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

Absorption spectra of intact β-LG (1) and TNB-β-LG (2) at pH 2. For comparison, the absorption spectra of the oxidized (dashed curves) and reduced (3) forms of DTNB are included, in which the spectrum of the DTNB represents one molecule of TNB.Figure optionsDownload as PowerPoint slide