Slow unfolded-state structuring in Acyl-CoA binding protein folding revealed by simulation and experiment.

Area of Science:

• Biophysics
• Computational Biology
• Molecular Biology

Background:

• Protein folding is crucial for biological function and understanding diseases.
• Discrepancies exist between simple experimental models and complex simulation-based network models of protein folding.
• Acyl-coenzyme A binding protein (ACBP) is a model system for studying protein folding dynamics.

Purpose of the Study:

• To reconcile experimental and simulation-based models of protein folding.
• To investigate the nature of unfolded-state structure and its role in ACBP folding.
• To characterize the kinetics and mechanisms of ACBP folding on microsecond timescales.

Main Methods:

• Single-molecule Förster Resonance Energy Transfer (smFRET) with side-chain mutagenesis.
• Ultrafast laminar-flow mixing experiments for microsecond kinetics.
• Trp-Cys quenching for unfolded-state dynamics.
• Molecular Dynamics (MD) simulations and Markov State Model (MSM) construction.

Main Results:

• The denatured state of ACBP exhibits compact, residual structure sensitive to mutations.
• Unfolded-state structure formation occurs on a slow timescale (~100 μs).
• Mutations impact both equilibrium and time-resolved smFRET measurements similarly.
• MSM predicts complex metastable states but no distinct early folding intermediate.

Conclusions:

• ACBP folding is not a simple two-state process but involves slow acquisition of unfolded-state structure.
• The fast kinetic phase in ACBP folding is attributed to heterogeneous unfolded-state dynamics, not a barrier-limited intermediate.
• This study reconciles experimental observations with complex network models of protein folding.