Trifluoroethanol-assisted protein folding: fragment 53--103 of bovine alpha-lactalbumin.
Fragment 53--103 of bovine alpha-lactalbumin, prepared by limited peptic digestion of the protein at low pH, is a 51-residue polypeptide chain crosslinked by two disulfide bonds encompassing helix C (residues 86--98) of the native protein. Refolding of the fully reduced fragment (four--SH groups) is expected to lead to three fully oxidized isomers, the native (61--77, 73--91) and the two misfolded species named ribbon (61--91, 73--77) and beads (61--73, 77--91) isomers. The fragment with correct disulfide bonds was formed in approx. 30% yield when refolding was conducted in aqueous solution at neutral pH in the presence of the redox system constituted by reduced and oxidized glutathione. On the other hand, when the reaction was conducted in 30% (v/v) trifluoroethanol (TFE), the oxidative refolding to the native isomer was almost quantitative. To provide an explanation of the beneficial effect of TFE in promoting the correct oxidative folding, the conformational features of the various fragment species were analyzed by far-UV circular dichroism measurements. The fully reduced fragment is largely unfolded in water, but it becomes helical in aqueous TFE. Correctly refolded fragment is produced most when the helical contents of the reduced and oxidized fragment in aqueous TFE are roughly equal. It is proposed that 30% TFE promotes a native-like format of the fragment and thus an efficient and correct pairing of disulfides. Higher concentrations of TFE, instead, promote some non-native helical secondary structure in the fragment species, thus hampering correct folding.
Polverino de Laureto
P
P
CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, 35121, Padua, Italy.
Donadi
M
M
Scaramella
E
E
Frare
E
E
Fontana
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D007768:Lactalbumin / Q000737:chemistry*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010434:Pepsin A; D010446:Peptide Fragments / Q000737:chemistry; D017510:Protein Folding; D014270:Trifluoroethanol / Q000737:chemistry*
D004220:Disulfides; D010446:Peptide Fragments; D014270:Trifluoroethanol; D007768:Lactalbumin; D010434:Pepsin A