title: Two-state folding of lysozyme versus multiple-state folding of alpha-lactalbumin illustrated by the technique of disulfide scrambling.
abstract: The folding of lysozyme and of alpha-lactalbumin exhibits vastly different kinetics and pathways. Existing evidence indicates that folding intermediates of alphaLA form a well-populated equilibrium molten globule state that is absent in the case of hen lysozyme. We demonstrate here such divergent folding mechanisms of lysozyme and alphaLA using the technique of disulfide scrambling. Two extensively unfolded homologous isomers (beads-form) of lysozyme (Cys6-Cys30, Cys64-Cys76, Cys80-Cys94, Cys115-Cys127) and alphaLA (Cys6-Cys28, Cys61-Cys73, Cys77-Cys91, Cys111-Cys120) were allowed to refold in parallel to form the native protein. Folding kinetics was measured by the recovery of the native structure. Folding intermediates, which illustrate the folding pathway, were trapped by quenching disulfide shuffling and were analyzed by reversed-phase high-pressure liquid chromatography. The results revealed that under identical folding conditions, the folding rate of lysozyme is about 30-fold faster than that of alphaLA. Folding intermediates of lysozyme are far less heterogeneous and sparsely populated than those of alphaLA. Numerous predominant on-pathway and off-pathway intermediates observed along the folding pathway of alphaLA are conspicuously absent in the case of lysozyme. The difference is most striking under fast folding conditions performed in the presence of protein disulfide isomerase. Under these conditions, folding of lysozyme undergoes a near two-state mechanism without accumulation of stable folding intermediates.
authors.affiliation: Research Center for Protein Chemistry, Institute of Molecular Medicine, Department of Biochemistry and Molecular Biology, The University of Texas, Houston, Texas 77030, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Jui-Yoa
authors.initials: JY
authors.lastname: Chang
mesh_terms: D000818:Animals; D002417:Cattle; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation
chemical_list: D004220:Disulfides; D007768:Lactalbumin; D009113:Muramidase; D003545:Cysteine