In vitro Determination of Rapamycin-triggered FKBP–FRB Interactions Using a Molecular Tension Probe

• Published in: Analytical Sciences Vol. 35; no. 1; pp. 71 - 78
• Main Authors: KIM, Sung Bae, NISHIHARA, Ryo, FUJII, Rika, PAULMURUGAN, Ramasamy, CITTERIO, Daniel, SUZUKI, Koji
• Format: Journal Article
• Language: English
• Published: Singapore The Japan Society for Analytical Chemistry 10.01.2019; Springer Nature Singapore; Japan Science and Technology Agency
• Subjects: Analytical Chemistry; artificial luciferase; bioluminescence; Catalysis; Chains; Chemistry; Circular Dichroism; Dichroism; E coli; imaging; in vitro assay; In vivo methods and tests; Kinetics; Luciferases - genetics; Luminescent Measurements; Molecular Probes; Molecular tension probe; Plasmids; Protein Binding; Protein interaction; Protein Interaction Mapping - methods; Proteins; protein–protein interactions; Rapamycin; Reaction kinetics; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Sirolimus - pharmacology; Substrates; Tacrolimus Binding Proteins - genetics; Tacrolimus Binding Proteins - metabolism; Tacrolimus-binding protein; TOR Serine-Threonine Kinases - genetics; TOR Serine-Threonine Kinases - metabolism; in vitro assay; Molecular tension probe; bioluminescence; imaging; protein-protein interactions; artificial luciferase; protein–protein interactions
• ISSN: 0910-6340; 1348-2246
• DOI: 10.2116/analsci.18SDP08

As protein–protein interactions (PPI) have been mostly investigated in cellulo or in vivo, it is unclear whether the PPI-based imaging schemes are practically valid in a bioanalytical means in vitro. The present study exemplifies the PPI in vitro inside a unique single-chain probe, named TP2.4, which carries a full-length artificial luciferase (ALuc) sandwiched in between two model proteins of interest, e.g., FKBP and FRB, expressed in E. coli, and purified. We found that the TP2.4 efficiently recognizes its ligand in vitro and varies its molecular kinetics: i.e., rapamycin boosts the enzymatic affinities (Km) of TP2.4 to its substrates, but does not or only weakly influences the turnover rates (Kcat) and the maximal velocity (Vmax). The corresponding circular dichroism (CD) study shows that rapamycin weakly contributes to the enhancement of the α-helical contents in TP2.4. Kinetic constants according to the substrates revealed that a coelenterazine derivative, 6-N3-CTZ, exerted the best catalytic efficiency and the greatest variance in the total photon counts. The present study is the first in vitro example that demonstrates how intramolecular PPI works in a purified single-chain bioluminescent probe and what factors practically influence the biochemistry.