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Anal Sci. 2019 Jan 10;35(1):71-78. doi: 10.2116/analsci.18SDP08. Epub 2018 Nov 30.

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

Author information

1
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST).
2
Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine.
3
Department of Applied Chemistry, Faculty of Science and Technology, Keio University.

Abstract

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.

KEYWORDS:

Molecular tension probe; artificial luciferase; bioluminescence; imaging; in vitro assay; protein–protein interactions

PMID:
30504653
DOI:
10.2116/analsci.18SDP08
[Indexed for MEDLINE]
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