Abstract
Bovine α-lactalbumin (αLA) has four disulfide (SS) bonds in the native form (N). On the oxidative folding pathways of this protein, two specific SS folding intermediates, i.e., (61–77, 73–91) and des[6–120], which have two and three native SS bonds, respectively, accumulate predominantly in the presence of Ca2+. In this study, we reinvestigated the pathways using a water-soluble cyclic selenoxide reagent, trans-3,4-dihydroxyselenolane oxide (DHSox), as a strong and quantitative oxidant to oxidize the fully reduced form (R). In the presence of ethylenediaminetetraacetic acid (EDTA) (under a metal-free condition), SS formation randomly proceeded, and N did not regenerate. On the other hand, two specific SS intermediates transiently generated in the presence of Ca2+. These intermediates could be assigned to (61–77, 73–91) and des[6–120] having two common SS bonds, i.e., Cys61-Cys77 and Cys73-Cys91, near the calcium binding pocket of the β-sheet domain. Much faster folding to N was observed in the presence of Mn2+, whereas Na+, K+, Mg2+, and Zn2+ did not affect the pathways. The two key intermediates were susceptible to temperature and a denaturant. The oxidative folding pathways revealed were significantly different from those of hen egg white lysozyme, which has the same SS-bonding pattern as αLA, suggesting that the folding pathways of SS-containing proteins can alter depending on the amino acid sequence and other factors, even when the SS-bond topologies are similar to each other.
Highlights
Folding of disulfide (SS)-rich proteins proceeds via sequential formation of the SS bonds between two cysteine (Cys) residues
The oxidative folding of a protein is catalyzed by various redox enzymes, such as protein disulfide isomerases [3,4,5], which effectively promote both oxidation (SS formation) and isomerization (SS rearrangement) in the endoplasmic reticulum to guide a nascent polypeptide chain to the native protein
For other proteins, such as bovine pancreatic ribonuclease A (RNase A) and hen egg white lysozyme (HEL), the folding proceeds through the pathways lying between these two extreme cases [13,14,15,16]
Summary
Folding of disulfide (SS)-rich proteins proceeds via sequential formation of the SS bonds between two cysteine (Cys) residues This folding process involves two chemical events: random SS formation to generate a mixture of various SS intermediates and SS rearrangement to produce distinct SS intermediates having partially native-like fold [1,2]. The other extreme is the folding of bovine pancreatic trypsin inhibitor (BPTI) having three SS bonds, for which only distinct SS intermediates with partially native-like fold sequentially generate from the fully reduced form (R) to N [12] For other proteins, such as bovine pancreatic ribonuclease A (RNase A) and hen egg white lysozyme (HEL), the folding proceeds through the pathways lying between these two extreme cases [13,14,15,16]. Incorrect protein folding (misfolding) may lead to the formation of protein deposits, inclusions, fibrils, etc., associated with various human diseases [17]
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