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In vitro unfolding of yeast multicopper oxidase Fet3p variants reveals unique role of each metal site
Umeå University, Faculty of Science and Technology, Chemistry.
2008 (English)In: PNAS Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, no 49, 19258-63 p.Article in journal (Refereed) Published
Abstract [en]

Fet3p from Saccharomyces cerevisiae is a multicopper oxidase (MCO) that contains 3 cupredoxin-like β-barrel domains and 4 copper ions located in 3 distinct metal sites (T1 in domain 3, T2, and the binuclear T3 at the interface between domains 1 and 3). To better understand how protein structure and stability is defined by cofactor coordination in MCO proteins, we assessed thermal unfolding of apo and metallated forms of Fet3p by using spectroscopic and calorimetric methods in vitro (pH 7). We find that unfolding reactions of apo and different holo forms of Fet3p are irreversible reactions that depend on the scan rate. The domains in apo-Fet3p unfold sequentially [thermal midpoint (Tm) of 45 °C, 62 °C, and 72 °C; 1 K/min]. Addition of T3 imposes strain in the apo structure that results in coupled domain unfolding and low stability (Tm of 50 °C; 1 K/min). Further inclusion of T2 (i.e., only T1 absent) increases overall stability by ≈5 °C but unfolding remains coupled in 1 step. Introduction of T1, producing fully-loaded holo-Fet3p (or in the absence of T2), results in stabilization of domain 3, which uncouples unfolding of the domains; unfolding of domain 2 occurs first along with Cu-site perturbations (Tm 50–55 °C; 1 K/min), followed by unfolding of domains 1 and 3 (≈65–70 °C; 1 K/min). Our results suggest that there is a metal-induced tradeoff between overall protein stability and metal coordination in members of the MCO family.

Place, publisher, year, edition, pages
2008. Vol. 105, no 49, 19258-63 p.
Keyword [en]
spectroscopy, calorimetry, cupredoxin, copper-binding protein
URN: urn:nbn:se:umu:diva-11206DOI: doi:10.1073/pnas.0806431105OAI: diva2:150877
Available from: 2008-12-11 Created: 2008-12-11 Last updated: 2011-01-10Bibliographically approved

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