title: Phosphorylation regulates proteolytic efficiency of TEV protease detected by a 5(6)-carboxyfluorescein-pyrene based fluorescent sensor.
abstract: TEV protease is of great importance for in vitro and in vivo site-specific cleavage of proteins. The proteolytic efficiency of TEV protease is often regulated by mutation of the substrate, which is irreversible and hard to be modulated. Herein, a facile and reversible method, based on phosphorylation in the substrate, is developed to regulate the cleavage capability of TEV protease. Phosphorylation at P3 tyrosine hinders the recognition of TEV protease to the substrate by using a robust fluorescent protease sensor. Moreover, the phosphate group can be easily removed by alkaline phosphatases for recovering the proteolytic efficiency of TEV protease. Additionally, 5(6)-carboxyfluorescein and pyrene have been used as high-efficiency mutual fluorophore-quencher pair in the peptide-based protease sensor for the first time, which provides a chance to simultaneously monitor the cleavage process in two respective fluorescence channels. Further studies indicated both dynamic and static components contributing to the mutual quenching system. The phosphorylation-regulated TEV protease proteolysis system can be used in conditional cleavage of protein or peptide tag.
authors.affiliation: Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China.
authors.forename: Yao-Hui
authors.initials: YH
authors.lastname: He
authors.affiliation: Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China.
authors.forename: Yan-Mei
authors.initials: YM
authors.lastname: Li
authors.affiliation: Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China. Electronic address: chen-yx@mail.tsinghua.edu.cn.
authors.forename: Yong-Xiang
authors.initials: YX
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D010450:Endopeptidases / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D059748:Proteolysis; D011721:Pyrenes / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D010455:Peptides; D011721:Pyrenes; C024098:6-carboxyfluorescein; C030984:pyrene; D010450:Endopeptidases; C099488:TEV protease
keywords: Mutual quenching; Phosphorylation; Pyrene; TEV protease