Alpha-lactalbumin Molten Globule Research Articles

Noncooperative Formation of the Off-Pathway Molten Globule during Folding of the α−β Parallel Protein Apoflavodoxin

During folding of many proteins, molten globules are formed. These partially folded forms of proteins have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristic of native structures. Molten globules are ensembles of interconverting conformers and are prone to aggregation, which can have detrimental effects on organisms. Consequently, molten globules attract considerable attention. The molten globule that is observed during folding of flavodoxin from Azotobacter vinelandii is a kinetically off-pathway species, as it has to unfold before the native state of the protein can be formed. This intermediate contains helices and can be populated at equilibrium using guanidinium hydrochloride as denaturant, allowing the use of NMR spectroscopy to follow molten globule formation at the residue level. Here, we track changes in chemical shifts of backbone amides, as well as disappearance of resonances of unfolded apoflavodoxin, upon decreasing denaturant concentration. Analysis of the data shows that structure formation within virtually all parts of the unfolded protein precedes folding to the molten globule state. This folding transition is noncooperative and involves a series of distinct transitions. Four structured elements in unfolded apoflavodoxin transiently interact and subsequently form the ordered core of the molten globule. Although hydrophobic, tryptophan side chains are not involved in the latter process. This ordered core is gradually extended upon decreasing denaturant concentration, but part of apoflavodoxin's molten globule remains random coil in the denaturant range investigated. The results presented here, together with those reported on the molten globule of alpha-lactalbumin, show that helical molten globules apparently fold in a noncooperative manner.

Equilibrium and Kinetic Studies on Folding of the Authentic and Recombinant Forms of Human α-Lactalbumin by Circular Dichroism Spectroscopy

The equilibrium and kinetics of the unfolding and refolding of authentic and recombinant human alpha-lactalbumin, the latter of which had an extra methionine residue at the N-terminus, were studied by circular dichroism spectroscopy, and the results were compared with the results for bovine and goat alpha-lactalbumins obtained in our previous studies. As observed in the bovine and goat proteins, the presence of the extra methionine residue in the recombinant protein remarkably destabilized the native state, and the destabilization was entirely ascribed to an increase in the rate of unfolding. The thermodynamic stability of the native state against the unfolded state was lower, and the thermodynamic stability of the molten globule state against the unfolded state was higher for the human protein than for the other alpha-lactalbumins previously studied. Thus, the population of the molten globule intermediate was higher during the equilibrium unfolding of human alpha-lactalbumin by guanidine hydrochloride. Unlike the molten globule states of the bovine and goat proteins, the human alpha-lactalbumin molten globule showed remarkably more intense circular dichroism ellipticity than the native state in the far-ultraviolet region below 225 nm. During refolding from the unfolded state, human alpha-lactalbumin thus exhibited overshoot kinetics, in which the alpha-helical peptide ellipticity exceeded the native value when the molten globule folding intermediate was formed in the burst phase. The subsequent folding involved reorganization of nonnative secondary structures. It should be noted that the rate constant of the major refolding phase was approximately the same among the three types of alpha-lactalbumin and that the rate constant of unfolding was accelerated 18-600 times in the human protein, and these results interpreted the lower thermodynamic stability of this protein.

Hydrophobic sequence minimization of the alpha-lactalbumin molten globule.

The molten globule, a widespread protein-folding intermediate, can attain a native-like backbone topology, even in the apparent absence of rigid side-chain packing. Nonetheless, mutagenesis studies suggest that molten globules are stabilized by some degree of side-chain packing among specific hydrophobic residues. Here we investigate the importance of hydrophobic side-chain diversity in determining the overall fold of the alpha-lactalbumin molten globule. We have replaced all of the hydrophobic amino acids in the sequence of the helical domain with a representative amino acid, leucine. Remarkably, the minimized molecule forms a molten globule that retains many structural features characteristic of a native alpha-lactalbumin fold. Thus, nonspecific hydrophobic interactions may be sufficient to determine the global fold of a protein.

Structural characterization of the molten globule of alpha-lactalbumin by solution X-ray scattering.

