Thermal unfolding of an intermediate is associated with non-arrhenius kinetics in the folding of hen lysozyme

A variety of techniques, including quenched-flow hydrogen exchange labellin g 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 r ange from 2 degrees C to 50 degrees C. Simple Arrhenius behaviour is not ob served, and although the overall rate of folding increases from 2 to 40 deg rees C, it decreases above 40 degrees C. In addition, the transient interme diate on the major folding pathway at 20 degrees C, in which the alpha-doma in is persistently structured in the absence of a stable beta-domain, is th ermally unfolded in a sigmoidal transition (T-m approximate to 40 degrees C ) indicative of a cooperatively folded state. At all temperatures, however, there is evidence for fast (similar to 25%) and slow (similar to 75%) popu lations of refolding molecules. By using transition state theory, the kinet ic 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 s pecies on the folding route between the denatured and native states, and wh ich accumulates as a consequence of its intrinsic stability. Our analysis s uggests that the temperature dependence of the rate constant for lysozyme f olding depends on both the total change in the heat capacity between the gr ound and transition states (the dominant factor at low temperatures) and th e heat-induced destabilization of the alpha-domain intermediate (the domina nt factor at high temperatures). Destablization of such kinetically compete nt intermediate species is Likely to be a determining factor in the non-Arr henius temperature dependence of the folding rate of those proteins for whi ch one or more intermediates are populated. (C) 2000 Academic Press.