Comparison of the conformational stability of the non-native α-helical intermediate of thiol-modified β-lactoglobulin upon interaction with sodium n-alkyl sulfates at two different pH

Bovine beta-lactoglobulin assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. beta-lactoglobulin has a free thiol at Cys121, which is buried between the beta-barrel and the C-terminal major or alpha-helix. This thiol group was specifically reacted with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) at pH 7.5 and 2, producing a modified beta-lactoglobulin containing a mix disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). beta-Lactoglobulin is a predominantly beta-sheet protein, although it has a markedly high intrinsic preference for alpha-helical structure. The formation of non-native alpha-helical intermediate of thiol modified beta-lactoglobulin (TNB-beta-LG) was induced by n-alkyl sulfates including sodium octyl sulfate, SOS; sodium decyl sulfate, SDeS; sodium dodecyl sulfate, SDS; and sodium tetradecyl sulfate, STS at pH 7.5 and 2. The conformation and stability of non-native a-helical intermediate (alpha I) state of TNB-beta-LG were studied by circular dichroism (M), fluorescence and differential scanning calorimetry (DSC) techniques. The effect of n-alkyl sulfates on the structure of alpha I state at both pH was utilized to investigate the contribution of hydrophobic interactions to the stability of alpha I intermediate. The present results suggest that the folding reaction of beta-LG follows a non-hierarchical mechanism and hydrophobic interactions play important roles in stabilizing the native state of beta-LG at pH 2 with more positive charges repulsion than at pH 7.5. Then TNB-beta-LG will become a useful model to analyze the conformation and stability of the intermediate of protein folding. (c) 2006 Elsevier Inc. All rights reserved.