Unveiling a hidden folding intermediate in c-type cytochromes by protein engineering.
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.
Borgia
Alessandro
A
Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche, Università di Roma "La Sapienza," P.le A. Moro 5, 00185 Rome, Italy.
Bonivento
Daniele
D
Travaglini-Allocatelli
Carlo
C
Di Matteo
Adele
A
Brunori
Maurizio
M
D018360:Crystallography, X-Ray; D045304:Cytochromes c / Q000737:chemistry*; D007700:Kinetics; D016296:Mutagenesis; D017354:Point Mutation; D015202:Protein Engineering; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology
D045304:Cytochromes c