The foldon substructure of staphylococcal nuclease

Area of Science:

• Protein dynamics and structural biology
• Biophysics and biochemical kinetics
• Molecular mechanisms of protein folding

Background:

• Proteins fold through intermediate states, but the precise nature of these substructures remains an active area of research.
• Understanding protein folding pathways is crucial for deciphering protein function and dysfunction in disease.
• Staphylococcal nuclease serves as a model system for studying protein folding due to its well-characterized structure.

Purpose of the Study:

• To identify and characterize submolecular folding units (foldons) in staphylococcal nuclease.
• To investigate the kinetic and thermodynamic properties of protein unfolding and refolding.
• To correlate structural units with specific folding and stability parameters.

Main Methods:

• Kinetic native-state hydrogen exchange (HX) mass spectrometry was employed to monitor unfolding.
• HX data provided measurements of equilibrium stability (ΔG(HX)) and kinetic rates (k(op), k(cl)).
• Analysis of HX parameters grouped residues into distinct foldon units based on their unfolding behavior.

Main Results:

• Thirty-four amide hydrogens were identified, exchanging via transient unfolding reactions under native conditions.
• Residues were classified into four distinct foldon groups (blue, red, green, yellow), representing structural units.
• The blue foldon, part of the β-barrel, exhibited the slowest unfolding rate and global protein stability.
• Other foldons (green, yellow, red) showed varying kinetic and thermodynamic properties, with some indicating potential nonnative interactions.

Conclusions:

• Staphylococcal nuclease appears to be composed of integrated foldon units that dictate folding pathways and functional properties.
• The identified foldons provide insights into the hierarchical nature of protein folding.
• Further studies are needed to fully delineate the extent and interactions of these foldon units.