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Synergistic Effects of Copper Sites on Apparent Stability of Multicopper Oxidase, Fet3p
- Source :
- International Journal of Molecular Sciences, Vol 19, Iss 1, p 269 (2018)
- Publication Year :
- 2018
- Publisher :
- MDPI AG, 2018.
-
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.
Details
- Language :
- English
- ISSN :
- 14220067
- Volume :
- 19
- Issue :
- 1
- Database :
- Directory of Open Access Journals
- Journal :
- International Journal of Molecular Sciences
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.87e7256718c54396bc0706be0f980b91
- Document Type :
- article
- Full Text :
- https://doi.org/10.3390/ijms19010269