Detection and characterization of the intermediate on the folding pathway of human .alpha.-lactalbumin

Nozaka, Makoto; Kuwajima, Kunihiro; Nitta, Katsutoshi; Sugai, Shintaro

doi:10.1021/bi00611a013

Cited by 106 publications

(61 citation statements)

References 33 publications

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“…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).…”

Section: Resultssupporting

confidence: 80%

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

et al. 1998

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The guanidine hydrochloride concentration dependence of the folding and unfolding rate constants of a derivative of a-lactalbumin, in which the 6-120 disulfide bond is selectively reduced and S-carboxymethylated, was measured and compared with that of disulfide-intact a-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 a-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 a-lactalbumin is discussed.

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Section: Resultssupporting

confidence: 80%

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

et al. 1998

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“…In addition, species 2, 4, 8, 9, and 10 have no proteolytic activity or tertiary structure. All of the characteristics of these forms of ervatamin B are typical of molten globule state (Ptitsyn, 1995), and the states seem to be molten globule-like as in some other proteins (Kuwajima et al, 1976;Nozaka et al, 1978;Kim and Baldwin 1982;Goto et al, 1990a, b;Zerovnik et al, 1999).…”

Section: Discussionmentioning

confidence: 88%

Acid and Chemical Induced Conformational Changes of Ervatamin B. Presence of Partially Structured Multiple Intermediates

Sundd¹,

Kundu²,

Jagannadham³

2002

BMB Reports

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The structural and functional aspects of ervatamin B were studied in solution. Ervatamin B belongs to the α + β class of proteins. The intrinsic fluorescence emission maximum of the enzyme was at 350 nm under neutral conditions, and at 355 nm under denaturing conditions. Between pH 1.0-2.5 the enzyme exists in a partially unfolded state with minimum or no tertiary structure, and no proteolytic activity. At still lower pH, the enzyme regains substantial secondary structure, which is predominantly a β-sheet conformation and shows a strong binding to 8-anilino-1-napthalene-sulfonic acid (ANS). In the presence of salt, the enzyme attains a similar state directly from the native state. Under neutral conditions, the enzyme was stable in urea, while the guanidine hydrochloride (GuHCl) induced equilibrium unfolding was cooperative. The GuHCl induced unfolding transition curves at pH 3.0 and 4.0 were non-coincidental, indicating the presence of intermediates in the unfolding pathway. This was substantiated by strong ANS binding that was observed at low concentrations of GuHCl at both pH 3.0 and 4.0. The urea induced transition curves at pH 3.0 were, however, coincidental, but non-cooperative. This indicates that the different structural units of the enzyme unfold in steps through intermediates. This observation is further supported by two emission maxima in ANS binding assay during urea denaturation. Hence, denaturant induced equilibrium unfolding pathway of ervatamin B, which differs from the acid induced unfolding pathway, is not a simple two-state transition but involves intermediates which probably accumulate at different stages of protein folding and hence adds a new dimension to the unfolding pathway of plant proteases of the papain superfamily.

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“…For example, in the case acid isolated molten globule of ␣-lactalbumin, there is nearly a 75% loss of tertiary structure, while the secondary structure is almost intact in addition to being highly compact in nature. Likewise, in cytochrome c this state is accompanied by a loss of approximately 23-30% of the secondary structure (20,21,(23)(24)(25)(26)). Yet, unusually, the PNA intermediate, despite the reduced tertiary structure, retains its carbohydrate binding activity to a considerable degree.…”

Section: Discussionmentioning

confidence: 99%

Molten Globule-like State of Peanut Lectin Monomer Retains Its Carbohydrate Specificity

Reddy¹,

Srinivas

Ahmad³

et al. 1999

Journal of Biological Chemistry

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A central question in biological chemistry is the minimal structural requirement of a protein that would determine its specificity and activity, the underlying basis being the importance of the entire structural element of a protein with regards to its activity vis à vis the overall integrity and stability of the protein. Although there are many reports on the characterization of protein folding/ unfolding intermediates, with considerable secondary structural elements but substantial loss of tertiary structure, none of them have been reported to show any activity toward their respective ligands. This may be a result of the conditions under which such intermediates have been isolated or due to the importance of specific structural elements for the activity. In this paper we report such an intermediate in the unfolding of peanut agglutinin that seems to retain, to a considerable degree, its carbohydrate binding specificity and activity. This result has significant implications on the molten globule state during the folding pathway(s) of proteins in general and the quaternary association in legume lectins in particular, where precise subunit topology is required for their biologic activities.Despite their preponderance in biologic systems, studies on the folding pathways of oligomeric proteins are less common and information regarding the same, meager. The folding process for oligomeric proteins is more complex as the acquisition of the quaternary structure entails both, the intramolecular refolding of the individual polypeptide chains and the simultaneous intermolecular interactions between the various subunits. Hence, elucidation of the hierarchy of events occurring during the denaturation of an oligomeric protein provides important means to delineate such a process.Legume lectins, a class of highly homologous, carbohydratebinding proteins exhibit the same jelly roll tertiary structural fold but differ considerably in their ligand specificity and quaternary structures (1, 3). They can hence be considered to be "natural mutants" of quaternary structures. However, apart from their agglutinating activity, the role of oligomerization in lectins is not clearly understood (1-11). Peanut (Arachis hypogea) agglutinin (PNA), 1 a homotetrameric nonglycosylated protein, violates an important principle of quaternary association in globular proteins, a unique case of a tetramer without a 4-fold or 222 symmetry (10, 11). Unlike most other legume lectins, which dissociate and unfold as single entities (12-14), we describe here its chaotrope (guanidine hydrochloride, GdnHCl) induced unfolding that consists of a substantially unfolded monomeric species, as an intermediate, which retains its carbohydrate specificity despite a considerable loss of tertiary structure. Existence of such a molten globule-like structure with retention of binding activity is perhaps unprecedented so far. Moreover, the occurrence of such a species for PNA suggests that the monomers of legume lectins are competent to bind sugars and oligomerization appear...

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Detection and characterization of the intermediate on the folding pathway of human .alpha.-lactalbumin

Cited by 106 publications

References 33 publications

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

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

Acid and Chemical Induced Conformational Changes of Ervatamin B. Presence of Partially Structured Multiple Intermediates

Molten Globule-like State of Peanut Lectin Monomer Retains Its Carbohydrate Specificity

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