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Chemical modification of proteins provides great opportunities to control and visualize living systems. The most common way to modify proteins is reaction of their abundant amines with N-hydroxysuccinimide (NHS) esters. Here we explore the impact of amine number and positioning on protein-conjugate behavior using streptavidin-biotin, a central research tool. Dye-NHS modification of streptavidin severely damaged ligand binding, necessitating development of a new streptavidin-retaining ultrastable binding after labeling. Exploring the ideal level of dye modification, we engineered a panel bearing 1-6 amines per subunit: "amine landscaping." Surprisingly, brightness increased as amine number decreased, revealing extensive quenching following conventional labeling. We ultimately selected Flavidin (fluorophore-friendly streptavidin), combining ultrastable ligand binding with increased brightness after conjugation. Flavidin enhanced fluorescent imaging, allowing more sensitive and specific cell labeling in tissues. Flavidin should have wide application in molecular detection, providing a general insight into how to optimize simultaneously the behavior of the biomolecule and the chemical probe.

Original publication




Journal article


Cell Chem Biol

Publication Date





1040 - 1047.e4


avidin, bioconjugation, flow cytometry, fluorescent probes, histochemistry, microscopy, photophysics, protein engineering, protein labeling, Amines, Antibodies, Biotin, CD3 Complex, Flow Cytometry, Fluorescent Dyes, HeLa Cells, Humans, Ligands, Microscopy, Confocal, Mutagenesis, Site-Directed, Protein Binding, Protein Stability, Spectrometry, Fluorescence, Streptavidin, Succinimides, Temperature