**3.3. Improved FlimPIA using a mutant acceptor (1)**

During another attempt to select paired cysteine residues for possible cross-linking of N-C domains, the introduction of S198C/S440C mutations on the background of original Acceptor was attempted. However, the obtained clone was later found to be contaminated with the S440C mutant retaining only one mutation. The resultant S440C mutant showed higher ability as the Acceptor, whereas the S198C/S440C mutant did not act as the Acceptor. To understand the effect of this mutation, we performed saturation mutagenesis of the S440 residue. The substitution of leucine, phenylalanine, and tryptophan, which have bulky and/ or large side chains, gave a higher maximal S/B ratio in FlimPIA (**Table 1**) [9]. Additionally, not all the mutants with bulky or long side chains showed higher S/B ratios. Although the precise reason is not known, it might be because mutations often affect protein stability and/ or aggregation.

We expected that the bulky and/or large side chains at this position could form steric hindrance with hinge region and the C-terminal domain from the structural modeling based on the adenylation conformation structure of *Luciola cruciata* Fluc with bound substrate analog (**Figure 6A**). On the other hand, there seemed no severe inhibition in the model of the oxidative luminescence conformation.

Then we examined the adenylation and oxidative luminescence activities of the S440L Acceptor. The amounts of LH<sup>2</sup> -AMP produced by the new and conventional Acceptors were examined according to the method using the N-terminal domain of Fluc as a selective detector of LH<sup>2</sup> -AMP [18]. The LH<sup>2</sup> -AMP produced by the new Acceptor was less than one-fifth of the LH<sup>2</sup> -AMP produced by the conventional Acceptor (**Figure 6B**). On the other hand, the kinetics against LH<sup>2</sup> -AMP are shown in **Table 2**. Because the concentration of the LH<sup>2</sup> -AMP that the

> Acceptor uses in FlimPIA is low, the *Vmax*/*Km* is the most important kinetics parameter. The value of the new Acceptor decreased to 33.6% of the value of the conventional Acceptor; therefore, the luminescence intensity in FlimPIA might decrease to some extent. Taken together, the balance of the adenylation and oxidative activities of the new Acceptor gave the highest

> **Figure 6.** Possible steric hindrance of adenylation conformation with the S440 L mutation. (A) Structure of Fluc (left) and a model of Fluc S440L (right), each at adenylation conformation. The Leu440 residue (shown in white) is enlarged in the inset. Drawn with PyMOL software. (B) Adenylation activity measured by the N-terminal domain method. Error bars

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

 **RLU\*/sec)** *K***m (μM)** *V***max/***K***m (× 106**

 **RLU/s μM−1)**

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

19

When the C-terminal domain of Fluc rotated to proceed from the adenylation step to the oxidative luminescence steps, the flexible hinge region between N- and C-terminal domains is considered highly important (**Figure 2**). Furthermore, the hinge region sits close to the active site in the adenylation conformation. To obtain suitable mutants for the Acceptor, semi-random mutations at the residues 436–439 in the hinge region were introduced [6]. The amino acid residues that enzymes in acyl-adenylate-forming enzyme superfamily contain at the corresponding positions were chosen in the semi-random library. The mutant R437K/L438I was selected from the library, because the mutants showed lower adenylation activity (~15% of the wildtype Fluc) and slightly higher oxidative luminescence activity (116% of the wild-type Fluc).

S/B ratio in the Acceptors, which we have developed.

*V***max (× 106**

K529Q 1.40 ± 0.16 0.513 ± 0.018 2.11 ± 0.01 K529Q/S440L 0.296 ± 0.031 0.321 ± 0.013 2.08 ± 0.41

**Table 2.** Oxidative luminescence activity of K529Q and S440L/K529Q (1 nM each).

mean ±SD (n = 3).

\*Relative light units

**3.4. Improved FlimPIA using mutated acceptor (2)**


**Table 1.** Comparison of maximum S/B ratios obtained by S440 mutants.

A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant… http://dx.doi.org/10.5772/intechopen.75644 19

**Figure 6.** Possible steric hindrance of adenylation conformation with the S440 L mutation. (A) Structure of Fluc (left) and a model of Fluc S440L (right), each at adenylation conformation. The Leu440 residue (shown in white) is enlarged in the inset. Drawn with PyMOL software. (B) Adenylation activity measured by the N-terminal domain method. Error bars mean ±SD (n = 3).


