title: Specifically and wash-free labeling of SNAP-tag fused proteins with a hybrid sensor to monitor local micro-viscosity.
abstract: Viscosity, as one of the major factors of intracellular microenvironment, influences the function of proteins. To detect local micro-viscosity of a protein, it is a precondition to apply a viscosity sensor for specifically target to proteins. However, all the reported small-molecule probes are just suitable for sensing/imaging of macro-viscosity in biological fluids of entire cells or organelles. To this end, we developed a hybrid sensor BDP-V BG by connecting a viscosity-sensitive boron-dipyrromethene (BODIPY) molecular rotor (BDP-V) to O6-benzylguanine (BG) for specific detection of local micro-viscosity of SNAP-tag fused proteins. We measured and calculated the reaction efficiency between the sensor and SNAP-tag protein in vitro to confirm the high labeling specificity. We also found that the labeling reaction results in a 53-fold fluorescence enhancement for the rotor, which qualifies it as a wash-free sensor with ignorable background fluorescence. The high sensitivity of protein labeled sensor (BDP-V-SNAP) to the changes of local viscosity was evaluated by detecting the enhancement of fluorescence lifetimes. Further, with the sensor BDP-V BG, we achieved high specific labeling of cells expressing two SNAP-tag fused proteins (nuclear histone H2B and mitochondrial COX8A). Two-photon excited fluorescence lifetime imaging revealed that, the micro-viscosities nearby the SNAP-tag fused two proteins are distinct. The different changes of local micro-viscosity of SNAP-tag fused histone protein in apoptosis induced by three nucleus-targeted drugs were also characterized for the first time.
authors.affiliation: State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China.
authors.forename: Xinbo
authors.initials: X
authors.lastname: Song
authors.affiliation: State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China.
authors.forename: Lingcheng
authors.initials: L
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China. Electronic address: xiaoyi@dlut.edu.cn.
authors.forename: Yi
authors.initials: Y
authors.lastname: Xiao
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000032:analysis; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D001896:Boron Compounds / Q000737:chemistry*; D019556:COS Cells; D002522:Chlorocebus aethiops; D003576:Electron Transport Complex IV / Q000032:analysis; D005456:Fluorescent Dyes / Q000737:chemistry*; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D006657:Histones / Q000032:analysis; D006801:Humans; D008958:Models, Molecular; D061848:Optical Imaging / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014783:Viscosity
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D006657:Histones; D011993:Recombinant Fusion Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; D003576:Electron Transport Complex IV; D019883:Alkyl and Aryl Transferases; C050956:DNA alkyltransferase
keywords: Fluorescent lifetime; Fluorescent rotor; Histone; Protein labeling; SNAP-tag; Viscosity