Streptavidins with intersubunit crosslinks have enhanced stability

Area of Science:

• Biochemistry
• Protein Engineering
• Structural Biology

Background:

• Streptavidin's tetrameric structure features a strong dimer interface and a weaker dimer-dimer interface.
• Enhancing structural stability is crucial for streptavidin applications.

Purpose of the Study:

• To investigate if strengthening the weak dimer-dimer interface improves streptavidin's structural stability.
• To assess the impact of intersubunit crosslinking on streptavidin's biotin-binding and stability.

Main Methods:

• Site-directed mutagenesis to introduce cysteine residues at position 127.
• Introduction of disulfide and irreversible covalent bonds across the dimer-dimer interface.
• Creation of a hybrid tetramer with crosslinked subunits.

Main Results:

• Crosslinked streptavidin variants exhibited enhanced biotin-binding after heat treatment.
• Crosslinked streptavidins demonstrated superior retention of bound biotin under harsh conditions (acidic pH, guanidine hydrochloride).

Conclusions:

• Covalent crosslinking across the dimer-dimer interface significantly enhances streptavidin's overall structural stability.
• Modified streptavidin shows improved performance in demanding environments.