Folding disulfide-containing proteins faster with an aromatic thiol

Area of Science:

• Biochemistry
• Protein Chemistry
• Chemical Biology

Background:

• In vitro protein folding of disulfide-containing proteins is typically slow.
• The traditional method uses a glutathione/glutathione disulfide redox buffer.

Purpose of the Study:

• To enhance the rate of in vitro protein folding.
• To investigate the protein folding process using a novel aromatic thiol.

Main Methods:

• Replaced glutathione with 4-mercaptobenzeneacetate (aromatic thiol 1).
• Investigated folding of reduced and scrambled RNase A at pH 7.0 and 7.7.
• Determined optimal thiol concentrations and measured folding rates concurrently.

Main Results:

• The aromatic thiol 4-mercaptobenzeneacetate increased folding rates 5-6 fold compared to glutathione.
• Folding rates were similar for both reduced and scrambled RNase A.
• The folding rate enhancement showed minimal variation with pH (7.0 vs 7.7).

Conclusions:

• Aromatic thiol 4-mercaptobenzeneacetate significantly accelerates in vitro protein folding.
• This thiol offers a more efficient alternative to traditional redox buffers for protein folding studies.
• The findings provide insights into disulfide bond formation during protein folding.