Insight into human insulin aggregation revisited using NMR derived translational diffusion parameters

Area of Science:

• Biochemistry
• Biophysics
• Pharmaceutical Science

Background:

• Human insulin aggregation is crucial for its pharmaceutical formulation and efficacy.
• Understanding insulin's oligomeric states in solution is key to controlling its behavior.
• Previous studies utilized various physicochemical methods, yielding diverse results.

Purpose of the Study:

• To investigate the aggregation behavior of human insulin using NMR-derived diffusion coefficients.
• To characterize the oligomeric ensemble of insulin under different conditions (pH, zinc ions, formulation).
• To provide a unified understanding of insulin misfolding and aggregation.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy to measure translational diffusion coefficients.
• Diffusion Ordered Spectroscopy (DOSY) experiments, including 3D HSQC-iDOSY, on 13C,15N-labeled insulin.
• Analysis using the Direct Exponential Curve Resolution Algorithm (DECRA).

Main Results:

• pH and concentration were identified as dominant factors in insulin aggregation.
• Two main components were resolved within the insulin oligomeric ensemble.
• 3D HSQC-iDOSY experiments provided detailed observation of individual components at different pH values (4 and 7.5).

Conclusions:

• NMR diffusion measurements offer valuable insights into insulin's aggregation state.
• The findings are crucial for optimizing pharmaceutical formulations and understanding insulin misfolding.
• Zinc ions play a significant role in insulin's behavior in solution.