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

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 oxidation conformation [10].

The luminescence of the cross-linked Acceptor was almost diminished compared to the noncross-linked Acceptor (**Figure 5B**). The Acceptor and Donor were fused to FRB and FKBP12, respectively. In a FlimPIA using the cross-linked Acceptor, the background signal was eliminated, and the signal induced by the interaction was significantly higher than the background signal (**Figure 5C**). Taken together, the results clearly showed that the Acceptor can be trapped into the oxidation conformation and the sensitive FlimPIA was successfully developed, giv-

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

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

17

As the substitution of the all cysteine residues considerably reduced the luminescence intensity, next, we tried to use the original Acceptor retaining the cysteine residues and put the cysteine residues at positions 108 and 447, which were then cross-linked by BMOE. As a result, one-fifth of the luminescence intensity of the cross-linked Acceptor was retained, probably due to miss- and/or incomplete cross-linking (**Figure 5D**). Although there was some background signal, an apparent improvement in luminescent intensity was observed. When the same concentration (50 nM each) of the probes and rapamycin were used, the maximum S/B

**Figure 5.** FlimPIA using the trapped Acceptor by bis-maleimide crosslinker (A–C) The trapping by bis-maleimide crosslinker (1). (D–E) The trapping by bis-maleimide crosslinker (2). (A) Scheme of the trapped Acceptors by BMOE. Residues shown in yellow were used for the N−C linkage. (B, D) Suppression of overall luminescent activity by

intensities with and without chemical modification by BMOE were compared (n = 3). (C, E) The mixture of FKBP/Donor and trapped FRB/Acceptor (50 nM each) was added with/without 50 nM rapamycin (n = 3). ©American Chemical Society.

and 10 mM ATP. The luminescent

chemical trapping of the Acceptor. The enzyme (10 nM) was reacted with 75 µM LH<sup>2</sup>

ratio was improved from 2.6 to 5.3, compared with the original system (**Figure 5E**).

ing a high S/B ratio.

**Figure 4.** Detection via FlimPIA in vitro. (A-D) Detection of FKBP-FRB association. (A) Luminescence time course at several rapamycin concentrations. A mixture of FKBP/Donor and FRB/Acceptor (50 nM each) was used. (n = 3). (B) Specific detection of FKBP12-FRB interaction. The four possible combinations of four Fluc mutants, namely, FKBP/Donor, FRB/Donor, FKBP/ Acceptor and FRB/Acceptor (50 nM each) were tested for their rapamycin dose-dependency. The relative luminescence integrated for 1.5−1.6 s after substrate addition is shown (n = 3). (C) Competition of PPI (protein−protein interaction) by FK506. Rapamycin (80 nM) and FK506 at indicated concentration were added to the mixture of FKBP/Donor and FRB/Acceptor (80 nM each). The luminescence integrated for 0.8−0.9 s after substrate addition is shown (n = 3). (D) Time course of S/B (signal/ background) ratio obtained with the mixture of FKBP/Donor and FRB/Acceptor with and without equimolar rapamycin. The ratio of the two light intensities at the indicated time point is shown. Sample with 40% fetal bovine serum and 750 nM proteins is also shown (n = 3). (E–F) Detection of p53-Mdm2 association. (E) Luminescence time course of the cognate (Mdm2/ Donor and p53/Acceptor) and control pairs (25 nM each) (n = 3). (F) Competition of PPI by a specific inhibitor. Nutlin-3 (bottom) at indicated concentration was added to the mixture of p53/Donor and Mdm2/Acceptor (25 nM each) (n = 3). The luminescence integrated for 0.8−0.9 s after substrate addition is shown. The ribbon model of Mdm2 (purple)-p53 peptide (light green) complex is also shown. ©American Chemical Society.

According to the report by Branchini et al. that the structure of Fluc could be fixed into the oxidative luminescence conformation by chemical trapping, we first took the same approach to entrap Acceptor mutant [21]. Specifically, all cysteine residues in the Acceptor were substituted with serine or alanine residues. Then, the residues at positions 108 and 447 were substituted with cysteine residues and cross-linked by 1,2-bis-(maleimide)ethane (BMOE) (**Figure 5A**).

