An “OFF–ON–OFF” fluorescence protein-labeling probe for real-time visualization of the degradation of short-lived proteins in cellular systems |
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| Authors: | Shahi Imam Reja Yuichiro Hori Takuya Kamikawa Kohei Yamasaki Miyako Nishiura Steven D Bull Kazuya Kikuchi |
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| Institution: | Division of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita Osaka 565-0871 Japan.; Immunology Frontier Research Center, Osaka University, Suita Osaka 565-0871 Japan ; Department of Chemistry, University of Bath, Claverton Down, Bath BA2 7AY UK |
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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.An “OFF–ON–OFF” fluorescence probe for real-time imaging of the expression (fluorescence ‘OFF’) and degradation (fluorescence ‘ON’) of short lived PYP-tag proteins in cellular systems. |
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