**Table 2.** Oxidative luminescence activity of K529Q and S440L/K529Q (1 nM each).

Acceptor uses in FlimPIA is low, the *Vmax*/*Km* is the most important kinetics parameter. The value of the new Acceptor decreased to 33.6% of the value of the conventional Acceptor; therefore, the luminescence intensity in FlimPIA might decrease to some extent. Taken together, the balance of the adenylation and oxidative activities of the new Acceptor gave the highest S/B ratio in the Acceptors, which we have developed.

#### **3.4. Improved FlimPIA using mutated acceptor (2)**

**3.3. Improved FlimPIA using a mutant acceptor (1)**

or aggregation.

18 Protein-Protein Interaction Assays

of LH<sup>2</sup>

against LH<sup>2</sup>

LH<sup>2</sup>

tive luminescence conformation.

Acceptor. The amounts of LH<sup>2</sup>


During another attempt to select paired cysteine residues for possible cross-linking of N-C domains, the introduction of S198C/S440C mutations on the background of original Acceptor was attempted. However, the obtained clone was later found to be contaminated with the S440C mutant retaining only one mutation. The resultant S440C mutant showed higher ability as the Acceptor, whereas the S198C/S440C mutant did not act as the Acceptor. To understand the effect of this mutation, we performed saturation mutagenesis of the S440 residue. The substitution of leucine, phenylalanine, and tryptophan, which have bulky and/ or large side chains, gave a higher maximal S/B ratio in FlimPIA (**Table 1**) [9]. Additionally, not all the mutants with bulky or long side chains showed higher S/B ratios. Although the precise reason is not known, it might be because mutations often affect protein stability and/

We expected that the bulky and/or large side chains at this position could form steric hindrance with hinge region and the C-terminal domain from the structural modeling based on the adenylation conformation structure of *Luciola cruciata* Fluc with bound substrate analog (**Figure 6A**). On the other hand, there seemed no severe inhibition in the model of the oxida-

Then we examined the adenylation and oxidative luminescence activities of the S440L

examined according to the method using the N-terminal domain of Fluc as a selective detector


**S440X S/B ratio S440X S/B ratio** L 7.93 ± 0.60 Q 2.11 ± 0.01 F 5.69 ± 0.12 R 2.08 ± 0.41 W 4.94 ± 0.06 S 1.87 ± 0.24 M 3.65 ± 0.35 N 1.86 ± 0.08 K 3.45 ± 0.20 V 1.85 ± 0.25 A 2.86 ± 0.22 D 1.80 ± 0.13 Y 2.81 ± 0.37 G 1.67 ± 0.26 H 2.57 ± 0.19 I 1.55 ± 0.21 C 2.32 ± 0.13 T 1.52 ± 0.22 E 2.31 ± 0.10 P 1.09 ± 0.11

**Table 1.** Comparison of maximum S/B ratios obtained by S440 mutants.





When the C-terminal domain of Fluc rotated to proceed from the adenylation step to the oxidative luminescence steps, the flexible hinge region between N- and C-terminal domains is considered highly important (**Figure 2**). Furthermore, the hinge region sits close to the active site in the adenylation conformation. To obtain suitable mutants for the Acceptor, semi-random mutations at the residues 436–439 in the hinge region were introduced [6]. The amino acid residues that enzymes in acyl-adenylate-forming enzyme superfamily contain at the corresponding positions were chosen in the semi-random library. The mutant R437K/L438I was selected from the library, because the mutants showed lower adenylation activity (~15% of the wildtype Fluc) and slightly higher oxidative luminescence activity (116% of the wild-type Fluc).

A single mutation, R437K, or a double mutation, R437K/L438I, was introduced into the conventional Acceptor (K529Q). The overall luminescence activity and the oxidative luminescence activity of the two new Acceptors were compared to that of the conventional Acceptor (**Figure 7A**, **B**). The overall activities of both new Acceptors decreased almost tenfold compared with that of the conventional Acceptor, whereas the oxidative luminescence activities were almost maintained. These results showed that R437K is a key residue for Acceptor activity.

