Issue 5, 2022

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

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.

Graphical abstract: An “OFF–ON–OFF” fluorescence protein-labeling probe for real-time visualization of the degradation of short-lived proteins in cellular systems

Supplementary files

Article information

Article type
Edge Article
Submitted
12 11 2021
Accepted
11 1 2022
First published
11 1 2022
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2022,13, 1419-1427

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

S. I. Reja, Y. Hori, T. Kamikawa, K. Yamasaki, M. Nishiura, S. D. Bull and K. Kikuchi, Chem. Sci., 2022, 13, 1419 DOI: 10.1039/D1SC06274C

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