The folding pathway of reduced lysozyme

Area of Science:

• Biochemistry
• Protein Folding
• Enzymology

Background:

• Hen egg lysozyme is a well-studied enzyme.
• Understanding protein folding mechanisms is crucial for biochemistry.
• Glutathione regeneration is key to disulfide bond formation in proteins.

Purpose of the Study:

• To elucidate the mechanism of glutathione regeneration in hen egg lysozyme.
• To identify the sequence of disulfide bond formation during lysozyme folding.
• To determine if lysozyme folding follows an all-or-none mechanism.

Main Methods:

• Investigated the kinetics of disulfide bond formation during lysozyme regeneration.
• Isolated and identified early disulfide-containing peptides.
• Analyzed S-alkylated derivatives of reduced cysteinyl residues.

Main Results:

• The first two disulfide bonds form significantly faster than the subsequent two.
• Enzymic activity is regained only after the initial disulfide bonds are formed.
• Early disulfide bonds involve cysteinyl residues III, IV, V, and VI.
• Cysteinyl residues I, II, VII, and VIII remain reduced in early folding intermediates.
• A peptide with a disulfide bond between cysteinyl residues II and VII was identified.

Conclusions:

• Lysozyme folding proceeds through a limited search of intermediates, not an all-or-none pathway.
• The formation of specific early disulfide bonds is essential for enzymatic activity.
• A potential folding pathway involving sequential disulfide bond formation is proposed.