The folding pathway of onconase is directed by a conserved intermediate

Area of Science:

• Protein biochemistry
• Structural biology
• Biophysics

Background:

• Understanding protein folding mechanisms is crucial for deciphering protein function and disease.
• Ribonuclease A (RNase A) is a well-studied model protein for folding studies.
• Homologous proteins with low sequence identity but similar structures offer insights into conserved folding principles.

Purpose of the Study:

• To investigate the folding pathway of onconase, a homologous protein to RNase A with potential therapeutic applications.
• To compare the folding mechanisms of onconase and RNase A to identify conserved folding determinants.

Main Methods:

• High-resolution techniques including rapid mixing, stopped-flow fluorescence spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy.
• Quenched-flow hydrogen/deuterium exchange experiments coupled with 2D NMR spectroscopy.

Main Results:

• Onconase folding proceeds through a slow peptidyl-prolyl cis-to-trans isomerization followed by an on-pathway intermediate to the native state.
• 31 amino acid residues were identified in the onconase folding intermediate, with 12 conserved residues shared with RNase A (39% conservation).
• The structure of the onconase intermediate closely resembles early intermediates observed during RNase A refolding.

Conclusions:

• Despite significant differences in primary sequence and stability, onconase and RNase A share comparable initial folding events.
• Conserved residues play a critical role in guiding the early stages of protein folding in homologous proteins.
• This study highlights the utility of comparing homologous proteins with distinct sequences but similar structures to understand fundamental folding mechanisms.