Bifunctional avidin with covalently modifiable ligand binding site.

Area of Science:

• Biochemistry
• Protein Engineering

Background:

• Avidin and streptavidin are widely used in life sciences due to their high affinity for biotin.
• Modifications to (strept)avidin's biotin-binding affinity have been explored to enhance existing applications.
• Despite modifications, (strept)avidin maintains a strong preference for biotin.

Purpose of the Study:

• To engineer the biotin-binding pocket of avidin to introduce a chemically active thiol group.
• To create bifunctional avidin molecules by selectively modifying one binding site.
• To develop novel tools for applications like surface biofunctionalization.

Main Methods:

• Introduced a single point mutation (S16C) in the avidin biotin-binding pocket to create a thiol group.
• Applied this mutation to a bivalent dual chain avidin, modifying one binding site while preserving the other.
• Covalently coupled maleimide to the modified binding site and explored coupling with other maleimide derivatives, including fluorescent labels.

Main Results:

• The introduction of the thiol group and maleimide coupling led to a decrease in biotin affinity.
• Demonstrated the covalent coupling of the thiol group to other maleimide derivatives, such as fluorescent labels.
• Enabled intratetrameric Förster Resonance Energy Transfer (FRET) through coupled fluorescent labels.

Conclusions:

• Engineered avidin with a single point mutation creates a versatile thiol group for covalent coupling.
• Bifunctional avidin molecules with selectively modified binding sites offer improved and novel functionalities.
• These engineered avidins serve as valuable tools for applications including the biofunctionalization of surfaces.