TY - JOUR
T1 - In vitro determination of Rapamycin-triggered FKBP-FRB interactions using a molecular tension probe
AU - Kim, Sung Bae
AU - Nishihara, Ryo
AU - Fujii, Rika
AU - Paulmurugan, Ramasamy
AU - Citterio, Daniel
AU - Suzuki, Koji
N1 - Funding Information:
This work was partly supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grants: Numbers 26288088, 15KK0029, 16K14051, 17H01215 and 24225001. We are grateful to Dr. Miller, Dr. Tanabe, and Prof. Senda for providing technical support on the purified TP2.4.
Publisher Copyright:
© The Japan Society for Analytical Chemistry.
PY - 2019
Y1 - 2019
N2 - 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 (K m ) of TP2.4 to its substrates, but does not or only weakly influences the turnover rates (K cat ) and the maximal velocity (V max ). 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-N 3 -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.
AB - 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 (K m ) of TP2.4 to its substrates, but does not or only weakly influences the turnover rates (K cat ) and the maximal velocity (V max ). 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-N 3 -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.
KW - Artificial luciferase
KW - Bioluminescence
KW - Imaging
KW - In vitro assay
KW - Molecular tension probe
KW - Protein-protein interactions
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U2 - 10.2116/analsci.18SDP08
DO - 10.2116/analsci.18SDP08
M3 - Article
C2 - 30504653
AN - SCOPUS:85059798970
SN - 0910-6340
VL - 35
SP - 71
EP - 78
JO - Analytical Sciences
JF - Analytical Sciences
IS - 1
ER -