A single mutation induces molten globule formation and a drastic destabilization of wild-type cytochrome c at pH 6.0

Area of Science:

• Biochemistry
• Structural Biology
• Protein Science

Background:

• Cytochromes c possess conserved residues crucial for structure and function.
• Four key positions, beyond heme binding, are conserved across seven subfamilies.
• These conserved residues are implicated in forming a common protein folding nucleus.

Purpose of the Study:

• To investigate the role of the conserved Leu94 residue in cytochrome c structure and stability.
• To characterize the structural and stability changes induced by mutating Leu94 to Glycine (L94G).

Main Methods:

• Circular dichroism spectroscopy (far-UV, near-UV, Soret).
• Fluorescence spectroscopy (intrinsic and 1-Anilino-8-naphthalene sulfonate).
• Dynamic light scattering.
• Thermal denaturation studies.
• Analysis of Protein Data Bank (PDB) coordinates.

Main Results:

• The L94G mutant displays molten globule characteristics at pH 6.0.
• NaCl induces a transition to a pre-molten globule state in the L94G mutant at pH 2.
• The L94G mutant shows a 28°C lower denaturation midpoint compared to wild-type horse cytochrome c.
• Structural analysis suggests reduced stability for the L94G mutant.

Conclusions:

• The Leu94 residue is critical for maintaining the native structure and stability of cytochrome c.
• The L94G mutation destabilizes the protein, leading to molten globule and pre-molten globule states.
• Conserved residues play a vital role in the protein folding nucleus and overall structural integrity.