In vitro unfolding of yeast multicopper oxidase Fet3p variants reveals unique role of each metal site.
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(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(m) of 50 degrees C; 1 K/min). Further inclusion of T2 (i.e., only T1 absent) increases overall stability by approximately 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(m) 50-55 degrees C; 1 K/min), followed by unfolding of domains 1 and 3 ( approximately 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.
Sedlák
Erik
E
Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251, USA.
Ziegler
Lynn
L
Kosman
Daniel J
DJ
Wittung-Stafshede
Pernilla
P
D001400:Azurin / Q000737:chemistry; D001665:Binding Sites; D002151:Calorimetry; D002570:Ceruloplasmin / Q000737:chemistry* / Q000378:metabolism*; D003300:Copper / Q000737:chemistry* / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006031:Glycosylation; D066298:In Vitro Techniques; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D013057:Spectrum Analysis
D029701:Saccharomyces cerevisiae Proteins; C050107:cupredoxin; D001400:Azurin; D003300:Copper; D002570:Ceruloplasmin; C085513:FET3 protein, S cerevisiae