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Specifically and wash-free labeling of SNAP-tag fused proteins with a hybrid sensor to monitor local micro-viscosity
- Source :
- Biosensors and Bioelectronics. 91:313-320
- Publication Year :
- 2017
- Publisher :
- Elsevier BV, 2017.
-
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.
- Subjects :
- Boron Compounds
Models, Molecular
Fluorescence-lifetime imaging microscopy
Guanine
Recombinant Fusion Proteins
Biomedical Engineering
Biophysics
Biosensing Techniques
02 engineering and technology
010402 general chemistry
01 natural sciences
Electron Transport Complex IV
Histones
chemistry.chemical_compound
Chlorocebus aethiops
Organelle
Electrochemistry
Histone H2B
Animals
Humans
Fluorescent Dyes
Alkyl and Aryl Transferases
biology
Viscosity
Optical Imaging
General Medicine
021001 nanoscience & nanotechnology
Fluorescence
In vitro
0104 chemical sciences
SNAP-tag
Histone
Biochemistry
chemistry
COS Cells
biology.protein
BODIPY
0210 nano-technology
Biotechnology
Subjects
Details
- ISSN :
- 09565663
- Volume :
- 91
- Database :
- OpenAIRE
- Journal :
- Biosensors and Bioelectronics
- Accession number :
- edsair.doi.dedup.....3d21889065b115a2569e1d8e3427cfd4