PDBsum entry 2exv

Electron transport

PDB id: 2exv

Contents

Protein chains

82 a.a.

Ligands

HEC ×2

ACY ×2

Waters ×128

References listed in PDB file

Key reference

Title

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

Authors

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

Ref.

J Biol Chem, 2006, 281, 9331-9336. [DOI no: 10.1074/jbc.M512127200]

PubMed id

16452476

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.

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.

Secondary reference #1

Title

Structure of cytochrome c551 from pseudomonas aeruginosa refined at 1.6 a resolution and comparison of the two redox forms.

Authors

Y.Matsuura, T.Takano, R.E.Dickerson.

Ref.

J Mol Biol, 1982, 156, 389-409. [DOI no: 10.1016/0022-2836(82)90335-7]

PubMed id

6283101

Figure 3.
FIG. 3. All theside-chains onan a-carbon skeleton. (a) Front view, and (b) view from Met61 aide. Not.e a long sequence of hydrophobic residues along the edge of heme crevice on the Met61 side.

Figure 9.
FIG. 9. Hydrogen-bod network among water molecules (WAT) 11. 23 and 25. LyslOCO Ile48CO. ro62C0, sn64Nd and 06 MetGlS, and Ala65NH in the heme crevice of the reduced frm. Probable ydrogens are indicated by thick lines on hydrogen bonds. Circles adjacent to water molecules 23 ad 25 indicate their positions in the oxidized form.

The above figures are reproduced from the cited reference with permission from Elsevier

Secondary reference #2

Title

Selected mutations in a mesophilic cytochrome c confer the stability of a thermophilic counterpart.

Authors

J.Hasegawa, S.Uchiyama, Y.Tanimoto, M.Mizutani, Y.Kobayashi, Y.Sambongi, Y.Igarashi.

Ref.

J Biol Chem, 2000, 275, 37824-37828. [DOI no: 10.1074/jbc.M005861200]

PubMed id

10918067

Figure 3.
Fig. 3. Structures of the quintuple mutant and wild-type PA c[551] proteins and HT c[552]. A, stereoview of the 20 structures of the quintuple mutant. B, schematic representation of main chain folding of the quintuple mutant (purple) overlaid with those of the wild-type PA c[551] (green) and HT c[552] (red).

Figure 4.
Fig. 4. Comparison of the side chain packing around the mutation sites in the quintuple mutant and the corresponding regions in the wild-type PA c[551] and HT c[552]. Amino acids mentioned throughout are designated with a one-letter code. Residues in HT c[552] are shown with the numbering used for those in PA c[551]. The mutated side chains of the quintuple mutant and the corresponding ones of the wild-type PA c[551] and HT c[552] are colored purple, green, and red, respectively. A, the hydrophobic region around Phe-7 and Val-13 of the wild-type PA c[551] and the corresponding regions in the quintuple mutant and HT c[552]. B, the loop and half of the third helix region from Phe-34 to Leu-44 of the wild-type PA c[551] and the corresponding regions in the quintuple mutant and HT c[552]. C, the internal hydrophobic region around Val-78 and the heme of the wild type and the corresponding regions in the quintuple mutant and HT c[552].

The above figures are reproduced from the cited reference with permission from the ASBMB

Secondary reference #3

Title

An obligatory intermediate in the folding pathway of cytochrome c552 from hydrogenobacter thermophilus.

Authors

C.Travaglini-Allocatelli, S.Gianni, V.K.Dubey, A.Borgia, A.Di matteo, D.Bonivento, F.Cutruzzolà, K.L.Bren, M.Brunori.

Ref.

J Biol Chem, 2005, 280, 25729-25734. [DOI no: 10.1074/jbc.M502628200]

PubMed id

15883159

Figure 1.
FIG. 1. X-ray crystal structure of the ferric derivative of H. thermophilus cytochrome c[552] (PDB code 1YNR [PDB] ). The heme group and the residues His-14, Met-59, Trp-54, and Trp-75 are shown in stick representation. The N- and C-terminal helices are highlighted in red and blue, respectively.

Figure 2.
FIG. 2. GdnHCl-induced equilibrium unfolding of H. thermophilus cytochrome c[552] (circles) and P. aeruginosa cytochrome c[551] (squares) monitored by Trp fluorescence at pH 4.7, 10 °C. Lines are the best fit to a two-state model as follows: [GdnHCl] = 4.0 ± 0.1 M and 2.1 ± 0.1 M for H. thermophilus cytochrome c[552] and P. aeruginosa cytochrome c[551], respectively; m-values 2.2 ± 0.1 kcal mol-1 M-1 and 2.4 ± 0.1 kcal mol-1 M-1 for H. thermophilus cytochrome c[552] and P. aeruginosa cytochrome c[551],respectively.

The above figures are reproduced from the cited reference with permission from the ASBMB