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Int. J. Mol. Sci. 2017, 18(9), 1996; doi:10.3390/ijms18091996

Effects of Metal Ions, Temperature, and a Denaturant on the Oxidative Folding Pathways of Bovine α-Lactalbumin

Department of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa 259-1292, Japan
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Received: 17 July 2017 / Revised: 11 September 2017 / Accepted: 13 September 2017 / Published: 16 September 2017
(This article belongs to the Collection Protein Folding)
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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. View Full-Text
Keywords: lactalbumin; protein folding; folding intermediate; disulfide bond; oxidation; reduction; calcium binding site; metal ion; temperature effect; selenoxide lactalbumin; protein folding; folding intermediate; disulfide bond; oxidation; reduction; calcium binding site; metal ion; temperature effect; selenoxide
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MDPI and ACS Style

Shinozaki, R.; Iwaoka, M. Effects of Metal Ions, Temperature, and a Denaturant on the Oxidative Folding Pathways of Bovine α-Lactalbumin. Int. J. Mol. Sci. 2017, 18, 1996.

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