Sedlák Erik, Ziegler Lynn, Kosman Daniel J, Wittung-Stafshede Pernilla
Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251, USA.
Proc Natl Acad Sci U S A. 2008 Dec 9;105(49):19258-63. doi: 10.1073/pnas.0806431105. Epub 2008 Nov 25.
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.
来自酿酒酵母的Fet3p是一种多铜氧化酶（MCO），它包含3个类铜蓝蛋白β桶结构域和4个铜离子，这些铜离子位于3个不同的金属位点（结构域3中的T1、T2以及结构域1和3之间界面处的双核T3）。为了更好地理解辅因子配位如何定义MCO蛋白的蛋白质结构和稳定性，我们在体外（pH 7）使用光谱和量热法评估了脱辅基和金属化形式的Fet3p的热解折叠。我们发现，脱辅基和不同全酶形式的Fet3p的解折叠反应是不可逆反应，且取决于扫描速率。脱辅基Fet3p中的结构域依次解折叠[热中点（T(m)）为45℃、62℃和72℃；升温速率1 K/min]。添加T3会在脱辅基结构中产生张力，导致结构域耦合解折叠且稳定性较低（T(m)为50℃；升温速率1 K/min）。进一步加入T2（即仅缺少T1）会使整体稳定性提高约5℃，但解折叠仍在一步中耦合进行。引入T1，产生完全负载的全酶Fet3p（或在缺少T2的情况下），会导致结构域3稳定，从而使结构域解折叠不耦合；结构域2的解折叠首先发生，同时伴随着铜位点的扰动（T(m) 50 - 55℃；升温速率1 K/min），随后是结构域1和3的解折叠（约65 - 70℃；升温速率1 K/min）。我们的结果表明，在MCO家族成员中，整体蛋白质稳定性和金属配位之间存在金属诱导的权衡。
