Comparison of the conformational stability of the non-native alpha-helical intermediate of thiol-modified beta-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 alpha-helical intermediate (alphaI) state of TNB-beta-LG were studied by circular dichroism (CD), fluorescence and differential scanning calorimetry (DSC) techniques. The effect of n-alkyl sulfates on the structure of alphaI state at both pH was utilized to investigate the contribution of hydrophobic interactions to the stability of alphaI 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.
Department of Biology, Faculty of Science, Islamic Azad University-Mashhad Branch, Mashhad, Iran. chamani@ibb.ut.ac.ir
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Chamani
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry*; D008968:Molecular Conformation; D012964:Sodium; D013053:Spectrophotometry; D013463:Sulfuric Acid Esters / Q000737:chemistry*
D007782:Lactoglobulins; D013463:Sulfuric Acid Esters; D004228:Dithionitrobenzoic Acid; D012964:Sodium