The luminescence of the cross-linked Acceptor was almost diminished compared to the noncross-linked Acceptor (**Figure 5B**). The Acceptor and Donor were fused to FRB and FKBP12, respectively. In a FlimPIA using the cross-linked Acceptor, the background signal was eliminated, and the signal induced by the interaction was significantly higher than the background signal (**Figure 5C**). Taken together, the results clearly showed that the Acceptor can be trapped into the oxidation conformation and the sensitive FlimPIA was successfully developed, giving a high S/B ratio.

As the substitution of the all cysteine residues considerably reduced the luminescence intensity, next, we tried to use the original Acceptor retaining the cysteine residues and put the cysteine residues at positions 108 and 447, which were then cross-linked by BMOE. As a result, one-fifth of the luminescence intensity of the cross-linked Acceptor was retained, probably due to miss- and/or incomplete cross-linking (**Figure 5D**). Although there was some background signal, an apparent improvement in luminescent intensity was observed. When the same concentration (50 nM each) of the probes and rapamycin were used, the maximum S/B ratio was improved from 2.6 to 5.3, compared with the original system (**Figure 5E**).

**Figure 5.** FlimPIA using the trapped Acceptor by bis-maleimide crosslinker (A–C) The trapping by bis-maleimide crosslinker (1). (D–E) The trapping by bis-maleimide crosslinker (2). (A) Scheme of the trapped Acceptors by BMOE. Residues shown in yellow were used for the N−C linkage. (B, D) Suppression of overall luminescent activity by chemical trapping of the Acceptor. The enzyme (10 nM) was reacted with 75 µM LH<sup>2</sup> and 10 mM ATP. The luminescent intensities with and without chemical modification by BMOE were compared (n = 3). (C, E) The mixture of FKBP/Donor and trapped FRB/Acceptor (50 nM each) was added with/without 50 nM rapamycin (n = 3). ©American Chemical Society.

According to the report by Branchini et al. that the structure of Fluc could be fixed into the oxidative luminescence conformation by chemical trapping, we first took the same approach to entrap Acceptor mutant [21]. Specifically, all cysteine residues in the Acceptor were substituted with serine or alanine residues. Then, the residues at positions 108 and 447 were substituted with cysteine residues and cross-linked by 1,2-bis-(maleimide)ethane (BMOE) (**Figure 5A**).

green) complex is also shown. ©American Chemical Society.

16 Protein-Protein Interaction Assays

**Figure 4.** Detection via FlimPIA in vitro. (A-D) Detection of FKBP-FRB association. (A) Luminescence time course at several rapamycin concentrations. A mixture of FKBP/Donor and FRB/Acceptor (50 nM each) was used. (n = 3). (B) Specific detection of FKBP12-FRB interaction. The four possible combinations of four Fluc mutants, namely, FKBP/Donor, FRB/Donor, FKBP/ Acceptor and FRB/Acceptor (50 nM each) were tested for their rapamycin dose-dependency. The relative luminescence integrated for 1.5−1.6 s after substrate addition is shown (n = 3). (C) Competition of PPI (protein−protein interaction) by FK506. Rapamycin (80 nM) and FK506 at indicated concentration were added to the mixture of FKBP/Donor and FRB/Acceptor (80 nM each). The luminescence integrated for 0.8−0.9 s after substrate addition is shown (n = 3). (D) Time course of S/B (signal/ background) ratio obtained with the mixture of FKBP/Donor and FRB/Acceptor with and without equimolar rapamycin. The ratio of the two light intensities at the indicated time point is shown. Sample with 40% fetal bovine serum and 750 nM proteins is also shown (n = 3). (E–F) Detection of p53-Mdm2 association. (E) Luminescence time course of the cognate (Mdm2/ Donor and p53/Acceptor) and control pairs (25 nM each) (n = 3). (F) Competition of PPI by a specific inhibitor. Nutlin-3 (bottom) at indicated concentration was added to the mixture of p53/Donor and Mdm2/Acceptor (25 nM each) (n = 3). The luminescence integrated for 0.8−0.9 s after substrate addition is shown. The ribbon model of Mdm2 (purple)-p53 peptide (light
