A Rhizavidin Monomer with Nearly Multimeric Avidin-Like Binding Stability Against Biotin Conjugates

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Developing stable monomeric avidin-like proteins for biotin-avidin linking has been a significant challenge.
• Existing multimeric avidin proteins have limitations in specific applications requiring monovalent interactions.

Purpose of the Study:

• To engineer a monomeric avidin-like protein with high biotin-binding stability.
• To utilize this engineered protein for advanced biological labeling and studying protein clustering effects.

Main Methods:

• Engineered enhanced monoavidin (eMA) from dimeric rhizavidin by preserving protein rigidity and modifying surface residues.
• Developed a 24-meric avidin probe by fusing eMA to a cage protein.
• Applied eMA for monovalent labeling of biotinylated lipids in membranes and studied protein clustering effects on live cells.

Main Results:

• eMA exhibits off-rates comparable to multimeric avidin proteins, demonstrating highly stable biotin binding.
• eMA enabled non-perturbing, monovalent labeling of lipids in bilayer membranes.
• Artificial clustering of cell-surface proteins using eMA and the 24-meric probe significantly enhanced protein internalization rates.

Conclusions:

• Enhanced monoavidin (eMA) represents a breakthrough in creating stable, monomeric avidin-like proteins.
• eMA facilitates precise molecular labeling and the construction of novel multimeric protein probes.
• Protein clustering dramatically influences cellular internalization dynamics, with implications for drug delivery and cell signaling research.