by ~140° to proceed from the adenylation step to the oxidative luminescence steps (**Figure 2**) [16, 17]. One reason for this hypothesis is that the active site of each step in acyl-adenylate-forming enzymes, including Fluc, is different. In the adenylation step, K529 is an important amino acid residue, and on the other hand, K443 and H245 are key residues for the oxidative luminescence steps [18–20].

Two mutant *Photinus pyralis* Flucs were designed for FlimPIA; one is H245D/K443A/L530R, which can produce LH<sup>2</sup> -AMP but cannot catalyze LH<sup>2</sup> -AMP to form OxL, and the other is K529Q, which very slowly produces LH<sup>2</sup> -AMP but maintains the catalytic steps in the oxidative luminescence half-reaction. Each mutant is fused to proteins that interact with each other. The interaction brings the mutants close together, and then LH<sup>2</sup> -AMP, which H245D/K443A/ L530R produces, is utilized by K529Q, resulting in OxL production (**Figure 3**). The mutant H245D/K443A/L530R acts as the "Donor" providing LH<sup>2</sup> -AMP, and the mutant K529Q works as the "Acceptor" of LH<sup>2</sup> -AMP [7].

rapamycin was up to 500 nM (**Figure 4D**). In addition, the association between FKBP12 and FRB could be detected in 40% fetal bovine serum diluted in phosphate buffered saline, sug-

**Figure 2.** The conformational change of Fluc. Fluc is composed of a large N-terminal domain and a smaller C-terminal domain, which rotates ∼140° according to the reactions to proceed from the adenylation reaction to the oxidative luminescent reactions: Key Lys residues (K529 and K443) are shown in light and dark blue, respectively. Another key

A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant…

http://dx.doi.org/10.5772/intechopen.75644

15

Next, p53 and Mdm2 were used as interacting proteins. The luminescence intensity of the mixture of the interacting pair (p53-Donor and Mdm2-Acceptor) was higher than the intensities of noninteracting pairs (p53-Donor and p53-Acceptor, Mdm2-Donor and Mdm2- Acceptor) (**Figure 4E**). The inhibition of the p53-Mdm2 interaction by Nutlin-3 was observed (**Figure 4F**). The result clearly shows that FlimPIA is a versatile system and can analyze tran-

The original FlimPIA had exhibited high background signal, which was mainly caused by the remaining adenylation activity of the Acceptor. As mentioned above, the C-terminal domain rotates according to the reactions proceeding from the adenylation to the oxidative luminescence reactions (**Figure 2**). Therefore, we tried to entrap the Acceptor conformation into the

gesting the applicability of the assay to clinical samples.

**Figure 3.** The working principle of FlimPIA. ©American Chemical Society.

residue H245 is shown in cyan. ©American Chemical Society.

**3.2. Improved FlimPIA by the entrapment of Fluc conformation**

sient interactions.

oxidation conformation [10].

When FKBP12 and FRB are fused to the Donor and Acceptor, respectively, the luminescence intensity increased depending on the concentration of rapamycin (**Figure 4A**, **B**). The EC50 values of the cognate pairs were 10.2 ± 0.6 and 16.0 ± 2.1 nM, respectively, which correspond well with the reported KD value of the association between FKBP12/rapamycin and FRB. FK506 (tacrolimus) is commonly used as an immunosuppressant to prevent the rejection of organ transplants and inhibits the rapamycin-dependent association between FKBP12 and FRB [14]. The luminescence intensity decreased upon FK506 addition (**Figure 4C**). The S/B ratio increased depending on the concentration of PPI when the concentration of probes and A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant… http://dx.doi.org/10.5772/intechopen.75644 15

**Figure 2.** The conformational change of Fluc. Fluc is composed of a large N-terminal domain and a smaller C-terminal domain, which rotates ∼140° according to the reactions to proceed from the adenylation reaction to the oxidative luminescent reactions: Key Lys residues (K529 and K443) are shown in light and dark blue, respectively. Another key residue H245 is shown in cyan. ©American Chemical Society.

