In vitro determination of Rapamycin-triggered FKBP-FRB interactions using a molecular tension probe

Sung Bae Kim, Ryo Nishihara, Rika Fujii, Ramasamy Paulmurugan, Daniel Citterio, Koji Suzuki

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)71-78
Number of pages8
JournalAnalytical Sciences
Volume35
Issue number1
DOIs
Publication statusPublished - 2019 Jan 1

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Tacrolimus Binding Proteins
Sirolimus
Proteins
Biochemistry
Kinetics
Substrates
Luciferases
Escherichia coli
Photons
Ligands
Derivatives
Imaging techniques

Keywords

  • Artificial luciferase
  • Bioluminescence
  • Imaging
  • In vitro assay
  • Molecular tension probe
  • Protein-protein interactions

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

In vitro determination of Rapamycin-triggered FKBP-FRB interactions using a molecular tension probe. / Kim, Sung Bae; Nishihara, Ryo; Fujii, Rika; Paulmurugan, Ramasamy; Citterio, Daniel; Suzuki, Koji.

In: Analytical Sciences, Vol. 35, No. 1, 01.01.2019, p. 71-78.

Research output: Contribution to journalArticle

Kim, Sung Bae ; Nishihara, Ryo ; Fujii, Rika ; Paulmurugan, Ramasamy ; Citterio, Daniel ; Suzuki, Koji. / In vitro determination of Rapamycin-triggered FKBP-FRB interactions using a molecular tension probe. In: Analytical Sciences. 2019 ; Vol. 35, No. 1. pp. 71-78.
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