Reversible unfolding of bovine beta-lactoglobulin mutants without a free thiol group.
Bovine beta-lactoglobulin (beta-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 beta-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, reversed-phase and gel-filtration high performance liquid chromatographies, and SDS-PAGE. The folded structure of each mutant was similar to that of wild-type beta-lg. Urea-induced 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 beta-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 beta-lg especially at neutral pH and that the mutation of Cys-121 improves the reversibility, enabling us to study the folding of beta-lg more exactly under various conditions.
Yagi
Masanori
M
Institute for Protein Research, Osaka University and CREST, Japan Science Corporation, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
Sakurai
Kazumasa
K
Kalidas
Chitkala
C
Batt
Carl A
CA
Goto
Yuji
Y
D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D003545:Cysteine; D004220:Disulfides; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D013438:Sulfhydryl Compounds / Q000032:analysis; D014508:Urea
D004220:Disulfides; D007782:Lactoglobulins; D013438:Sulfhydryl Compounds; D014508:Urea; D003545:Cysteine