The prosegment catalyzes pepsin folding to a kinetically trapped native state

Area of Science:

• Biochemistry
• Protein Folding
• Enzymology

Background:

• Irreversible protein unfolding is often attributed to changes in the unfolded state.
• Pepsin, an aspartic peptidase, exhibits irreversible denaturation.
• The role of the native state's intrinsic properties in irreversibility is less explored.

Purpose of the Study:

• To investigate an alternative explanation for pepsin's irreversible denaturation.
• To elucidate the contribution of the native state's stability and folding barriers to irreversibility.
• To understand the role of the prosegment (PS) domain in pepsin folding and stability.

Main Methods:

• Analysis of protein unfolding and refolding pathways.
• Thermodynamic and kinetic characterization of native and refolded states.
• Investigation of the prosegment's influence on folding energetics.

Main Results:

• Native pepsin is thermodynamically metastable with a high folding barrier (24.6 kcal/mol).
• Without the prosegment (PS), pepsin refolds into a stable but inactive state.
• The PS stabilizes the folding transition state, facilitating thermodynamically driven folding.

Conclusions:

• Pepsin's irreversibility stems from a combination of a high folding barrier and a metastable native state.
• The prosegment (PS) is crucial for achieving thermodynamically stable native pepsin folding.
• Kinetic stability, rather than thermodynamic stability, may be a common protein design strategy in aspartic peptidases.