Swine pepsinogen folding intermediates are highly structured, motile molecules

Area of Science:

• Biochemistry
• Protein folding
• Structural biology

Background:

• Protein refolding from denatured states is crucial for biological function.
• Swine pepsinogen unfolds in urea or alkali, refolding through intermediate states.
• Proline isomerization is a known rate-limiting step in some protein folding pathways.

Purpose of the Study:

• To investigate the kinetics and structural properties of swine pepsinogen refolding intermediates.
• To elucidate the role of proline isomerization in the slow phase of pepsinogen refolding.
• To compare the structural characteristics of intermediate (Is) and native (N) swine pepsinogen.

Main Methods:

• Kinetic studies using absorbance and circular dichroism (CD) spectroscopy.
• Fluorescence spectroscopy to probe protein structure and solvent accessibility.
• Hydrogen exchange measurements to assess structural stability.
• Fluorescent probe binding assays to analyze surface hydrophobicity.

Main Results:

• Refolding proceeds via rapid formation of partially folded intermediates (Is) and a slow conversion to native protein (N).
• The slow refolding step is attributed to proline residue isomerization.
• Intermediates (Is) exhibit structural similarity to native protein (N) but are more open to solvent and have a more hydrophobic surface.
• Is are identified as more dynamic and fluctuating species compared to the stable native form.

Conclusions:

• Incorrect proline configurations in intermediates do not halt pepsinogen folding but prevent the formation of a stable, tightly packed native structure.
• Partially folded intermediates are structurally similar on average to the native state but possess greater flexibility.
• Proline isomerization is critical for achieving the final, cooperative, and stable native conformation of swine pepsinogen.