1. Structure-Based Rational Design of Two Enhanced Bacterial Lipocalin Blc Tags for Protein-PAINT Super-resolution Microscopy.
- Author
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Muslinkina L, Gavrikov AS, Bozhanova NG, Mishin AS, Baranov MS, Meiler J, Pletneva NV, Pletnev VZ, and Pletnev S
- Subjects
- Amino Acid Sequence, Boron Compounds chemistry, Fluorescence, Fluorescent Dyes chemistry, HEK293 Cells, HeLa Cells, Humans, Lipocalins genetics, Microscopy, Fluorescence methods, Mutation, Protein Binding, Boron Compounds metabolism, Fluorescent Dyes metabolism, Keratins metabolism, Lipocalins metabolism, Vimentin metabolism
- Abstract
Super-resolution fluorescent imaging in living cells remains technically challenging, largely due to the photodecomposition of fluorescent tags. The recently suggested protein-PAINT is the only super-resolution technique available for prolonged imaging of proteins in living cells. It is realized with complexes of fluorogen-activating proteins, expressed as fusions, and solvatochromic synthetic dyes. Once photobleached, the dye in the complex is replaced with a fresh fluorogen available in the sample. With suitable kinetics, this replacement creates fluorescence blinking required for attaining super-resolution and overcomes photobleaching associated with the loss of an irreplaceable fluorophore. Here we report on the rational design of two protein-PAINT tags based on the 1.58 Å crystal structure of the DiB1:M739 complex, an improved green-emitting DiB3/F74V:M739 and a new orange-emitting DiB3/F53L:M739. They outperform previously reported DiB-based tags to become best in class biomarkers for protein-PAINT. The new tags advance protein-PAINT from the proof-of-concept to a reliable tool suitable for prolonged super-resolution imaging of intracellular proteins in fixed and living cells and two-color PAINT-like nanoscopy with a single fluorogen.
- Published
- 2020
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