Probing the non-native H helix translocation in apomyoglobin folding intermediates

Area of Science:

• Protein folding dynamics
• Biophysical characterization of protein structure

Background:

• Apomyoglobin folding proceeds through intermediate states.
• Non-native packing of G and H helices may impede folding.
• Understanding these intermediates is crucial for protein folding research.

Purpose of the Study:

• To investigate the role of H helix translocation in apomyoglobin folding intermediates.
• To provide direct evidence for H helix movement using fluorescence spectroscopy.
• To explore the impact of engineered disulfide bonds on folding pathways.

Main Methods:

• Site-directed mutagenesis to introduce cysteine residues.
• Fluorescence spectroscopy to monitor W14 fluorescence quenching.
• Förster resonance energy transfer (FRET) measurements with a naphthalene sulfonate acceptor.
• Disulfide bond formation (S108C-L135C) to constrain helix movement.

Main Results:

• Differential quenching of W14 fluorescence confirmed H helix translocation in kinetic and equilibrium intermediates.
• FRET measurements indicated H helix sliding by approximately one helical turn in the equilibrium molten globule.
• An engineered disulfide bond (S108C-L135C) prevented H helix translocation and resolved local energetic frustration.
• Disulfide bond formation had minimal impact on the overall refolding rate.

Conclusions:

• Apomyoglobin folding landscape is rugged with multiple bottlenecks.
• H helix translocation is a key event in folding intermediates.
• Relieving individual bottlenecks is insufficient to significantly accelerate folding to the transition state.