| Autor: |
Matagne, Andre´, Jamin, Marc, Chung, Evonne W., Robinson, Carol V., Radford, Sheena E., Dobson, Christopher M. |
| Zdroj: |
JMB Online (Journal of Molecular Biology); March 17, 2000, Vol. 297 Issue: 1 p193-210, 18p |
| Abstrakt: |
A variety of techniques, including quenched-flow hydrogen exchange labelling monitored by electrospray ionization mass spectrometry, and stopped-flow absorbance, fluorescence and circular dichroism spectroscopy, has been used to investigate the refolding kinetics of hen lysozyme over a temperature range from 2 °C to 50 °C. Simple Arrhenius behaviour is not observed, and although the overall rate of folding increases from 2 to 40 °C, it decreases above 40 °C. In addition, the transient intermediate on the major folding pathway at 20 °C, in which the α-domain is persistently structured in the absence of a stable β-domain, is thermally unfolded in a sigmoidal transition (Tm≈40 °C) indicative of a cooperatively folded state. At all temperatures, however, there is evidence for fast (∼25 %) and slow (∼75 %) populations of refolding molecules. By using transition state theory, the kinetic data from various experiments were jointly fitted to a sequential three-state model for the slow folding pathway. Together with previous findings, these results indicate that the α-domain intermediate is a productive species on the folding route between the denatured and native states, and which accumulates as a consequence of its intrinsic stability. Our analysis suggests that the temperature dependence of the rate constant for lysozyme folding depends on both the total change in the heat capacity between the ground and transition states (the dominant factor at low temperatures) and the heat-induced destabilization of the α-domain intermediate (the dominant factor at high temperatures). Destablization of such kinetically competent intermediate species is likely to be a determining factor in the non-Arrhenius temperature dependence of the folding rate of those proteins for which one or more intermediates are populated. |
| Databáze: |
Supplemental Index |
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