期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 136, 期 44, 页码 15551-15558出版社
AMER CHEMICAL SOC
DOI: 10.1021/ja5055862
关键词
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资金
- National Science Foundation [MCB-1330746, CHE-0848398]
- University of Pittsburgh
- National Institutes of Health [P30CA047904]
- Medical Research Council [U105181009, UD99999908]
- Trinity College
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1330746] Funding Source: National Science Foundation
The site-specific incorporation of three new coumarin lysine analogues into proteins was achieved in bacterial and mammalian cells using an engineered pyrrolysyl-tRNA synthetase system. The genetically encoded coumarin lysines were successfully applied as fluorescent cellular probes for protein localization and for the optical activation of protein function. As a proof-of-principle, photoregulation of firefly luciferase was achieved in live cells by caging a key lysine residue, and excellent OFF to ON light-switching ratios were observed. Furthermore, two-photon and single-photon optochemical control of EGFP maturation was demonstrated, enabling the use of different, potentially orthogonal excitation wavelengths (365, 405, and 760 nm) for the sequential activation of protein function in live cells. These results demonstrate that coumarin lysines are a new and valuable class of optical probes that can be used for the investigation and regulation of protein structure, dynamics, function, and localization in live cells. The small size of coumarin, the site-specific incorporation, the application as both a light-activated caging group and as a fluorescent probe, and the broad range of excitation wavelengths are advantageous over other genetically encoded photocontrol systems and provide a precise and multifunctional tool for cellular biology.
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