Ancient thioredoxins evolved to modern-day stability-function requirement by altering native state ensemble.

Area of Science:

• Biochemistry
• Molecular Evolution
• Protein Dynamics

Background:

• Thioredoxins (THRXs) are essential proteins found across all life forms.
• Ancestral and extant THRXs share structural similarities but exhibit distinct functional activities and stability.
• Evolutionary changes in THRXs present a puzzle regarding their altered properties.

Purpose of the Study:

• To investigate the evolutionary puzzle of differing functional activity and stability between ancient and modern thioredoxins.
• To compare the native state ensemble and dynamic flexibility index (DFI) of ancient and extant THRXs.
• To understand how protein flexibility changes correlate with THRX evolution and function.

Main Methods:

• Comparative analysis of ancient and modern thioredoxin native state ensembles.
• Quantification of protein flexibility using the dynamic flexibility index (DFI).
• Clustering proteins based on DFI profiles to classify them by activity and stability.

Main Results:

• DFI profiles reveal significant differences in extant proteins around α3, α4 helices, and catalytic regions compared to ancient forms.
• Allosteric coupling between the active site and the protein differs between ancient and extant THRXs, potentially explaining reduced activity at low pH.
• Evolution correlates with an increase in low-flexibility (hinge) and high-flexibility sites, and increased DFI heterogeneity with decreased melting temperature.

Conclusions:

• DFI profiles provide a classification scheme for THRXs that aligns with activity and stability.
• Evolutionary changes in protein flexibility and allosteric coupling explain functional divergence in thioredoxins.
• Increased flexibility heterogeneity and changes in hinge regions are key features of thioredoxin evolution towards modern forms.