The kinetics properties of the conventional and the new Acceptors fused to FRB are shown in **Table 3**. The lower overall activities and the similar oxidative luminescence activities are probably due to the remarkably lower *Vmax* values for LH<sup>2</sup> and ATP and similar *V*max and *K*<sup>m</sup> values for LH<sup>2</sup> -AMP. Moreover, in the structural model of the adenylation conformation, the mutated residue K437 sits close to the active site residues such as K529, suggesting some inhibition of the adenylation activity (**Figure 7C**).

**3.5. Optimization of assay conditions**

**Table 3.** Kinetics properties of Acceptors fused to FRB.

*K***m for LH2** *V***max (× 104**

**RLU/s) for LH<sup>2</sup>**

was reacted with (1) LH<sup>2</sup>

of the probes and substrates.

LH<sup>2</sup>

K529A/R437K/ L438I

The overall activities of the improved Acceptor (R4437K/K529Q) mentioned in Section 3.4 showed a tenfold decrease, and the oxidative luminescence activities were almost maintained. However, the S/B ratio increased only 1.6-fold. To investigate this discrepancy, the Acceptor

*K***m for ATP** *V***max (104**

62.7 ± 4.1 35.1 ± 0.7 306 ± 25 39.8 ± 1.1 0.710 ± 0.093 1.28 ± 0.06

K529Q 95.0 ± 12.1 3.49 ± 0.20 424 ± 55 2.50 ± 0.11 0.412 ± 0.055 1.04 ± 0.04 K529Q/R437L 115 ± 4.0 5.52 ± 0.08 307 ± 25 3.94 ± 0.11 0.605 ± 0.063 0.737 ± 0.027

**RLU/s) for ATP**

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

The luminescence intensity in the case of (3) should be equal to the sum of the intensities of (1) and (2). However, the intensity in (3) was remarkably lower than the sum. Therefore, we thought that some competition may exist in the oxidative luminescence steps. It was reported


**Figure 8.** Optimization of assay condition in vitro. (A) An experimental simulation of FlimPIA using the conventional Acceptor. (B) The responses with and without 50 nM rapamycin in the presence of 1 mM CoA and 20 mM ATP. (C) The responses in the presence of 1 mM CoA and 1 mM ATP. (D) The results of tube-based luminometer with rapid mixing


+ ATP + LH<sup>2</sup>

*K***m for LH2 -AMP** *V***max (×106**

**LH2 -AMP**

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

 **RLU/s) for** 

21




+ ATP, (2) LH<sup>2</sup>

that dehydroluciferyl-AMP (L-AMP), which is converted from LH<sup>2</sup>

dehydroluciferyl-coenzyme A, which is a less potent competitor of LH<sup>2</sup>

When the FKBP12-FRB interaction was detected by FlimPIA, the maximum S/B ratio reached approximately 4, whereas it was approximately 2.5 in the conventional assay (**Figure 7D**). Taken together, we succeeded in finding a suitable mutant for the Acceptor in the semirandom library of the hinge region. Furthermore, these results suggest that the hinge region is important for controlling the two half-reactions of Fluc and supports the hypothesis that the C-terminal domain rotates to accomplish the half-reactions.

**Figure 7.** FlimPIA in vitro using the new Acceptor mutated in the hinge region. (A) Overall luminescent activity of the conventional Acceptor and the two new Acceptors. Reactions with LH<sup>2</sup> and ATP (n = 3). (B) Luminescent activity of the Acceptors with LH<sup>2</sup> -AMP as a substrate (n = 3). (C) 3D models of the Acceptors at adenylation conformation. The wildtype Fluc (left), the conventional Acceptor (middle), and the mutant M1 (right) are shown. In the conventional Acceptor, the shortest distance between the active site against LH<sup>2</sup> (529Q) and R437 was ~3.8 Å, which was shorter in the mutant (~1.6 Å). (D) FlimPIA with 50 nM each of FKBP/Donor and FRB/the new Acceptor with/ without 50 nM rapamycin (n = 3).


**Table 3.** Kinetics properties of Acceptors fused to FRB.

#### **3.5. Optimization of assay conditions**

A single mutation, R437K, or a double mutation, R437K/L438I, was introduced into the conventional Acceptor (K529Q). The overall luminescence activity and the oxidative luminescence activity of the two new Acceptors were compared to that of the conventional Acceptor (**Figure 7A**, **B**). The overall activities of both new Acceptors decreased almost tenfold compared with that of the conventional Acceptor, whereas the oxidative luminescence activities were almost maintained.

