Tracing a protein's folding pathway over evolutionary time using ancestral sequence reconstruction and hydrogen...

Area of Science:

• Biochemistry
• Evolutionary Biology
• Structural Biology

Background:

• Protein folding mechanisms are extensively studied, but their evolutionary significance is less understood.
• Ancestral sequence reconstruction and advanced biophysical techniques offer new ways to study protein evolution and structure.

Purpose of the Study:

• To investigate the evolutionary conservation and divergence of protein folding pathways.
• To explore how evolutionary processes shape protein energy landscapes.

Main Methods:

• Utilized ancestral sequence reconstruction to access ancient protein forms.
• Employed pulsed-labeling hydrogen exchange coupled with mass spectrometry (pL-HX-MS) to determine folding intermediate structures.
• Studied the ribonuclease H (RNase H) family from T. thermophilus and E. coli lineages.

Main Results:

• All homologous and ancestral RNase H proteins studied consistently populate a similar folding intermediate.
• This conserved folding conformation persists across billions of years of evolution.
• While the intermediate structure is conserved, the folding pathways leading to it have diverged.
• Rational mutations were shown to alter the protein folding trajectory.

Conclusions:

• Evolutionary processes can preserve specific protein folding conformations.
• Evolutionary trajectories can also modify protein folding pathways and associated energy landscapes.
• The study highlights the interplay between evolutionary history and protein biophysics.