A compact denatured state is often observed under a mild denaturation condition for various proteins. A typical example is the alpha-lactalbumin molten globule. Although the molecular compactness and shape are the essential properties for defining the molten globule, there have been ambiguities of these properties for the molten globule of alpha-lactalbumin. Using solution X-ray scattering, we have examined the structural properties of two types of molten globule of alpha-lactalbumin, the apo-protein at neutral pH and the acid molten globule. The radius of gyration for the native holo-protein was 15.7 A, but the two different molten globules both had a radius of gyration of 17.2 A. The maximum dimension of the molecule was also increased from 50 A for the native state to 60 A for the molten globule. These values clearly indicate that the molten globule is not as compact as the native state. The increment in the radius of gyration was less than 10% for the alpha-lactalbumin molten globule, compared with up to 30% for the molten globules of other globular proteins. Intramolecular disulfide bonds restrict the molecular expansion of the molten globule. The distance distribution function of the alpha-lactalbumin molten globule is composed of a single peak suggesting a globular shape, which is simply swollen from the native state. The scattering profile in the high Q region of the molten globule indicates the presence of a significant amount of tertiary fold. Based on the structural properties obtained by solution X-ray scattering, general and conceptual structural images for the molten globules of various proteins are described and compared with the individual, detailed structural model obtained by nuclear magnetic resonance.

Pathway of disulfide-coupled unfolding and refolding of bovine alpha-lactalbumin.

alpha-Lactalbumin's four disulfide bonds have been used to probe the nature of its native, molten globule, and unfolded states. The Ca2+ concentration could be used to vary the stability of the native state because it binds a single calcium ion with high affinity. In the Ca(2+)-bound native state, the Cys6-120 and Cys28-111 disulfides were reduced sequentially, reflecting their relative accessibilities. The native disulfides do not stabilize the native conformation as much as in other proteins, nor was the unfolding process as cooperative. After two disulfides were reduced, only part of the protein molecule appeared to remain folded. In the absence of Ca2+, the native state was much less stable, and a molten globule-type conformation tended to be adopted. Formation of disulfide bonds in the reduced protein was initially noncooperative and nonspecific. The molten globule state increased the rate at which disulfides were formed, by tending to keep close in space cysteine residues that are distant in the sequence. A large number of nonnative disulfide pairings were preferred, and the disulfides were not stabilized to any great extent. The alpha-lactalbumin molten globule seems to be stabilized by nonspecific interactions and without a well-defined topology. The native protein was regenerated only when Ca2+ bound to and stabilized the three-disulfide intermediate lacking the Cys6-120 disulfide, which then rapidly formed the fourth disulfide bond. The rate-limiting intramolecular steps were rearrangement of disulfide bonds to native combinations in species with two and three disulfide bonds.

Structural characterization of the disulfide folding intermediates of bovine alpha-lactalbumin.

Specific three- and two-disulfide intermediates that accumulate transiently during reduction of the disulfide bonds of Ca(2+)-bound bovine alpha-lactalbumin have been trapped, isolated, and characterized. The three-disulfide intermediate was shown to lack the Cys6-120 disulfide bond, confirming the observations of others. The newly-recognized two-disulfide form has been shown to lack the Cys6-120 and Cys28-111 native disulfide bonds. The remaining native disulfide bonds in the two partially reduced derivatives of alpha-lactalbumin are stable only when the proteins are in a Ca(2+)-bound state. Otherwise, they adopt an equilibrium between molten globule and unfolded conformations, and rapid thiol-disulfide interchange occurs, at a rate as high as when the proteins are fully unfolded in 8 M urea, to generate distinct mixtures of rearranged products. Urea gradient electrophoresis, circular dichroism, fluorescence, and ANS binding have been combined to give a detailed structural picture of alpha-lactalbumin, its derivatives with native and with nonnative disulfide bonds, and the fully reduced protein. The native structure of alpha-lactalbumin appears to be split by selective disulfide bond cleavage into at least one subdomain, which retains the Ca(2+)-binding site. The alpha-lactalbumin molten globule state is shown largely to result from nonspecific hydrophobic collapse, to be devoid of cooperative or specific tertiary interactions, and not to be stabilized substantially by the native or rearranged disulfide bonds.