title: Hierarchical unfolding of the alpha-lactalbumin molten globule: presence of a compact intermediate without a unique tertiary fold.
abstract: The difference between the framework model and the hydrophobic collapse model of protein folding largely rests on whether a secondary-structure framework can exist independently of native tertiary interactions. Here, we used circular dichroism and disulfide exchange experiments to examine the unfolding mechanism of alpha-LA(alpha), a two- disulfide variant of human alpha-lactalbumin (alpha-LA) that adopts a molten globule conformation under near physiological conditions. Our results show that as the concentration of denaturant increases, the alpha-LA molten globule first loses its ability to form a specific, native-like tertiary fold. Subsequently, at a higher denaturant concentration, the protein loses its secondary structure and adopts an extended conformation. A compact, non-native disulfide bond isomer, which does not form significantly under both native and strongly denaturing conditions, was found to be moderately populated in approximately 2 M guanidine hydrochloride (GuHCl). Qualitatively the same result was also obtained in urea. These results suggest that formation of secondary structure is a necessary, but not sufficient condition for formation of the native-like tertiary fold and support a hierarchical model of protein folding.
authors.affiliation: Department of Biochemistry, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA.
authors.forename: S
authors.initials: S
authors.lastname: Chakraborty
authors.forename: Z
authors.initials: Z
authors.lastname: Peng
mesh_terms: D019943:Amino Acid Substitution; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D007536:Isomerism; D007768:Lactalbumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D014508:Urea / Q000494:pharmacology
chemical_list: D004220:Disulfides; D014508:Urea; D007768:Lactalbumin; D019791:Guanidine; D003545:Cysteine