Evidence for the underlying cause of diversity of the disulfide folding pathway.
The pathways of oxidative folding of disulfide proteins exhibit a high degree of diversity, which is illustrated by the varied extent of (a) the heterogeneity of folding intermediates, (b) the predominance of intermediates containing native disulfide bonds, and (c) the level of accumulation of fully oxidized scrambled isomers as intermediates. BPTI and hirudin exemplify two extreme cases of such divergent folding pathways. We previously proposed that the underlying cause of this diversity is associated with the degree of stability of protein subdomains. Here we present compelling evidence that substantiates this hypothesis by studying the folding pathway of alphaLA-IIA. alphaLA-IIA is a partially folded intermediate of alpha-lactalbumin (alphaLA). It comprises a structured beta-sheet (calcium-binding) domain linked by two native disulfide bonds (Cys(61)-Cys(77) and Cys(73)-Cys(91)) and a disordered alpha-helical domain with four free cysteines (Cys(6), Cys(28), Cys(111), and Cys(120)). Purified alphaLA-IIA was allowed to refold without and with stabilization of its structured beta-sheet domain by calcium. In the absence of calcium, the folding pathway of alphaLA-IIA resembles that of hirudin, displaying a highly heterogeneous population of folding intermediates, including fully oxidized scrambled species. Upon stabilization of its beta-sheet domain by bound calcium, oxidative folding of alphaLA-IIA undergoes a pathway conspicuously similar to that of BPTI, exhibiting limited species of folding intermediates containing mostly native disulfide bonds.
Chang
Jui-Yoa
JY
Research Center for Protein Chemistry, Institute of Molecular Medicine, and Department of Biochemistry and Molecular Biology, The University of Texas, Houston, Texas 77030, USA. Rowen.Chang@uth.tmc.edu
D000818:Animals; D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D007768:Lactalbumin / Q000737:chemistry*; D008956:Models, Chemical; D010084:Oxidation-Reduction; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics
D002135:Calcium-Binding Proteins; D004220:Disulfides; D007768:Lactalbumin; D003545:Cysteine; D002118:Calcium