Engineering mouse apolipoprotein A-I into a monomeric, active protein useful for structural determination

Area of Science:

• Biochemistry
• Structural Biology
• Cardiovascular Research

Background:

• Apolipoprotein AI (apoAI) is a key component of high-density lipoprotein (HDL) and a predictor of coronary artery disease (CAD).
• Lipid-free apoAI naturally forms oligomers, a property potentially linked to its lipoprotein-binding activity, but this link remains unclear.

Purpose of the Study:

• To investigate whether the self-association property of apoAI is essential for its lipoprotein-binding activity.
• To develop a method for engineering oligomeric proteins into active monomers.
• To generate a monomeric apoAI mutant for structural determination.

Main Methods:

• Engineered a monomeric mouse apoAI mutant by replacing six hydrophobic residues with polar or smaller hydrophobic residues.
• Utilized cross-linking, Circular Dichroism (CD), Differential Scanning Calorimetry (DSC), and Nuclear Magnetic Resonance (NMR) to characterize the mutant.
• Performed lipid-binding assays to compare the mutant's activity with wild-type apoAI.

Main Results:

• The engineered apoAI mutant was over 90% monomeric at 8 mg/mL.
• The mutant exhibited identical secondary, tertiary structure, stability, and lipoprotein-binding activity compared to wild-type apoAI.
• Both monomeric mutant and wild-type apoAI formed similar reconstituted HDL (rHDL) particles.
• High-quality NMR data was obtained from the monomeric apoAI, facilitating structural determination.

Conclusions:

• The self-association of apoAI is not required for its biological activity, including lipoprotein binding.
• The engineered monomeric apoAI mutant is biologically active and suitable for high-resolution structural studies.
• This work provides a novel approach for engineering active monomeric forms of oligomeric proteins.