**Figure 3.** The working principle of FlimPIA. ©American Chemical Society.

of p53 in certain cancer cells. In the assay of p53-Mdm2 interaction using p53-C and Mdm2-N, the signal intensity and S/B ratio rose with higher concentrations of the probes of the interacting pair. To investigate the reversibility of the PCA, an inhibitor of the p53-Mdm2 interaction, Nutlin-3, was added to the mixture of p53-C and Mdm2-N. The luminescence intensity

The invitro PCA opens the way to study PPIs of cytotoxic proteins, which is impossible to perform in cells. Furthermore, the possibility that the cellular components affect PPIs can be excluded.

In this section, we describe a novel PPI assay, FlimPIA, which we recently developed and

In contrast to PCA, in which the structure of Fluc is divided into two domains as the probes, FlimPIA divides the reaction catalyzed by Fluc into two half-reactions. Fluc catalyzes the

catalysis, namely, an adenylation step and oxidative luminescence steps. In the adenylation

posed that Fluc, which consists of a large N-terminal domain and a small C-terminal domain connected by a flexible hinge region, rotates its C-terminal domain by ~140° to proceed from the adenylation step to the oxidative luminescence steps (**Figure 2**) [16, 17]. One reason for this hypothesis is that the active site of each step in acyl-adenylate-forming enzymes, including Fluc, is different. In the adenylation step, K529 is an important amino acid residue, and on the other hand, K443 and H245 are key residues for the oxidative luminescence steps [18–20]. Two mutant *Photinus pyralis* Flucs were designed for FlimPIA; one is H245D/K443A/L530R,

tive luminescence half-reaction. Each mutant is fused to proteins that interact with each other.

L530R produces, is utilized by K529Q, resulting in OxL production (**Figure 3**). The mutant

When FKBP12 and FRB are fused to the Donor and Acceptor, respectively, the luminescence intensity increased depending on the concentration of rapamycin (**Figure 4A**, **B**). The EC50 values of the cognate pairs were 10.2 ± 0.6 and 16.0 ± 2.1 nM, respectively, which correspond well with the reported KD value of the association between FKBP12/rapamycin and FRB. FK506 (tacrolimus) is commonly used as an immunosuppressant to prevent the rejection of organ transplants and inhibits the rapamycin-dependent association between FKBP12 and FRB [14]. The luminescence intensity decreased upon FK506 addition (**Figure 4C**). The S/B ratio increased depending on the concentration of PPI when the concentration of probes and



) to the excited state oxyluciferin (OxL) by a two-step






decreased depending on the concentration of Nutlin-3.

**3. Development of a novel PPI assay FlimPIA**

is converted to d-luciferyl adenylate (LH<sup>2</sup>

The interaction brings the mutants close together, and then LH<sup>2</sup>

H245D/K443A/L530R acts as the "Donor" providing LH<sup>2</sup>


continue to improve.

14 Protein-Protein Interaction Assays

step, LH<sup>2</sup>

steps, LH<sup>2</sup>

**3.1. Principle of FlimPIA**

which can produce LH<sup>2</sup>

as the "Acceptor" of LH<sup>2</sup>

K529Q, which very slowly produces LH<sup>2</sup>

conversion of firefly d-luciferin (LH<sup>2</sup>

rapamycin was up to 500 nM (**Figure 4D**). In addition, the association between FKBP12 and FRB could be detected in 40% fetal bovine serum diluted in phosphate buffered saline, suggesting the applicability of the assay to clinical samples.

Next, p53 and Mdm2 were used as interacting proteins. The luminescence intensity of the mixture of the interacting pair (p53-Donor and Mdm2-Acceptor) was higher than the intensities of noninteracting pairs (p53-Donor and p53-Acceptor, Mdm2-Donor and Mdm2- Acceptor) (**Figure 4E**). The inhibition of the p53-Mdm2 interaction by Nutlin-3 was observed (**Figure 4F**). The result clearly shows that FlimPIA is a versatile system and can analyze transient interactions.
