2EXV

Crystal structure of the F7A mutant of the cytochrome c551 from Pseudomonas aeruginosa

Summary for 2EXV

• Entry DOI: 10.2210/pdb2exv/pdb
• Related: 1DVV 1YNR 351C
• Descriptor: Cytochrome c-551, HEME C, ACETIC ACID, ... (4 entities in total)
• Functional Keywords: cytochrome c, alpha helix, heme c, electron transport
• Biological source: Pseudomonas aeruginosa
• Cellular location: Periplasm: P00099
• Total number of polymer chains: 2
• Total formula weight: 18614.70

Authors

Bonivento, D.,Di Matteo, A.,Borgia, A.,Travaglini-Allocatelli, C.,Brunori, M. (deposition date: 2005-11-09, release date: 2006-02-07, Last modification date: 2024-10-09)

Primary citation

Borgia, A.,Bonivento, D.,Travaglini-Allocatelli, C.,Di Matteo, A.,Brunori, M.
Unveiling a Hidden Folding Intermediate in c-Type Cytochromes by Protein Engineering
J.Biol.Chem., 281:9331-9336, 2006

PubMed 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.

PubMed: 16452476
DOI: 10.1074/jbc.M512127200

Experimental method

X-RAY DIFFRACTION (1.86 Å)