Increasing stability reduces conformational heterogeneity in a protein folding intermediate ensemble

Area of Science:

• Biochemistry
• Structural Biology
• Physical Chemistry

Background:

• Protein folding intermediates represent transient states during the complex process of protein structure formation.
• Understanding the heterogeneity of these intermediates is crucial for elucidating folding mechanisms and protein stability.
• The energy landscape theory posits that protein folding involves navigating a complex energy surface with multiple conformational states.

Purpose of the Study:

• To investigate the structural heterogeneity within a late folding intermediate ensemble (IL) of the small protein barstar.
• To determine how varying stability conditions influence the structural composition of the folding intermediate.
• To provide experimental evidence for key tenets of the energy landscape theory of protein folding.

Main Methods:

• Utilized a multi-site, time-resolved fluorescence resonance energy transfer (TR-FRET) methodology.
• Measured four distinct intra-molecular distances within the structural components of the barstar IL.
• Analyzed sub-populations of molecules within the IL ensemble under different stability conditions.

Main Results:

• The IL ensemble comprises sub-populations with varying native-like and unfolded-like distances.
• Under stable conditions, most molecules in IL exhibit native-like distances.
• Decreased stability leads to an increased proportion of molecules with unfolded-like distances, indicating higher conformational entropy.

Conclusions:

• Protein folding intermediates are structurally heterogeneous ensembles, not single conformations.
• Conformational entropy increases as protein structures become less stable.
• Experimental results support the energy landscape theory's predictions regarding conformational space restriction and condition-dependent folding pathways.