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Int. J. Mol. Sci. 2018, 19(1), 269; https://doi.org/10.3390/ijms19010269

Synergistic Effects of Copper Sites on Apparent Stability of Multicopper Oxidase, Fet3p

1
Center for Interdisciplinary Biosciences, Technology and Innovation Park P.J. Šafárik University, Jesenna 5, 041 54 Košice, Slovakia
2
Department of Biology and Biological Engineering, Division of Chemical Biology, Chalmers University of Technology, 412 96 Gothenburg, Sweden
*
Authors to whom correspondence should be addressed.
Received: 28 November 2017 / Revised: 19 December 2017 / Accepted: 12 January 2018 / Published: 16 January 2018
(This article belongs to the Collection Protein Folding)
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Abstract

Saccharomyces cerevisiae Fet3p is a multicopper oxidase that contains three cupredoxin-like domains and four copper ions located in three distinct metal sites (T1 in domain 3; T2 and the binuclear T3 at the interface between domains 1 and 3). To probe the role of the copper sites in Fet3p thermodynamic stability, we performed urea-induced unfolding experiments with holo-, apo- and three partially-metallated (T1, T2 and T1/T2 sites depleted of copper) forms of Fet3p. Using a combination of spectroscopic probes (circular dichroism, fluorescence intensity and maximum, 8-anilinonaphthalene-1-sulfonic acid (ANS) emission, oxidase activity and blue color), we reveal that all forms of Fet3p unfold in a four-state reaction with two partially-folded intermediates. Using phase diagrams, it emerged that Fet3p with all copper sites filled had a significantly higher stability as compared to the combined contributions of the individual copper sites. Hence, there is long-range inter-domain communication between distal copper sites that contribute to overall Fet3p stability. View Full-Text
Keywords: multicopper oxidases MCO; cupredoxin-like domain; phase diagram method; multidomain protein stability; Fet3p multicopper oxidases MCO; cupredoxin-like domain; phase diagram method; multidomain protein stability; Fet3p
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Sedlák, E.; Žoldák, G.; Wittung-Stafshede, P. Synergistic Effects of Copper Sites on Apparent Stability of Multicopper Oxidase, Fet3p. Int. J. Mol. Sci. 2018, 19, 269.

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