Distinguishing between two-state and three-state models for ubiquitin folding

Area of Science:

• Protein folding kinetics
• Biophysics
• Molecular biology

Background:

• The folding mechanism of mammalian ubiquitin remains debated, with conflicting evidence for two-state versus three-state kinetic models.
• Understanding ubiquitin folding is crucial for comprehending protein dynamics and cellular function.

Purpose of the Study:

• To definitively determine the kinetic folding pathway of mammalian ubiquitin at 25°C.
• To investigate the influence of denaturant concentration on folding rates.
• To assess the impact of stabilizing additives on the folding mechanism.

Main Methods:

• Utilized conventional rapid mixing techniques, including single-jump, double-jump, and continuous-flow modes.
• Measured protein folding rate constants up to approximately 1000 s⁻¹.
• Analyzed fluorescence signal changes to detect kinetic intermediates.

Main Results:

• Demonstrated a linear dependence of folding rates on denaturant concentration.
• Observed no unaccounted "burst-phase" fluorescence signal changes, indicative of a lack of kinetic intermediates.
• Confirmed two-state folding behavior in the presence of stabilizing additives like sodium sulfate and sodium chloride.

Conclusions:

• The folding of mammalian ubiquitin at 25°C is adequately described by a simple two-state kinetic model.
• Deviations from ideal two-state "chevron" behavior require careful interpretation, especially with heterogeneous folding or near detection limits.
• These findings provide clarity on ubiquitin's folding pathway and highlight methodological considerations.