A high-resolution probe of protein folding

Area of Science:

• Biochemistry
• Biophysics
• Structural Biology

Background:

• Protein folding is a fundamental process in biology, crucial for protein function.
• Understanding the mechanisms of protein folding, including intermediate states, remains a significant challenge.

Purpose of the Study:

• To investigate the equilibrium folding pathway of cytochrome c with residue-specific detail.
• To develop a method for visualizing protein folding intermediates using infrared spectroscopy.

Main Methods:

• Semisynthesis of cytochrome c incorporating specifically deuterated residues.
• Utilizing infrared (IR) spectroscopy to monitor changes in C-D bond vibrations.
• Analyzing IR spectra as a function of guanidine hydrochloride denaturant concentration.

Main Results:

• Deuterated residues allowed for clear visualization of spectral changes even at high protein and denaturant concentrations.
• A conformational change was identified at the Met80 residue, a protein-based ligand.
• Global unfolding of cytochrome c was observed at 2.3 M guanidine hydrochloride.

Conclusions:

• The study demonstrates a powerful technique for obtaining residue-specific information on protein folding.
• The findings provide detailed insights into the folding pathway of cytochrome c, highlighting key conformational transitions.
• This approach can be extended to study the folding of other proteins and resolve transient intermediates.