Multiple parallel-pathway folding of proline-free Staphylococcal nuclease

Area of Science:

• Biochemistry and Molecular Biology
• Protein Folding Dynamics

Background:

• Complex protein refolding kinetics are often attributed to slow cis/trans isomerization of peptidyl prolyl bonds in the denatured state.
• The extent to which other factors contribute to complex folding pathways remains an open question in protein science.

Purpose of the Study:

• To investigate the folding kinetics of a proline-free variant of staphylococcal nuclease to isolate folding mechanisms independent of prolyl isomerization.
• To determine if complex folding kinetics can arise from factors other than prolyl isomerization.

Main Methods:

• Utilized stopped-flow and double-jump techniques to monitor the refolding process of the proline-free staphylococcal nuclease variant.
• Analyzed the kinetic data to identify the number and nature of the folding pathways involved.

Main Results:

• The proline-free staphylococcal nuclease refolds into its native state via at least two distinct, accessible parallel pathways.
• The folding process exhibits multi-exponential kinetics with a lag phase, indicative of intermediates on individual pathways.
• These findings demonstrate that a single amino acid sequence can adopt multiple parallel folding pathways.

Conclusions:

• Complex protein folding kinetics are not solely due to prolyl isomerization and can arise from multiple parallel pathways.
• The observed multiple parallel-pathway folding in staphylococcal nuclease suggests this phenomenon may be common in other proteins, even those with simpler observed kinetics.