A stable, molten-globule-like cytochrome c

Area of Science:

• Biochemistry
• Protein Science
• Molecular Biology

Background:

• Cytochrome c is a crucial protein in electron transport.
• Understanding protein folding and stability is vital for biotechnology.
• Thermus thermophilus cytochrome c is a thermophilic protein.

Purpose of the Study:

• To characterize Thermus thermophilus cytochrome c expressed in Escherichia coli.
• To investigate the structural and stability differences between native and recombinant cytochrome c.
• To explore the implications of altered protein structure on folding pathways.

Main Methods:

• Recombinant protein expression in E. coli.
• Guanidine hydrochloride-induced unfolding studies.
• Spectroscopic analysis (Soret, far-UV CD, tryptophan fluorescence).
• Amino-acid sequencing.

Main Results:

• E. coli-expressed cytochrome c adopted a molten globule-like state.
• Recombinant protein showed lower stability and cooperativity than native cytochrome c.
• Spectroscopic data indicated a distorted heme environment and increased side-chain dynamics.
• A single cysteine-heme linkage was identified in the recombinant protein, unlike the native form.

Conclusions:

• The altered structure of E. coli-expressed cytochrome c is attributed to the reduced number of heme linkages.
• The molten globule state of recombinant cytochrome c may serve as a model for folding intermediates.
• Findings offer insights into protein folding kinetics and the role of specific structural features.