Lysozyme/Alginate Interactions: Structural and Thermodynamic Insights through ITC and SAXS

Area of Science:

• Biomolecular interactions
• Phase separation phenomena
• Protein-polysaccharide complexation

Background:

• Coacervation and aggregation are distinct phase separation processes.
• Physicochemical conditions like pH and ionic strength influence these phenomena.
• Lysozyme (LYS)-alginate (ALG) mixtures serve as a model system to study these interactions.

Purpose of the Study:

• To investigate the role of ionic strength in determining liquid-liquid phase separation (LLPS) versus liquid-solid phase separation (LSPS) in LYS-ALG mixtures.
• To characterize the structural and energetic differences between LSPS and LLPS under varying salt concentrations.
• To elucidate the electrostatic driving forces behind LYS-ALG phase behavior.

Main Methods:

• Isothermal Titration Calorimetry (ITC) to measure binding thermodynamics.
• Small-Angle X-ray Scattering (SAXS) to analyze structural properties.
• Systematic variation of sodium chloride (NaCl) concentration at fixed pH (7).

Main Results:

• Low salt (0-50 mM NaCl) induced compact fractal aggregates (LSPS).
• Intermediate salt (100-150 mM NaCl) resulted in coacervates with swollen globules (LLPS).
• High salt (200 mM NaCl) led to soluble complexes, inhibiting phase separation.
• Binding strength decreased significantly with increasing salt concentration, correlating with structural changes.

Conclusions:

• Ionic strength is a critical tunable parameter controlling LYS-ALG phase behavior.
• LSPS is associated with stronger electrostatic interactions than LLPS.
• Distinct structural and energetic signatures differentiate LSPS and LLPS, providing insights into biomolecular complex formation.