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In vitro unfolding of yeast multicopper oxidase Fet3p variants reveals unique role of each metal site

Authors :
Sedlak, Erik
Ziegler, Lynn
Kosman, Daniel J.
Wittung-Stafshede, Pernilla
Source :
Proceedings of the National Academy of Sciences of the United States. Dec 9, 2008, Vol. 105 Issue 49, p19258, 6 p.
Publication Year :
2008

Abstract

Fet3p from Saccharomyces cerevisiae is a multicopper oxidase (MCO) that contains 3 cupredoxin-like [beta]-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 ([T.sub.m]) of 45 [degrees]C, 62[degrees]C, and 72[degrees]C; 1 K/min]. Addition of T3 imposes strain in the apo structure that results in coupled domain unfolding and low stability ([T.sub.m] of 50[degrees]C; 1 K/min). Further inclusion of T2 (i.e., only T1 absent) increases overall stability by [approximately equal to]5[degrees]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 ([T.sub.m] 50-55[degrees]C; 1 K/min), followed by unfolding of domains 1 and 3 ([approximately equal to]65-70[degrees]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. spectroscopy | calorimetry | cupredoxin | copper-binding protein

Details

Language :
English
ISSN :
00278424
Volume :
105
Issue :
49
Database :
Gale General OneFile
Journal :
Proceedings of the National Academy of Sciences of the United States
Publication Type :
Academic Journal
Accession number :
edsgcl.191315473