Evidence for identity between the equilibrium unfolding intermediate and a transient folding intermediate: a comparative study of the folding reactions of .alpha.-lactalbumin and lysozyme

Abstract

The refolding kinetics of alpha-lactalbumin at different concentrations of guanidine hydrochloride have been investigated by means of kinetic circular dichroism and stopped-flow absorption measurements. The refolding reaction consists of at least two stages, the instantaneous accumulation of the transient intermediate that has peptide secondary structure and the subsequent slow process associated with formation of tertiary structure. The transient intermediate is compared with the well-characterized equilibrium intermediate observed during the denaturant-induced unfolding. Stabilities of the secondary structures against the denaturant, affinities for Ca2+, and tryptophan absorption properties of the transient and equilibrium intermediates were investigated. In all of these respects, the transient intermediate is identical with the equilibrium one, demonstrating the validity of the use of the equilibrium intermediate as a model of the folding intermediate. Essentially the same transient intermediate was also detected in the folding of lysozyme, the protein known to be homologous to alpha-lactalbumin but whose equilibrium unfolding is represented as a two-state reaction. The stability and cooperativity of the secondary structure of the intermediate of lysozyme are compared with those of alpha-lactalbumin. The results show that the protein folding occurring via the intermediate is not limited to the proteins that show equilibrium intermediates. Although the unfolding equilibria of most proteins are well approximated as a two-state reaction, the two-state hypothesis may not be applicable to the folding reaction under the native condition. Two models of protein folding, intermediate-controlled folding model and multiple-pathway folding model, which are different in view of the role of the intermediate in determining the pathway of folding, are also discussed.

Transition state in the folding of α‐lactalbumin probed by the 6‐120 disulfide bond

“…In this model, the observed folding-unfolding rate constant is related to kf and k, as k,,, = k f + k, which is based on the apparently two-state mechanism. This model was originally proposed by Nozaka et al (1978) and was previously called the multiple-pathway folding model (Ikeguchi et al, 1986). This model is also consistent with a recent energy landscape theory (Bryngelson et al, 1995;Dill & Chan, 1997).…”

“…In Figure 1, the GdnHCl concentration dependence of the apparent folding-unfolding rate constant (kapp) of 2CM-3SS-BLA is compared with that obtained previously for disulfide-intact BLA in the absence and presence of Ca2+ (Ikeguchi et al, 1986). Typical V-shape dependence of the apparent folding-unfolding rate on GdnHCl concentration is observed except for apo 2CM-3SS-BLA.…”

“…Two alternative models of the BLA folding It has been known that the molten globule intermediate accumulates at an early stage of the folding of both BLA and 2CM-3SS-BLA and that the subsequent folding process is explained by the two-state process without any other stable intermediates, except for a minor slow folding phase observed in the presence of Ca2+ (Kuwajima et al, 1985Ikeguchi et al, 1986Ikeguchi et al, , 1992Balbach et al, 1995Balbach et al, , 1996Arai & Kuwajima, 1996). In this study, we ignore this minor slow phase, because its amplitude is only 10% of the total observable change during the folding and it is ascribable to a slow isomerization process in the unfolded state .…”

“…In this study, we ignore this minor slow phase, because its amplitude is only 10% of the total observable change during the folding and it is ascribable to a slow isomerization process in the unfolded state . In our previous studies, the accumulation of the molten globule state of disulfide-intact BLA has been interpreted in terms of the two alternative models (Ikeguchi et al, 1986;Kuwajima et al, 1989 (Ikeguchi et al, 1986).…”

“…For analysis of the denaturant concentration dependence of the folding-unfolding rate constants, it is important to determine which of the above two models should be used. Because the interconversion between the A and U states is rapid 41n our previous report (Ikeguchi et al, 1986), "D' was used to denote the unfolded state. However, we had changed the abbreviation to indicate the unfolded state from "D' to " U (Ikeguchi et al, 1992).…”