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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >In Vitro Unfolding Of Yeast Multicopper Oxidase Fet3p Variants Reveals Unique Role Of Each Metal Site
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In Vitro Unfolding Of Yeast Multicopper Oxidase Fet3p Variants Reveals Unique Role Of Each Metal Site

机译：酵母多铜氧化酶Fet3p变体的体外展开揭示了每种金属位点的独特作用

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摘要

Fet3p from Saccharomyces cerevisiae is a multicopper oxidase (MCO) that contains 3 cupredoxin-like /S-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 spectro-scopic 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 ℃, 62 ℃, and 72 ℃; 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 ℃; 1 K/min). Further inclusion of T2 (i.e., only T1 absent) increases overall stability by ≈5 ℃ 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 ≈; 1 K/min), followed by unfolding of domains 1 and 3 (≈65-70 ≈; 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.
机译：酿酒酵母（Saccharomyces cerevisiae）的Fet3p是一种多铜氧化酶（MCO），包含3个类似铜氧还蛋白的/ S-桶结构域和4个铜离子，它们位于3个不同的金属位点（结构域3，T2中的T1和结构域1之间的界面处的双核T3）和3）。为了更好地了解MCO蛋白质中辅因子的协同作用如何定义蛋白质的结构和稳定性，我们使用体外分光光度法和量热法评估了Fet3p的载脂蛋白和金属化形式的热解折叠。我们发现载脂蛋白和Fet3p的不同整体形式的展开反应是不可逆的反应，这取决于扫描速率。 apo-Fet3p中的结构域依次展开[温度中点（T_m）分别为45℃，62℃和72℃； 1 K / min]，T3的添加会在apo结构中施加应变，从而导致偶联结构域展开和低稳定性（T_m为50℃； 1 K / min）。进一步包含T2（即仅不存在T1）可将整体稳定性提高≈5℃，但展开仍需1步。引入T1，产生满载的holo-Fet3p（或在没有T2的情况下），导致结构域3稳定，从而解开结构域的解偶联；域2的展开首先与Cu位置扰动（T_m 50-55≈; 1 K / min）一起发生，然后是域1和3的展开（≈65-70≈; 1 K / min）。我们的结果表明，在MCO家族成员的整体蛋白质稳定性和金属配位之间存在金属诱导的权衡。

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