Molecular tension-indexed bioluminescent probe for determining protein-protein interactions

Bioconjugate Chemistry
Sung Bae KimHiroaki Tao

Abstract

This study demonstrates a unique, nontranscriptional assay system based on molecular tension of a luciferase artificially appended by protein-protein binding. We hypothesized that an artificially appended molecular tension to a full-length luciferase may diversify the enzymatic activity through a modification of the active site. For the basic probe design, a full-length luciferase was sandwiched between two component proteins of interest. The length of flexible linkers between the components was minimized to exert an efficient molecular tension to the sandwiched luciferase. When N- and C-terminal ends of Renilla luciferase 8 were flanked by the ligand-binding domain of human estrogen receptor alpha (ER LBD) and SH2 domain of Src, named ERS, this simple probe was surprisingly sensitive to estrogens. The luminescence spectra by ERS were largely enhanced by an addition of 4-hydroxytamoxifen (OHT), 17beta-estradiol, and genistein. The detection limit of ERS reached 1 nM OHT. Quantum yield (QY) and Michaelis-Menten constant of ERS were found to be 6.3% and 94.3 muM, respectively. The enzymatic activities of ERS are also governed by different types of coelenterazine (CTZ) variants. The two hydroxy groups in CTZ are critical for the e...Continue Reading

References

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Apr 25, 2009·Analytical Chemistry·Sung Bae KimHiroaki Tao

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Citations

Apr 20, 2012·Protein Engineering, Design & Selection : PEDS·Sung Bae Kim
Apr 23, 2013·Chemical & Pharmaceutical Bulletin·Sung Bae KimAkira Kimura
Dec 14, 2012·International Journal of Molecular Sciences·Sung Bae KimTakeaki Ozawa
Jun 6, 2013·International Journal of Endocrinology·Gwenneg KerdivelFarzad Pakdel
May 26, 2016·MethodsX·Sung Bae Kim, Rika Fujii
Nov 2, 2012·Bioconjugate Chemistry·Sung Bae KimMasaki Torimura
Apr 21, 2017·Scientific Reports·Ryo NishiharaSung Bae Kim
Sep 24, 2020·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Sung-Bae Kim, Ramasamy Paulmurugan

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