Trifluoroethanol-assisted protein folding: fragment 53-103 of bovine alpha-lactalbumin

Fragment 53-103 of bovine cc-lactalbumin. prepared by limited peptic digest ion 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 expe cted 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 appro x. 30% yield when refolding was conducted in aqueous solution at neutral pH in the presence of the redox system constituted by reduced and oxidized gl utathione. On the other hand, when the reaction was conducted in 30% (v/v) trifluoroethanol (TFE), the oxidative refolding to the native isomer was al most quantitative. To provide an explanation of the beneficial effect of TF E in promoting the correct oxidative folding, the conformational features o f the various fragment species were analyzed by far-UV circular dichroism m easurements. The fully reduced fragment is largely unfolded in water, but i t becomes helical in aqueous TFE. Correctly refolded fragment is produced m ost when the helical contents of the reduced and oxidized fragment in aqueo us TFE are roughly equal, It is proposed that 30% TFE promotes a native-lik e format of the fragment and thus an efficient and correct pairing of disul fides. Higher concentrations of TFE, instead, promote some non-native helic al secondary structure in the fragment species, thus hampering correct fold ing. (C) 2001 Elsevier Science B.V. All rights reserved.