Solution structure of a protein denatured state and folding intermediate

Area of Science:

• Protein folding and biophysics
• Structural biology

Background:

• The earliest stages of protein folding remain controversial due to limited high-resolution structural data.
• Distinguishing between true folding intermediates and denatured states is challenging.

Purpose of the Study:

• To determine the solution structure of a true denatured state under folding-favoring conditions.
• To investigate the equilibrium and kinetic behavior of this denatured state.

Main Methods:

• Engineered a mutant Drosophila melanogaster Engrailed homeodomain (L16A) that reversibly folds/unfolds with ionic strength.
• Utilized nuclear magnetic resonance (NMR) to solve the solution structure of the denatured state.

Main Results:

• The L16A mutant exists in a well-ordered, native-like denatured state at physiological ionic strength.
• This denatured state is poised for folding via helix docking and disruption of non-native interactions.
• Unfolding exhibited progressive characteristics, mimicking features of early folding intermediates.

Conclusions:

• The studied denatured state represents a true folding intermediate.
• The observed progressive unfolding explains controversies surrounding intermediates versus compact denatured states.