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Gianni S , Travaglini-Allocateli C , Cutruzzolla F , Brunori M , Shastry MCR , Roder H
Parallel pathways in cytochrome c(551) folding
Journal of Molecular Biology. 2003 Jul 25;330(5) :1145-1152
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Abstract
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 similar to50% 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,,, (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 mus, show evidence for a rapid chain collapse within 100 mus 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. (C) 2003 Elsevier Ltd. All rights reserved.
Notes
Brunori, M,Univ Roma La Sapienza, Ist Pasteur, Fdn Cenci Bolognetti, Dipartimento Sci Biochim,CNR, Piazzale Aldo Moro 5, I-00185 Rome, Italy Article English