Reversible Unfolding of Bovine β-Lactoglobulin Mutants without a Free Thiol Group

Abstract

Bovine ␤-lactoglobulin (␤-lg) has been used extensively as a model for studying protein folding. One of the problems preventing clarification of the folding mechanism is the incomplete reversibility from the unfolded state, probably caused by the thiol-disulfide exchange between a free thiol at Cys-121 and two disulfide bonds. We constructed and expressed three ␤-lg subtype A mutants in which Cys-121 was replaced by Ala, Ser, or Val (i.e. C121A, C121S, and C121V). We studied the reversibilities of these mutants from urea denaturation using circular dichroism, tryptophan fluorescence, reversedphase and gel-filtration high performance liquid chromatographies, and SDS-PAGE. The folded structure of each mutant was similar to that of wild-type ␤-lg. Ureainduced unfolding at pH 7.0 and 3.0 showed that although the C121S mutation notably decreases the stability, the destabilizing effects of the C121A and C121V mutations are less severe. For all of the mutants, complete refolding from the unfolded state in 8 M urea at both pH 7.0 and 3.0 was observed. Kinetics of the formation of the irreversibly unfolded species of wild-type ␤-lg in 8 M urea at pH 7.0 indicated that, first, an intramolecular thiol-disulfide exchange occurs to produce a mixture of species with non-native disulfide bonds followed by the intermolecular thiol-disulfide exchange producing the oligomers. These results indicate that intramolecular and intermolecular thiol-disulfide exchange reactions cause the low reversibility of wild-type ␤-lg especially at neutral pH and that the mutation of Cys-121 improves the reversibility, enabling us to study the folding of ␤-lg more exactly under various conditions.

Comparative analysis of refolding of chemically denatured β-lactoglobulin types A and B using the dilution additive mode

“…Although BLG is a predominantly ␤-sheet protein, the addition of trifluoroethanol (TFE) induces a drastic conformational change resulting in a predominantly ␣-helical structure [15]. This nonnative ␣-state has also been called the "TFE-state" because this ␤ → ␣ transition has been widely studied in the presence of TFE [14].…”

“…One of the factors preventing the use of natural or alkaline pH conditions is the low reversibility of the unfolded state [15].…”

Structure and stability of Gyuba, a β‐lactoglobulin chimera

“…However, influence of Cys121Ala mutation on the cooperativity (m value) seems to be less than that of Cys121Ser or Cys121Val mutation. 25 An interaction between Pro126 (in the loop that connects the H-strand and a-helix) and Tyr20 (in the A-strand) is found in BLG, whereas a corresponding interaction is absent in Gyuba because residue 126 is Gln. That interactions between proline and aromatic residues contribute to protein stability have been suggested by studies on the HP domain 30 and exendin-4.…”

“…In previous report, it was revealed that a Cys121Ala mutation reduced the stability of BLG by 6.5 kJ/mol, although it improved the reversibility. 25 Because Gyuba has the same substitution, it must be at least partly responsible for destabilization of Gyuba. However, influence of Cys121Ala mutation on the cooperativity (m value) seems to be less than that of Cys121Ser or Cys121Val mutation.…”

“…Indeed, it has been shown that thermal and urea denaturation is reversible for BLG mutants in which C121 was replaced by Ala, Ser, or Val. 24,25 Thermodynamic parameters were calculated using the thermal denaturation curves and assuming two-state transitions for the denaturations [Eqs.…”