The molten globule state of alpha-lactalbumin

The molten globule state of alpha-lactalbumin is the best-characterized folding intermediate of globular proteins and has been studied intensively by various spectroscopic and physiochemical techniques, including stopped-flow CD and fluorescence spectroscopies, a hydrogen-exchange technique, 1H-NMR spectroscopy, disulfide-exchange chemistry, site-directed mutagenesis, and calorimetric techniques. This review summarizes recent studies. Major findings about the structure of the molten globule state are: 1) It is highly heterogeneous, having a highly structured alpha-helical domain with the beta-sheet domain being significantly unfolded; and 2) it is not a nonspecific, collapsed polypeptide but already has a native-like tertiary fold. These structural characteristics are essential to fully understand the thermodynamic properties of the molten globule state which are described in connection with a recently proposed computational approach to predict the structure of the molten globule state of a protein. Mutant proteins in which the stability of the molten globule state was changed were constructed. Studies of the equilibrium unfolding and kinetic refolding of the mutant proteins will provide further insight into the molten globule state as a folding intermediate. In spite of an initial expectation that the structure recognized by an Escherichia coli chaperone, GroEL, is the molten globule, the interaction of GroEL with alpha-lactalbumin in the molten globule state is much weaker than the interaction with more unfolded states of alpha-lactalbumin, a disulfide-reduced form, and disulfide rearranged species

Reversible and Fast Association Equilibria of a Molecular Chaperone, gp57A, of Bacteriophage T4

The association of a molecular chaperone, gp57A, of bacteriophage T4, which facilitates formation of the long and short tail fibers, was investigated by analytical ultracentrifugation, differential scanning microcalorimetry, and stopped-flow circular dichroism (CD) to establish the association scheme of the protein. Gp57A is an oligomeric α-helix protein with 79 amino acids. Analysis of the sedimentation velocity data by direct boundary modeling with Lamm equation solutions together with a more detailed boundary analysis incorporating association schemes led us to conclude that at least three oligomeric species of gp57A are in reversible and fast association equilibria and that a 3mer-6mer-12mer model described the data best. On the other hand, differential scanning microcalorimetry revealed a highly reversible two-step transition of dissociation/denaturation, both of which accompanied decrease in CD at 222 nm. The melting curve analysis revealed that it is consistent with a 6mer-3mer-1mer model. The refolding/association kinetics of gp57A measured by stopped-flow CD was consistent with the interpretation that the bimolecular reaction from trimer to hexamer was preceded by a fast α-helix formation in the dead-time. Trimer or hexamer is likely the functional oligomeric state of gp57A

The pro117 to glycine mutation of staphylococcal nuclease simplifies the unfolding folding kinetics

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro117 is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed

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

The guanidine hydrochloride concentration dependence of the folding and unfolding rate constants of a derivative of α-lactalbumin, in which the 6-120 disulfide bond is selectively reduced and S-carboxymethylated, was measured and compared with that of disulfide-intact α-lactalbumin. The concentration dependence of the folding and unfolding rate constants was analyzed on the basis of the two alternative models, the intermediate-controlled folding model and the multiple-pathway folding model, that we had proposed previously. All of the data supported the multiple-pathway folding model. Therefore, the molten globule state that accumulates at an early stage of folding of α-lactalbumin is not an obligatory intermediate. The cleavage of the 6-120 disulfide bond resulted in acceleration of unfolding without changing the refolding rate, indicating that the loop closed by the 6-120 disulfide bond is unfolded in the transition state. It is theoretically shown that the chain entropy gain on removing the cross-link from a random coil chain with helical stretches can be comparable to that from an entirely random chain. Therefore, the present result is not inconsistent with the known structure in the molten globule intermediate. Based on this result and other knowledge obtained so far, the structure in the transition state of the folding reaction of α-lactalbumin is discussed

Secondary structure of globular proteins at the early and the final stages in protein folding

The ellipticities for an early transient intermediate in refolding observed by kinetic circular dichroism measurements at 220–225 nm for 14 different proteins are summarized, and the ellipticity values are compared with those for the final native proteins and also with the ellipticities expected from a physical theory of protein and polypeptide secondary structure. The results show that a substantial part of the protein secondary structure is in general formed in the earliest detectable intermediate in refolding and that the ellipticities in both the native and the intermediate states are consistent with the physical theory of protein secondary structure

An early immunoreactive folding intermediate of the tryptophan synthase β2 subunit is a ‘molten globule’

The refolding kinetics of the tryptophan synthase β2 subunit have been investigated by circular dichroism (CD) and binding of a fluorescent hydrophobic probe (ANS), using the stopped-flow technique. The kinetics of regain of the native far UV CD signal show that, upon refolding of urea denatured β2, more than half of the protein secondary structure is formed within the dead time of the CD stopped-flow apparatus (0.013 s). On the other hand, upon refolding of guanidine unfolded β2 the fluorescence of ANS passes through a maximum after about 1 s and then ‘slowly’ decreases. These results show the accumulation, in the 1–10 s time range, of an early transient folding intermediate which has a pronounced secondary structure and a high affinity for ANS. In this time range, the near UV CD remains very low. This transient intermediate thus appears to have all the characteristics of the ‘molten globule’ state [(1987) FEBS Lett. 224, 9-13]. Moreover, by comparing the intrinsic time of the disappearance of this transient intermediate (t 35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633-7640] early imuno-reactive intermediate recognized by a monoclonal antibody (t 12 s), it is shown that this native-like epitope forms within the ‘molten globule’, before the tight packing of the protein side chains

Oligomeric Hsp33 with enhanced chaperone activity

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress

A folding model of α-lactalbumin deduced from the three-state denaturation mechanism

It has been shown that α-lactalbumin undergoes a three-state denaturation, involving a helical intermediate state, on treatment with guanidine hydrochloride. The unfolding of the protein and the characteristics of the intermediate state are examined by means of circular dichroism, difference spectra and pH-jump measurements to investigate the temperature dependence and kinetic properties of the unfolding and refolding, the pH dependence of the transition between the intermediate and the fully unfolded states, and the effect of disulphide bond reduction on the stabilization of the intermediate.The results show that the long-range specific interactions such as specific electrostatic interactions and disulphide linkages are not important for stabilizing the intermediate, and that the transition between the intermediate and the fully unfolded states is extremely rapid (a relaxation time of less than one millisecond) and may correspond to the helix-coil transition of a polypeptide backbone. On the other hand, the activation parameters of the transition between the native and the intermediate states have suggested that the final stabilization by charge-pair interactions is preceded by hydrophobic interactions in the process of going from the intermediate to the native state.The mechanism of folding of the protein is discussed, and the folding process from the fully unfolded to the native state is apparently divided into at least three main steps: (1) the formation of incipient helical structures dictated by local interactions; (2) the packing of the helical segments accompanied with hydrophobic interactions; (3) the final stabilization by the electrostatic interactions. The relevance to the current theoretical results on protein folding is also discussed