Structural determinants of oxidative folding in proteins.

Area of Science:

• Biochemistry
• Protein Folding
• Biophysical Chemistry

Background:

• Disulfide bonds are crucial for protein structure and function.
• Understanding the oxidative folding pathways of proteins with multiple disulfide bonds is complex.
• Distinguishing productive intermediates from dead-end species is essential for elucidating folding mechanisms.

Purpose of the Study:

• To introduce a novel method for determining the kinetic fate of disulfide species during oxidative folding.
• To differentiate between on-pathway intermediates and dead-end species.
• To investigate the oxidative folding pathway of bovine pancreatic ribonuclease A (RNase A).

Main Methods:

• Utilizing the differential sensitivity of structured versus unstructured disulfide species to low concentrations of reducing agents like dithiothreitol (DTT).
• Applying a small concentration of reduced DTT (260 microM) to distinguish between productive and dead-end intermediates.
• Analyzing the accessibility of thiol groups and disulfide bonds in various disulfide species.

Main Results:

• Identified des[40-95] and des[65-72] as productive intermediates in RNase A folding.
• Characterized des[26-84] and des[58-110] as metastable dead-end species that preferentially reshuffle.
• Demonstrated that the accessibility of thiol groups and disulfide bonds dictates the kinetic fate of disulfide species.
• Proposed four generic types of oxidative folding pathways based on disulfide-secure and disulfide-insecure intermediates.

Conclusions:

• The developed method effectively distinguishes productive intermediates from dead-end species.
• Productive intermediates are disulfide-secure, while dead-end species are disulfide-insecure.
• A general three-stage model for oxidative folding of RNase A and similar proteins is suggested.