Protein vivisection reveals elusive intermediates in folding

Area of Science:

• Biochemistry
• Structural Biology
• Protein Dynamics

Background:

• Protein folding mechanisms are complex and often involve transient intermediates that are difficult to detect.
• Understanding these folding intermediates is crucial for elucidating the complete protein folding pathway.

Purpose of the Study:

• To develop a novel strategy for populating and characterizing transient protein folding intermediates.
• To investigate the folding pathway of ubiquitin by trapping a specific intermediate.

Main Methods:

• Protein engineering: substitution of a buried aliphatic residue with a charged residue (e.g., Leu to Glu) to destabilize a specific region.
• Biophysical characterization: Nuclear Magnetic Resonance (NMR) spectroscopy and hydrogen exchange methods to analyze the trapped intermediate.
• Reversible trapping: utilizing mildly acidic conditions for charge neutralization and refolding.

Main Results:

• Successfully trapped a partially unfolded intermediate of ubiquitin, with the beta5-strand specifically unfolded.
• The trapped intermediate refolded to a native-like structure upon charge neutralization.
• NMR and hydrogen exchange identified a second folding intermediate and provided insights into the order and free energies of folding events.

Conclusions:

• The developed strategy provides a powerful tool for trapping and studying high-energy protein folding intermediates.
• This method offers broad applicability for investigating protein folding mechanisms and other reactions involving transient states.
• Detailed characterization of intermediates refines our understanding of the protein folding landscape and kinetics.