PDBsum entry 2exv

Electron transport

PDB id: 2exv

Contents

Protein chains

82 a.a. *

Ligands

HEC ×2

ACY ×2

Waters ×128

* Residue conservation analysis

PDB id:

2exv

Name:

Electron transport

Title:

Crystal structure of the f7a mutant of the cytochrome c551 from pseudomonas aeruginosa

Structure:

CytochromE C-551. Chain: a, c. Synonym: cytochrome c551, cytochrome c8. Engineered: yes. Mutation: yes

Source:

Pseudomonas aeruginosa. Organism_taxid: 287. Gene: nirm. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.86Å R-factor: 0.178 R-free: 0.233

Authors:

D.Bonivento,A.Di Matteo,A.Borgia,C.Travaglini-Allocatelli,M.Brunori

Key ref:

A.Borgia et al. (2006). Unveiling a hidden folding intermediate in c-type cytochromes by protein engineering. J Biol Chem, 281, 9331-9336. PubMed id: 16452476 DOI: 10.1074/jbc.M512127200

Date:

09-Nov-05 Release date: 07-Feb-06

Protein chains

P00099  (CY551_PSEAE) -  Cytochrome c-551 from Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)

Seq: 104 a.a.

Struc: 82 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1074/jbc.M512127200

J Biol Chem 281:9331-9336 (2006)

PubMed id: 16452476

Unveiling a hidden folding intermediate in c-type cytochromes by protein engineering.

A.Borgia, D.Bonivento, C.Travaglini-Allocatelli, A.Di Matteo, M.Brunori.

ABSTRACT

Several investigators have highlighted a correlation between the basic features of the folding process of a protein and its topology, which dictates the folding pathway. Within this conceptual framework we proposed that different members of the cytochrome c (cyt c) family share the same folding mechanism, involving a consensus partially structured state. Pseudomonas aeruginosa cyt c(551) (Pa cyt c(551)) folds via an apparent two-state mechanism through a high energy intermediate. Here we present kinetic evidence demonstrating that it is possible to switch its folding mechanism from two to three state, stabilizing the high energy intermediate by rational mutagenesis. Characterization of the folding kinetics of one single-site mutant of the Pa cyt c(551) (Phe(7) to Ala) indeed reveals an additional refolding phase and a fast unfolding process which are explained by the accumulation of a partially folded species. Further kinetic analysis highlights the presence of two parallel processes both leading to the native state, suggesting that the above mentioned species is a non obligatory on-pathway intermediate. Determination of the crystallographic structure of F7A shows the presence of an extended internal cavity, which hosts three "bound" water molecules and a H-bond in the N-terminal helix, which is shorter than in the wild type protein. These two features allow us to propose a detailed structural interpretation for the stabilization of the native and especially the intermediate states induced by a single crucial mutation. These results show how protein engineering, x-ray crystallography and state-of-the-art kinetics concur to unveil a folding intermediate and the structural determinants of its stability.

Selected figure(s)

Figure 1.
FIGURE 1. A, structural superimposition of the N-terminal -helix of wt Pa cyt c[551] (green) and F7A mutant (cyan). Interatomic distances between hydrogen bonded main chain atoms are shown. B, electron density map at 1.86 Å resolution of the F7A mutant showing details of the cavity generated by the mutation. The three water molecules that fill the cavity together with the H-bonds (dashed lines) established with the protein are shown.

Figure 3.
FIGURE 3. Folding kinetics of F7A cyt c[551] followed by fluorescence at pH 4.7, 10 °C. All data points are from single mixing experiments with the exception of the fast unfolding limb (empty circles), which has been obtained by double-mixing experiments. Continuous and dashed lines represent the best global fit for the on- and off-pathway model, respectively (15).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 9331-9336) copyright 2006.

Figures were selected by an automated process.