An Obligatory Intermediate in the Folding Pathway of Cytochromec552 from Hydrogenobacterthermophilus

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 c 552 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 c 552 shares high sequence identity and structural homology with its homologue from the mesophilic bacterium Pseudomonas aeruginosa cytochrome c 551 , 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.

Identification and characterization of protein folding intermediates

“…The folding of c 552 from Thermus thermophylus [29] and Hydrogenobacter thermophilus [36], the FF domain [30] and Im7 protein [28] In all cases, global analysis of observed kinetics led to the conclusion that the observed folding intermediate was an on-pathway species on the route between the denatured and native states.…”

Heme Attachment Motif Mobility Tunes CytochromecRedox Potential

“…The proximal His16 in Ht cyt c 552 and Pa cyt c 551 donates a hydrogen bond to the backbone carbonyl of Pro25, with an oxygen-nitrogen distance of ~2.8 Å ( Figure 1A) (21,22). Hydrogen bonding involving the axial His in heme proteins has been shown to affect the ligand's electrondonating properties, which in turn tunes redox potential (13,14,44,46,47,57).…”

Cytochrome c552Mutants: Structure and Dynamics at the Active Site Probed by Multidimensional NMR and Vibration Echo Spectroscopy

“…24 Unlike its homologues, Ht-cyt c 552 has a glutamine at position 64 (Gln64), which is not oriented toward the axial Met61, but is instead localized over a methyl group (heme 3-CH 3 using the Fischer numbering system) at the heme edge, near the protein surface ( Figure 1b). [15][16][17] Zhong and co-workers have shown that the absence of a hydrogen bond to the axial Met affects the dynamics, and presumably the functionality, of the active site by imparting a fluxional character to Met61 and influencing heme redox potential. 17,[22][23][24] Multidimensional spectroscopic techniques have provided a wealth of information in the field of biology.…”

“…[1][2][3] Due to their wide availability, robust structure, and ease of handling, these proteins have served as model systems to study the interplay of protein structure, dynamics, and function. [4][5][6][7][8][9][10][11][12][13] Cytochrome c 552 from Hydrogenobacter thermophilus (Ht-cyt c 552 ) [14][15][16][17] was recently shown to be a structurally unique example within the cyt c 8 family of class I 18 cyt c's. Proteins in the cyt c 8 structural family, for example, Pseudomonas cyt c 551 's, [19][20][21] typically have an asparagine residue at position 64 (Asn64) that is situated to donate a hydrogen bond to a methionine at position 61 (Met61), which occupies the sixth coordination site to the heme ( Figure 1a; sequence numbering is based on the Pseudomonas aeruginosa cyt c 551 sequence).…”

“…The first mutant studied (Ht-M61A) was characterized by only the single M61A mutation and retained an axial Gln64, which was expected to be oriented out of the heme pocket (Figure 1b) as it is in the native protein. 16,17,22 The second mutant (Ht-M61A/Q64N) was further modified to have Gln64 replaced by an Asn in an effort to generate an active site structure similar to that seen in most cyt c 8 's, [19][20][21][22][23] with Asn64 positioned to interact with the heme-bound CO (Figure 1a).…”