The kinetics properties of the conventional and the new Acceptors fused to FRB are shown in **Table 3**. The lower overall activities and the similar oxidative luminescence activities are

mutated residue K437 sits close to the active site residues such as K529, suggesting some inhi-

When the FKBP12-FRB interaction was detected by FlimPIA, the maximum S/B ratio reached approximately 4, whereas it was approximately 2.5 in the conventional assay (**Figure 7D**). Taken together, we succeeded in finding a suitable mutant for the Acceptor in the semirandom library of the hinge region. Furthermore, these results suggest that the hinge region is important for controlling the two half-reactions of Fluc and supports the hypothesis that the

**Figure 7.** FlimPIA in vitro using the new Acceptor mutated in the hinge region. (A) Overall luminescent activity of the

type Fluc (left), the conventional Acceptor (middle), and the mutant M1 (right) are shown. In the conventional Acceptor,

(~1.6 Å). (D) FlimPIA with 50 nM each of FKBP/Donor and FRB/the new Acceptor with/ without 50 nM rapamycin



and ATP and similar *V*max and *K*<sup>m</sup>

and ATP (n = 3). (B) Luminescent activity of the

(529Q) and R437 was ~3.8 Å, which was shorter in the mutant

These results showed that R437K is a key residue for Acceptor activity.

probably due to the remarkably lower *Vmax* values for LH<sup>2</sup>

C-terminal domain rotates to accomplish the half-reactions.

conventional Acceptor and the two new Acceptors. Reactions with LH<sup>2</sup>

the shortest distance between the active site against LH<sup>2</sup>

bition of the adenylation activity (**Figure 7C**).

values for LH<sup>2</sup>

20 Protein-Protein Interaction Assays

Acceptors with LH<sup>2</sup>

(n = 3).

The overall activities of the improved Acceptor (R4437K/K529Q) mentioned in Section 3.4 showed a tenfold decrease, and the oxidative luminescence activities were almost maintained. However, the S/B ratio increased only 1.6-fold. To investigate this discrepancy, the Acceptor was reacted with (1) LH<sup>2</sup> + ATP, (2) LH<sup>2</sup> -AMP, and (3) LH<sup>2</sup> + ATP + LH<sup>2</sup> -AMP (**Figure 8A**). The luminescence intensity in the case of (3) should be equal to the sum of the intensities of (1) and (2). However, the intensity in (3) was remarkably lower than the sum. Therefore, we thought that some competition may exist in the oxidative luminescence steps. It was reported that dehydroluciferyl-AMP (L-AMP), which is converted from LH<sup>2</sup> -AMP, competes with LH<sup>2</sup> -AMP in the oxidative luminescence steps, and coenzyme A (CoA) converts L-AMP to dehydroluciferyl-coenzyme A, which is a less potent competitor of LH<sup>2</sup> -AMP. First, we added

**Figure 8.** Optimization of assay condition in vitro. (A) An experimental simulation of FlimPIA using the conventional Acceptor. (B) The responses with and without 50 nM rapamycin in the presence of 1 mM CoA and 20 mM ATP. (C) The responses in the presence of 1 mM CoA and 1 mM ATP. (D) The results of tube-based luminometer with rapid mixing of the probes and substrates.

CoA to the mixture of FlimPIA (**Figure 8B**). In the presence of CoA, the maximum S/B ratio reached 8, representing a twofold improvement, when 50 nM of each probe was used.

Next, we optimized the concentration of ATP, as it was designed so that the *Km* value of the Acceptor for ATP was lower than that of the wild type to suppress the adenylation activity, but the *Km* value of the Donor for ATP was maintained to provide LH<sup>2</sup> -AMP. The optimal concentration of ATP was 1 mM, and the maximal S/B ratio reached approximately 40, representing a fivefold improvement, when 50 nM of each probe was used (**Figure 8C**).

Finally, we had optimized the reaction conditions. As the increase of luminescence occurred as soon as substrates were added, a luminometer equipped with a stirrer was used to mix and react the substrates quickly (**Figure 8D**). The luminescence intensity increased quasi-linearly from 0.2 to 0.6 s after the reaction start and then reached a plateau. The maximal S/B ratio reached more than 60 when 100 nM of each probe was used.

Taken together, these improvements achieved a remarkably higher S/B ratio and sensitivity [6].
