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.

Parallel pathways in cytochrome c551 folding / Gianni, Stefano; TRAVAGLINI ALLOCATELLI, Carlo; Cutruzzola', Francesca; Brunori, Maurizio; RAMACHANDRA SHASTRY M., C; Roder, H.. - In: JOURNAL OF MOLECULAR BIOLOGY. - ISSN 0022-2836. - STAMPA. - 330:5(2003), pp. 1145-1152. [10.1016/S0022-2836(03)00689-2]

Parallel pathways in cytochrome c551 folding

GIANNI, STEFANO;TRAVAGLINI ALLOCATELLI, Carlo;CUTRUZZOLA', Francesca;BRUNORI, Maurizio;
2003

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 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.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11573/363340
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