Lower Protein Stability Does Not Necessarily Increase Local Dynamics.

Area of Science:

• Biochemistry
• Protein Dynamics
• Enzyme Kinetics

Background:

• Overall protein stability impacts enzyme function on millisecond timescales.
• Mitochondrial cytochrome c's alkaline conformational transition involves heme ligand exchange.
• The conserved Ω-loop D (residues 67-87) is crucial for heme crevice stability and function.

Purpose of the Study:

• To investigate the effect of a destabilizing L85A mutation on the kinetics and thermodynamics of mitochondrial cytochrome c's alkaline transition.
• To understand how overall protein stability influences substructure dynamics within the heme crevice.

Main Methods:

• Guanidine-HCl unfolding monitored by circular dichroism for global unfolding thermodynamics.
• pH titrations at 695 nm for local unfolding thermodynamics.
• pH-jump stopped-flow methods and gated electron transfer to measure alkaline transition kinetics and heme crevice dynamics.

Main Results:

• The L85A mutation destabilized yeast iso-1-cytochrome c.
• Contrary to expectations, heme crevice dynamics were not faster in the destabilized variant.
• Below pH 7, the WT*/K73H/L85A variant exhibited slower heme crevice dynamics.

Conclusions:

• Destabilizing mutations in cytochrome c do not necessarily lead to faster substructure dynamics.
• The relationship between protein stability and dynamics is complex and may not follow the psychrophilic/mesophilic enzyme analogy.
• The Ω-loop D region's role in modulating stability and dynamics warrants further investigation.