Chitin-binding protein 21 (CBP21) from Serratia marcescens is a lytic polysaccharide monooxygenase that contains a copper ion as a cofactor. We aimed to elucidate the unfolding mechanism of CBP21 and the effects of Cu2+ on its structural stability at pH 5.0. Thermal unfolding of both apo- and holoCBP21 was reversible. ApoCBP21 unfolded in a simple two-state transition manner. The peak temperature of the DSC curve, tp , for holoCBP21 (74.4°C) was about nine degrees higher than that for apoCBP21 (65.6°C). The value of tp in the presence of excess Cu2+ was around 75°C, indicating that Cu2+ does not dissociate from the protein molecule during unfolding. The unfolding mechanism of holoCBP21 was considered to be as follows: N∙Cu2+ ⇌ U∙Cu2+ , where N and U represent the native and unfolded states, respectively. Urea-induced equilibrium unfolding analysis showed that holoCBP21 was stabilized by 35 kJ mol-1 in terms of the Gibbs energy change for unfolding (pH 5.0, 25°C), compared with apoCBP21. The increased stability of holoCBP21 was considered to result from the structural stabilization of the protein-Cu2+ complex itself.

中文翻译：

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展开 CBP21，一种裂解性多糖单加氧酶，不解离其铜离子辅因子

来自粘质沙雷氏菌的几丁质结合蛋白 21 (CBP21) 是一种溶解性多糖单加氧酶，含有铜离子作为辅助因子。我们旨在阐明 CBP21 的展开机制以及 Cu2+ 在 pH 5.0 下对其结构稳定性的影响。apo-和holoCBP21 的热解折叠是可逆的。ApoCBP21 以简单的两态转换方式展开。HoloCBP21（74.4°C）的DSC曲线峰值温度tp比apoCBP21（65.6°C）高约9度。存在过量 Cu2+ 时的 tp 值约为 75°C，表明 Cu2+ 在解折叠过程中不会与蛋白质分子解离。HoloCBP21 的展开机制被认为如下： N∙Cu2+ ⇌ U∙Cu2+ ，其中 N 和 U 分别代表原始状态和展开状态。尿素诱导的平衡解折叠分析表明，与 apoCBP21 相比，holoCBP21 在解折叠的 Gibbs 能量变化（pH 5.0，25°C）方面稳定了 35 kJ mol-1。holoCBP21 稳定性的增加被认为是由于蛋白质-Cu2+ 复合物本身的结构稳定性。