Abstract
SummaryChemical 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.
Highlights
Derivatizing proteins allows huge expansion of their functional and translational potential
We discovered that dye modification resulted in a significant impairment to biotin-conjugate binding, which we overcame by structure-based engineering
Biotin-4-fluorescein fluorescence is quenched by 90% upon streptavidin binding
Summary
Derivatizing proteins allows huge expansion of their functional and translational potential. There is a wide literature on the use of NHS-dye conjugates, with some examples where labeling interferes with binding properties and examples of excess dye labeling reducing overall fluorescence (Vira et al, 2010; Zanetti-Domingues et al, 2013). In such systems, amine modification sites have rarely been changed, which would enable precise control of the potential reaction sites and optimization of molecular properties, e.g., ligand-binding kinetics, protein stability, and fluorescent brightness
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