Refolding kinetics of two homologous proteins, lysozyme and alpha-lactalbumin, were studied by following the time-dependent changes in the circular dichroism spectra in the aromatic and the peptide regions. The refolding was initiated by 20-fold dilution of the protein solutions originally unfolded at 6 M guanidine hydrochloride, at pH 1.5 for lysozyme and pH 7.0 for alpha-lactalbumin at 4.5 degrees C. In the aromatic region, almost full changes in ellipticity that were expected from the equilibrium differences in the spectra between the native and unfolded proteins were observed kinetically. The major fast phase of lysozyme folding has a decay time of 15 s. The decay time of alpha-lactalbumin depends on the presence or absence of bound Ca2+: 10 s for the holoprotein and 100 s for the apoprotein. In the peptide region, however, most of the ellipticity changes of the two proteins occur within the dead time (less than 3 s) of the present measurements. This demonstrates existence of an early folding intermediate which is still unfolded when measured by the aromatic bands but has folded secondary structure as measured by the peptide bands. Extrapolation of the ellipticity changes to zero time at various wavelengths gives a spectrum of the folding intermediate. Curve fitting of the peptide spectra to estimate the secondary structure fractions has shown that the two proteins assume a similar structure at an early stage of folding and that the intermediate has a structure similar to that of partially unfolded species produced by heat and, for alpha-lactalbumin, also by acid and a moderate concentration of guanidine hydrochloride.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

No abstract

Kinetic refolding reactions of ferricytochrome c and β‐lactoglobulin have been studied by stopped‐flow circular dichroism by monitoring rapid ellipticity changes of peptide backbone and side‐chain chromophores. In both proteins, a transient intermediate accumulates within the dead time of stopped‐flow mixing (18 ms), and the intermediate has an appreciable amount of secondary structure but possesses an unfolded tertiary structure. It is suggested that the rapid formation of a secondary structure framework in protein folding is a common property observed in a variety of globular proteins.

The kinetics of the reversible folding and unfolding of Escherichia coli dihydrofolate reductase have been studied by stopped-flow circular dichroism in the peptide region at pH 7.8 and 15 degrees C. The reactions were induced by concentration jumps of a denaturant, urea. The method can detect various intermediates transiently populated in the reactions although the equilibrium unfolding of the protein is apparently approximated by a two-state reaction. The results can be summarized as follows. (1) From transient circular dichroism spectra measured as soon as the refolding is started, a substantial amount of secondary structure is formed in the burst phase, i.e., within the dead time of stopped-flow mixing (18 ms). (2) The kinetics from this burst-phase intermediate to the native state are multiphasic, consisting of five phases designated as tau 1, tau 2, tau 3, tau 4, and tau 5 in increasing order of the reaction rate. Measurements of the kinetics at various wavelengths have provided kinetic difference circular dichroism spectra for the individual phases. (3) The tau 5 phase shows a kinetic difference spectrum consistent with an exciton contribution of two aromatic residues in the peptide CD region. The absence of the tau 5 phase in a mutant protein, in which Trp 74 is replaced by leucine, suggests that Trp 74 is involved in the exciton pair and that the tau 5 phase reflects the formation of a hydrophobic cluster around Trp 74. From the similarity of the kinetic difference spectrum to the difference between the native spectra of the mutant and wild-type proteins, it appears that Trp 47 is the partner in the exciton pair and that the structure formed in the tau 5 phase persists during the later stages of folding. (4) The later stages of folding show kinetic difference spectra that can be interpreted by rearrangement of secondary structure, particularly the central beta sheet of the protein. The pairwise similarities in the spectrum between the tau 3 and tau 4 phases, and between the tau 1 and tau 2 phases, also suggest the presence of two parallel folding channels for refolding. (5) The unfolding kinetics show three to four phases and are interpreted in terms of the presence of multiple native species. The total ellipticity change in kinetic unfolding reaction, however, agrees with the ellipticity difference between the native and unfolding states, indicating the absence of the burst phase in unfolding.(ABSTRACT TRUNCATED AT 400 WORDS)