Evidence for sequential barriers and obligatory intermediates in apparent two-state protein folding

Area of Science:

• Biochemistry
• Physical Chemistry
• Structural Biology

Background:

• Small proteins often exhibit rapid folding without observable intermediates, contrasting with larger proteins that may involve transient partially folded states.
• The interpretation of non-linear activation free-energy relationships in protein folding has been debated, with some models proposing broad transition barrier regions.

Purpose of the Study:

• To investigate the properties of free energy barriers in protein folding.
• To challenge the prevailing interpretation of non-linear free energy relationships as indicative of broad barrier regions.
• To propose an alternative model for protein folding pathways.

Main Methods:

• Analysis of experimental data from 23 proteins exhibiting non-linear activation free-energy relationships.
• Comparison of a sequential folding model with a broad barrier model.

Main Results:

• Results argue against a single broad barrier region, suggesting instead sequential folding pathways with distinct barriers and a few obligatory high-energy intermediates.
• The sequential model consistently explains folding barriers across protein variants and varying solvent conditions.
• This model accounts for transitions between linear and non-linear free energy relationships and from two-state to multi-state folding upon mutations or experimental condition changes.

Conclusions:

• Non-linearities in protein folding free energy relationships arise from sequential pathways with distinct barriers and intermediates, not broad barriers.
• The sequential model provides a unified explanation for diverse protein folding behaviors, including apparent two-state and multi-state folding.
• The relative stability of intermediates is key to understanding the discrepancy between two-state and multi-state folding, underscoring the significance of partially folded states.