Changing the transition state for protein (Un) folding

Area of Science:

• Biochemistry and Molecular Biology
• Protein Folding and Stability
• Spectroscopy

Background:

• Cytochromes c are crucial electron transport proteins involved in cellular respiration.
• Understanding protein folding and unfolding mechanisms is vital for comprehending protein function and dysfunction.
• The Saccharomyces cerevisiae iso-1-cytochrome c system provides a model for studying these processes.

Purpose of the Study:

• To investigate the (un)folding transition states of wild-type and N52I variant Saccharomyces cerevisiae iso-1-ferri- and ferrocytochromes c.
• To compare the effectiveness of heat and guanidinium chloride (GdmCl) as denaturants.
• To analyze the role of oxidation state and specific mutations on protein stability and transition state characteristics.

Main Methods:

• Equilibrium and kinetic denaturation experiments were employed.
• Denaturation was induced using guanidinium chloride (GdmCl).
• Circular dichroism (CD) spectropolarimetry, particularly Soret CD spectra, was used to monitor denaturation.

Main Results:

• Thermal and GdmCl denaturation yield different states, with heat being a more potent denaturant.
• Ferrocytochrome c stability is linked to burying the positive charge upon oxidation.
• Perturbations like reduction and the N52I mutation alter transition state free energy and solvent accessibility, with specific effects observed in the reduced N52I variant.

Conclusions:

• The stability of ferrocytochrome c is essential for its function under physiological conditions.
• The transition state of unfolding is sensitive to protein reduction and specific amino acid substitutions.
• Detailed characterization of transition states provides insights into the folding pathways and stability determinants of cytochromes c.