An "oFF-ON-OFF" fluorescence protein-labeling probe for real-time visualization of the degradation of short-lived proteins in cellular systems

Shahi Imam Reja; Yuichiro Hori; Takuya Kamikawa; Kohei Yamasaki; Miyako Nishiura; Steven D. Bull; Kazuya Kikuchi

doi:10.1039/d1sc06274c

An "oFF-ON-OFF" fluorescence protein-labeling probe for real-time visualization of the degradation of short-lived proteins in cellular systems

Shahi Imam Reja, Yuichiro Hori, Takuya Kamikawa, Kohei Yamasaki, Miyako Nishiura, Steven D. Bull, Kazuya Kikuchi

Department of Chemistry

Osaka University

Research output: Contribution to journal › Article › peer-review

10 Citations (SciVal)

Abstract

The ability to monitor proteolytic pathways that remove unwanted and damaged proteins from cells is essential for understanding the multiple processes used to maintain cellular homeostasis. In this study, we have developed a new protein-labeling probe that employs an 'OFF-ON-OFF' fluorescence switch to enable real-time imaging of the expression (fluorescence ON) and degradation (fluorescence OFF) of PYP-tagged protein constructs in living cells. Fluorescence switching is modulated by intramolecular contact quenching interactions in the unbound probe (fluorescence OFF) being disrupted upon binding to the PYP-tag protein, which turns fluorescence ON. Quenching is then restored when the PYP-tag-probe complex undergoes proteolytic degradation, which results in fluorescence being turned OFF. Optimization of probe structures and PYP-tag mutants has enabled this fast reacting 'OFF-ON-OFF' probe to be used to fluorescently image the expression and degradation of short-lived proteins.

Original language	English
Pages (from-to)	1419-1427
Number of pages	9
Journal	Chemical Science
Volume	13
Issue number	5
Early online date	11 Jan 2022
DOIs	https://doi.org/10.1039/d1sc06274c
Publication status	Published - 7 Feb 2022

Bibliographical note

Funding Information:
This research was supported by the JSPS KAKENHI (Grant Numbers: JP17H06409 “Frontier Research on Chemical Communications”, JP18H03935 and 19K22255 to K. K.; and JP17H02210, JP18K19402, and JP20H02879 to Y. H.), JSPS A3 Foresight Program, JSPS Asian CORE Program, “Asian Chemical Biology Initiative”, Japan (JSPS)–UK (RSC) Research Cooperative Program (JPJSBP120195705 to K. K.), AMED-CREST, and Toray Science Foundation (19-6008). S. I. R. would like to thank JSPS (P18116) and TBRF (TBRF-RF-123) for postdoctoral fellowship. S. D. B. would like to thank the Royal Society for an International Exchange Grant (IEC\R3\183068).

ASJC Scopus subject areas

General Chemistry

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abstract = "The ability to monitor proteolytic pathways that remove unwanted and damaged proteins from cells is essential for understanding the multiple processes used to maintain cellular homeostasis. In this study, we have developed a new protein-labeling probe that employs an 'OFF-ON-OFF' fluorescence switch to enable real-time imaging of the expression (fluorescence ON) and degradation (fluorescence OFF) of PYP-tagged protein constructs in living cells. Fluorescence switching is modulated by intramolecular contact quenching interactions in the unbound probe (fluorescence OFF) being disrupted upon binding to the PYP-tag protein, which turns fluorescence ON. Quenching is then restored when the PYP-tag-probe complex undergoes proteolytic degradation, which results in fluorescence being turned OFF. Optimization of probe structures and PYP-tag mutants has enabled this fast reacting 'OFF-ON-OFF' probe to be used to fluorescently image the expression and degradation of short-lived proteins. ",

author = "Reja, {Shahi Imam} and Yuichiro Hori and Takuya Kamikawa and Kohei Yamasaki and Miyako Nishiura and Bull, {Steven D.} and Kazuya Kikuchi",

note = "Funding Information: This research was supported by the JSPS KAKENHI (Grant Numbers: JP17H06409 “Frontier Research on Chemical Communications”, JP18H03935 and 19K22255 to K. K.; and JP17H02210, JP18K19402, and JP20H02879 to Y. H.), JSPS A3 Foresight Program, JSPS Asian CORE Program, “Asian Chemical Biology Initiative”, Japan (JSPS)–UK (RSC) Research Cooperative Program (JPJSBP120195705 to K. K.), AMED-CREST, and Toray Science Foundation (19-6008). S. I. R. would like to thank JSPS (P18116) and TBRF (TBRF-RF-123) for postdoctoral fellowship. S. D. B. would like to thank the Royal Society for an International Exchange Grant (IEC\R3\183068). ",

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AU - Bull, Steven D.

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An "oFF-ON-OFF" fluorescence protein-labeling probe for real-time visualization of the degradation of short-lived proteins in cellular systems

Abstract

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