Protein secondary structure determines the temporal relationship between folding and disulfide formation

Area of Science:

• Cell Biology
• Protein Folding
• Biochemistry

Background:

• The timing of disulfide bond formation relative to protein folding is crucial for cellular function.
• Two models, the folded precursor and quasi-stochastic models, propose different mechanisms for disulfide bond formation.
• Understanding these mechanisms in a cellular context is essential for comprehending protein maturation.

Purpose of the Study:

• To investigate the mechanisms of oxidative folding for diverse protein substrates in a cellular context.
• To determine the timing of disulfide bond formation relative to protein folding stages and endoplasmic reticulum translocation.
• To identify factors influencing native versus non-native disulfide bond formation.

Main Methods:

• Utilized a eukaryotic cell-free translation system to study oxidative folding.
• Analyzed disulfide isomers in stalled translation intermediates.
• Examined three structurally diverse substrates: β2-microglobulin, prolactin, and the ADAM10 disintegrin domain.

Main Results:

• Disulfide bonds formed prior to conformational folding in protein domains lacking secondary structure.
• Non-native disulfide bond formation was observed in unstructured domains.
• Native disulfide bonds formed after partial folding in proteins with defined secondary structure.
• Nascent protein structure was found to promote correct disulfide formation during cotranslational folding.

Conclusions:

• Protein structure dictates the mechanism and timing of disulfide bond formation.
• Nascent protein structure plays a key role in ensuring the fidelity of disulfide bond formation.
• Findings reconcile the folded precursor and quasi-stochastic models, suggesting context-dependent mechanisms.