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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 degrees C to 50 degrees C. Simple Arrhenius behaviour is not observed, and although the overall rate of folding increases from 2 to 40 degrees C, it decreases above 40 degrees C. In addition, the transient intermediate on the major folding pathway at 20 degrees C, in which the alpha-domain is persistently structured in the absence of a stable beta-domain, is thermally unfolded in a sigmoidal transition (T(m) approximately 40 degrees C) indicative of a cooperatively folded state. At all temperatures, however, there is evidence for fast ( approximately 25 %) and slow ( approximately 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 alpha-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 alpha-domain intermediate (the dominant factor at high temperatures). Destabilization 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.

Original publication




Journal article


J Mol Biol

Publication Date





193 - 210


Allosteric Regulation, Animals, Chickens, Circular Dichroism, Deuterium, Disulfides, Enzyme Stability, Female, Fluorescence, Hydrogen, Kinetics, Mass Spectrometry, Muramidase, Protein Denaturation, Protein Folding, Protein Renaturation, Protein Structure, Tertiary, Temperature, Thermodynamics, Tryptophan