Parallel pathways in cytochrome c(551) folding.
The folding of cytochrome c(551) from Pseudomonas aeruginosa was previously thought to follow a simple sequential mechanism, consistent with the lack of histidine residues, other than the native His16 heme ligand, that can give rise to mis-coordinated species. However, further kinetic analysis reveals complexities indicative of a folding mechanism involving parallel pathways. Double-jump interrupted refolding experiments at low pH indicate that approximately 50% of the unfolded cytochrome c(551) population can reach the native state via a fast (10 ms) folding track, while the rest follows a slower folding path with populated intermediates. Stopped-flow experiments using absorbance at 695 nm to monitor refolding confirm the presence of a rapidly folding species containing the native methionine-iron bond while measurements on carboxymethylated cytochrome c(551) (which lacks the Met-Fe coordination bond) indicate that methionine ligation occurs late during folding along the fast folding track, which appears to be dominant at physiological pH. Continuous-flow measurements of tryptophan-heme energy transfer, using a capillary mixer with a dead time of about 60 micros, show evidence for a rapid chain collapse within 100 micros preceding the rate-limiting folding phase on the milliseconds time scale. A third process with a time constant in the 10-50 ms time range is consistent with a minor population of molecules folding along a parallel channel, as confirmed by quantitative kinetic modeling. These findings indicate the presence of two or more slowly inter-converting ensembles of denatured states that give rise to pH-dependent partitioning among fast and slow-folding pathways.
Gianni
Stefano
S
Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche e Istituto di Biologia e Patologia Molecolari del CNR, Università di Roma La Sapienza, Piazzale A. Moro 5, 00185 Rome, Italy.
Travaglini-Allocatelli
Carlo
C
Cutruzzolà
Francesca
F
Brunori
Maurizio
M
Shastry
M C Ramachandra
MC
Roder
Heinrich
H
D001426:Bacterial Proteins; D003574:Cytochrome c Group / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008715:Methionine / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D011994:Recombinant Proteins; D014508:Urea; C017167:cytochrome C(551); D008715:Methionine; D007501:Iron