Abstract
The folding mechanism of many proteins involves the population of partially organized structures en route to the native state. Identification and characterization of these intermediates is particularly difficult, as they are often only transiently populated and may play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. Following different spectroscopic probes, and employing state-of-the-art kinetic analysis, we present evidence that the folding mechanism of the thermostable cytochrome c552 from Hydrogenobacter thermophilus does involve the presence of an elusive, yet compact, on-pathway intermediate. Characterization of the folding mechanism of this cytochrome c is particularly interesting for the purpose of comparative folding studies, because H. thermophilus cytochrome c552 shares high sequence identity and structural homology with its homologue from the mesophilic bacterium Pseudomonas aeruginosa cytochrome c551, which refolds through a broad energy barrier without the accumulation of intermediates. Analysis of the folding kinetics and correlation with the three-dimensional structure add new evidence for the validity of a consensus folding mechanism in the cytochrome c family.
Highlights
As for any other chemical process, a meaningful description of the protein folding mechanism demands careful analysis of its kinetics
It has been shown that differences in local propensity to form elements of secondary structure may modulate the stabilities of transient species, such as intermediates and/or transition state(s), and introduce variations in the order whereby structural motifs are formed during folding [11, 12]
To obtain additional experimental evidence to strengthen the hypothesis of a consensus folding mechanism in this protein family, we report an analysis of the folding kinetics of cytochrome c552 from the thermophilic bacterium Hydrogenobacter thermophilus
Summary
As for any other chemical process, a meaningful description of the protein folding mechanism demands careful analysis of its kinetics. It has been shown that differences in local propensity to form elements of secondary structure may modulate the stabilities of transient species, such as intermediates and/or transition state(s), and introduce variations in the order whereby structural motifs are formed during folding [11, 12]. Such effects may result in different topologies of folding transition states in homologous proteins, as demonstrated by Baker and co-workers [13] for the IgG binding domains of proteins G and L. Starting from the identification of an on-pathway intermediate observed in the folding of Thermus thermophilus cytochrome c552 [16], a critical re-evaluation of published kinetic data on different prokaryotic and eukaryotic c-type cytochromes led to the proposal [17] that proteins belonging to the cytochrome c family share a consensus folding mechanism involving, in all cases, the population of an on-pathway intermediate with conserved structural features
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