Deswapping bovine odorant binding protein

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• Bovine Odorant Binding Protein (bOBP) naturally forms a domain-swapped dimer.
• Unlike other mammalian OBPs, bOBP lacks cysteines and disulfide bridges, contributing to its dimeric state.

Purpose of the Study:

• To engineer a monomeric form of bOBP.
• To stabilize the monomeric protein using a disulfide bridge.
• To investigate the ligand-binding properties of the engineered monomeric bOBP.

Main Methods:

• Site-directed mutagenesis was used to introduce a glycine insertion (bOBP-121Gly+) to disrupt domain swapping.
• Further mutations (Trp64Cys and His155Cys) were introduced to create a stabilizing disulfide bridge.
• X-ray crystallography was employed to determine the structure of the triple mutant at 1.65 Å resolution.

Main Results:

• A monomeric triple mutant bOBP protein was successfully produced and stabilized by a disulfide bridge between Cys64 and Cys155.
• The overall backbone structure of the mutant was similar to native bOBP, with minor variations at the hinge region and C-terminus.
• The monomeric mutant exhibited comparable microM Kd values for binding ligands such as 1-amino-anthracene and 1-octen-3-ol as the native dimeric form.

Conclusions:

• Engineering a disulfide bridge can effectively stabilize a monomeric Odorant Binding Protein.
• The monomeric bOBP mutant retains significant ligand-binding affinity, suggesting structural plasticity.
• This work provides a novel tool for studying OBP structure-function relationships and ligand interactions.