Probing residue-level unfolding during lysozyme precipitation

Area of Science:

• Protein Biochemistry
• Structural Biology
• Biophysical Chemistry

Background:

• Hen egg white lysozyme (HEWL) is a model protein extensively studied for its structural properties.
• Salt-induced precipitation is a common technique in protein purification and manipulation.
• Understanding protein conformational changes during precipitation is crucial for maintaining structural integrity.

Purpose of the Study:

• To investigate residue-level conformational changes in HEWL induced by different salt precipitation conditions.
• To compare the effects of potassium thiocyanate (KSCN) and ammonium sulfate ((ND4)2SO4) on HEWL structure.
• To evaluate the suitability of these precipitants for preserving protein structural integrity.

Main Methods:

• Nuclear magnetic resonance (NMR) spectroscopy utilizing hydrogen exchange measurements.
• Deuteration of HEWL in phosphate buffer.
• Controlled precipitation at pH 2.1 followed by redissolution at pH 3.8.
• Purification and subsequent NMR analysis of refolded HEWL.

Main Results:

• Potassium thiocyanate (KSCN) at 1.0 M induced significant hydrogen exchange in 14 residues, primarily in the beta-sheet/loop domain of HEWL.
• Lower KSCN concentrations (0.2 M) showed a less pronounced effect.
• Ammonium sulfate ((ND4)2SO4) at 1.2 M did not induce detectable conformational changes, preserving HEWL's structural integrity.
• The core of HEWL, including alpha-helices, remained intact even at high KSCN concentrations.

Conclusions:

• Potassium thiocyanate (KSCN) causes partial unfolding in HEWL, likely due to preferential interactions, disrupting the beta-sheet/loop domain.
• Ammonium sulfate ((ND4)2SO4) is a preferred precipitant for maintaining the structural integrity of HEWL.
• The intact alpha-helical core may aid in the dissolution of KSCN-precipitated HEWL.