Structural Transitions of Papain-like Cysteine Proteases: Implications for Sensor Development

Area of Science:

• Biochemistry
• Biotechnology
• Enzymology

Background:

• Papain-like cysteine proteases are crucial in biomedicine and biotechnology.
• Their free thiol group is key for sensor development and inhibitor binding.
• Preserving native protein structure is vital for inhibitor interactions and molecular imprinting.

Purpose of the Study:

• To investigate the impact of reversible thiol group blocking on papain denaturation.
• To assess how blocking affects papain's activity loss and aggregation.
• To understand the reversibility of denaturation and activity upon inhibitor removal.

Main Methods:

• Utilized S-Methyl methanethiosulfonate (MMTS) to reversibly block the free thiol group of papain.
• Employed biophysical techniques: fluorimetry and high-resolution infrared spectroscopy.
• Quantified changes in protein structure, including secondary structure content and aggregation.

Main Results:

• MMTS binding increased hydrophobic residue exposure, indicated by enhanced 8-Anilinonaphthalene-1-sulfonic acid fluorescence.
• Significant secondary structure changes observed: decreased alpha-helices and unordered structures, increased beta-sheets (25% to 52%).
• Reversible inhibition correlated with reversible denaturation, but high inhibitor excess caused irreversible activity loss.

Conclusions:

• Reversible thiol blocking in papain leads to denaturation and aggregation, which can be reversed upon inhibitor removal.
• High concentrations of blocking agents can cause irreversible denaturation, impacting enzyme activity.
• The stable, beta-sheet-rich domain of papain-like proteases offers potential for foldamer-based sensor development.