Solution structure of an engineered insulin monomer at neutral pH

Area of Science:

• Biochemistry
• Structural Biology
• Protein Chemistry

Background:

• Insulin, a crucial hormone, circulates as a monomer but readily self-associates into dimers and higher oligomers.
• This oligomerization significantly hinders the study of monomeric insulin structure and dynamics in solution.
• Understanding monomeric insulin structure is vital for its biological function and therapeutic applications.

Purpose of the Study:

• To create a stable, monomeric insulin mutant suitable for structural determination at neutral pH.
• To elucidate the solution structure of monomeric insulin using advanced spectroscopic techniques.

Main Methods:

• Site-directed mutagenesis was employed to modify dimer- and hexamer-forming surfaces of insulin.
• Nuclear magnetic resonance (NMR) spectroscopy and near-UV circular dichroism (CD) were used to assess monomeric state and structural features.
• Structure calculations were performed using NOE-derived distance restraints and torsion restraints.

Main Results:

• A novel insulin mutant, (B1, B10, B16, B27) Glu, des-B30 insulin, was generated, retaining 47% biological potency.
• This mutant remained monomeric at millimolar concentrations in aqueous solution at neutral pH (6.5-7.5).
• NMR analysis provided residue assignments and identified NOE cross-peaks, enabling detailed structural calculations.

Conclusions:

• The engineered insulin mutant is the first to allow for detailed structure determination in solution at neutral pH.
• The calculated solution structure reveals distinct helical and loop regions in the A- and B-chains, with some disordered segments.
• The findings provide critical insights into the structural basis of monomeric insulin function and self-association behavior.