A Disordered Loop Mediates Heterogeneous Unfolding of an Ordered Protein by Altering the Native Ensemble

Area of Science:

• Protein dynamics and folding
• Structural biology
• Biophysics

Background:

• Flexible loops contribute to protein stability through entropic effects.
• Disrupting these loops can destabilize native protein structures.
• Downhill folding systems are sensitive to perturbations due to weak unfolding cooperativity.

Purpose of the Study:

• To investigate the impact of perturbations on the native ensemble of a helical, downhill folding protein domain (PDD).
• To understand the role of a partially structured loop in protein conformational stability and folding.
• To explore the relationship between loop flexibility, mutational effects, and protein folding pathways.

Main Methods:

• Combined experimental and computational approaches.
• Statistical mechanical modeling to analyze structural changes.
• Long time-scale molecular dynamics simulations.
• Fluorescence spectroscopy to validate computational predictions.

Main Results:

• Perturbations induce continuous structural changes driven by entropic stabilization.
• Mutations cause a conformational switch from parallel to antiparallel helix orientation.
• The flexible loop and C-terminal helix exhibit increased disorder and non-native interactions in mutants.

Conclusions:

• Long loops play a critical role in protein unfolding mechanisms and sensitivity to mutations.
• Protein native ensembles are dynamic and susceptible to conformational switching.
• Findings provide testable predictions for protein folding studies, including two-state systems.