Initial hydrophobic collapse is not necessary for folding RNase A

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Protein folding theories differ on the sequence of secondary structure formation and molecular compaction.
• Existing experimental data cannot definitively distinguish between secondary structure preceding compaction or hydrophobic collapse driving secondary structure formation.

Purpose of the Study:

• To investigate the refolding process of chemically denatured ribonuclease A (RNase A).
• To monitor the kinetics of secondary structure formation and molecular compaction during RNase A refolding.

Main Methods:

• Utilized stopped-flow dynamic light scattering to monitor protein compaction.
• Employed stopped-flow circular dichroism (CD) to track secondary structure formation.
• Studied disulfide-intact ribonuclease A (RNase A) refolding.

Main Results:

• Significant secondary structure formation occurred early in the refolding of the slow-folding species of RNase A.
• This initial secondary structure formation was not accompanied by substantial molecular compaction.
• A subsequent rate-limiting step showed simultaneous secondary structure formation and compaction.

Conclusions:

• The refolding of RNase A does not exhibit initial global hydrophobicity, contradicting a universal requirement for protein folding.
• This observed folding mechanism may be characteristic of moderately hydrophobic proteins.