Long-Lived Intermediates in a Cooperative Two-State Folding Transition

Area of Science:

• Biomolecular dynamics
• Protein folding mechanisms
• Spectroscopic analysis

Background:

• Biomolecular folding often proceeds via a two-state transition, obscuring intermediate structures.
• Ion mobility spectrometry (IMS) previously identified six intermediates in polyproline-13 (Pro13) folding.
• Understanding folding pathways is crucial for protein function and drug design.

Purpose of the Study:

• To investigate the effect of histidine addition on the folding pathway of polyproline-13 (Pro13).
• To characterize the folding transition of HisPro13 from polyproline I (PPI) in propanol to polyproline II (PPII) in aqueous solution.
• To elucidate the mechanism behind the observed induction period in HisPro13 folding.

Main Methods:

• Ion mobility spectrometry (IMS) was used to monitor the folding transition of HisPro13.
• Temperature-dependent studies were conducted to analyze the induction period.
• Equilibrium studies were performed to determine thermodynamic properties (endothermic, entropically favored).

Main Results:

• HisPro13 folding transitions directly from PPI to PPII, lacking observable intermediates seen in Pro13.
• A significant, temperature-dependent induction period precedes the two-state transition.
• Equilibrium analysis indicates an endothermic but entropically favorable folding process.

Conclusions:

• Histidine incorporation dramatically alters the Pro13 folding pathway, eliminating observable intermediates.
• The induction period suggests a sequential mechanism involving approximately 13-17 short-lived, entropically favorable intermediates with high activation barriers.
• A new model for HisPro13 folding is proposed, highlighting the role of histidine in modulating protein dynamics.