title: Selective and ratiometric fluorescent trapping and quantification of protein vicinal dithiols and in situ dynamic tracing in living cells.
abstract: Protein vicinal dithiols play fundamental roles in intracellular redox homeostasis due to their involvement in protein synthesis and function through the reversible vicinal dithiol oxidation to disulfide. To provide quantitative information about the global distribution and dynamic changes of protein vicinal dithiols in living cells, we have designed and synthesized a ratiometric fluorescent probe (VTAF) for trapping of vicinal dithiol-containing proteins (VDPs) in living cells. VTAF exhibits a ratiometric fluorescence signal upon single excitation, which enables self-calibration of the fluorescence signal and quantification of endogenous vicinal dithiols of VDPs. Its potential for in situ dynamic tracing of changes of protein vicinal dithiols under different cellular redox conditions was exemplified. VTAF facilitated the direct observation of subcellular distribution of endogenous VDPs via ratiometric fluorescence imaging and colocalization assay. And the results suggested that there are abundant VDPs in mitochondria. Moreover, some redox-sensitive VDPs are also present on cell surface which can respond to redox stimulus. This ratiometric fluorescence technique presents an important extension to previous fluorescence intensity-based probes for trapping and quantifying protein vicinal dithiols in living cells, as well as its visible dynamic tracing of VDPs.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology , 130 Meilong Road, Shanghai 200237, China.
authors.forename: Chusen
authors.initials: C
authors.lastname: Huang
authors.forename: Ti
authors.initials: T
authors.lastname: Jia
authors.forename: Mengfang
authors.initials: M
authors.lastname: Tang
authors.forename: Qin
authors.initials: Q
authors.lastname: Yin
authors.forename: Weiping
authors.initials: W
authors.lastname: Zhu
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.forename: Nengqin
authors.initials: N
authors.lastname: Jia
authors.forename: Yufang
authors.initials: Y
authors.lastname: Xu
authors.forename: Xuhong
authors.initials: X
authors.lastname: Qian
mesh_terms: D000818:Animals; D002417:Cattle; D002470:Cell Survival; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D061986:MCF-7 Cells; D008928:Mitochondria / Q000378:metabolism; D057054:Molecular Imaging; D055620:Optical Phenomena; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D014050:Toluene / Q000031:analogs & derivatives*
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D014050:Toluene; C004848:dithiol