Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A -: How important is the S-S bond for folding and stability?
Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A -: How important is the S-S bond for folding and stability?
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DOI：
10.1046/j.1432-1033.2000.01501.x
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发表时间：
2000-07-01
期刊：
影响因子：
--
通讯作者：
Sjölin, L
中科院分区：
文献类型：
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作者：
Bonander, N;Leckner, J;Sjölin, L
天青蛋白具有包含 8 个 β 链和 1 个 α 螺旋的 β 桶折叠。残基 3 和 26 之间的二硫键连接 β 链 β 1 和 β 3 的 N 末端。构建了三种缺乏二硫键的突变蛋白：C3A/C26A、C3A/C26I 以及 C3A/S25R/C26A/K27R 突变体中的推定盐桥 (SB)。所有三种突变体都表现出与野生型蛋白质相似的光谱特性。此外，C3A/C26A 突变体的晶体结构以 2.0 埃分辨率测定，与野生型蛋白相比，唯一的差异在于突变的邻近性。突变体在比野生型蛋白质低 10-22 摄氏度的温度下失去 628 nm 电荷转移带。通过荧光和 CD 光谱监测盐酸胍 (GdnHCl) 诱导的变性来研究负载锌的 C3A/C26A 突变体的折叠。使用 CD 和荧光光谱观察 1.3 m GdnHCl 处的折叠平衡中点。 CD测定的折叠自由能为-24.9 kJ.mol(-1)，与携带完整二硫键的野生型Zn2+蛋白相比，不稳定程度约为20 kJ.mol(-1)，表明二硫键对于赋予天青蛋白稳定的结构很重要。无论是在折叠能还是热变性方面，C3A/C26I突变体都比C3A/C26A更稳定，而SB突变体则不太稳定。对于二硫键缺陷的 C3A/C26A 突变体，未观察到野生型 Zn2+-天青蛋白的折叠中间体。 C3A/C26A 突变体的解折叠速率与野生型蛋白相似，表明突变位点不参与早期解折叠反应。
Azurin has a beta-barrel fold comprising eight beta-strands and one alpha helix. A disulfide bond between residues 3 and 26 connects the N-termini of beta strands beta 1 and beta 3. Three mutant proteins lacking the disulfide bond were constructed, C3A/C26A, C3A/C26I and a putative salt bridge (SB) in the C3A/S25R/C26A/K27R mutant. All three mutants exhibit spectroscopic properties similar to the wild-type protein. Furthermore, the crystal structure of the C3A/C26A mutant was determined at 2.0 Angstrom resolution and, in comparison to the wild-type protein, the only differences are found in the immediate proximity of the mutation. The mutants lose the 628 nm charge-transfer band at a temperature 10-22 degrees C lower than the wild-type protein. The folding of the zinc loaded C3A/C26A mutant was studied by guanidine hydrochloride (GdnHCl) induced denaturation monitored both by fluorescence and CD spectroscopy. The midpoint in the folding equilibrium, at 1.3 m GdnHCl, was observed using both CD and fluorescence spectroscopy. The free energy of folding determined from CD is -24.9 kJ.mol(-1), a destabilization of approximate to 20 kJ.mol(-1) compared to the wild-type Zn2+-protein carrying an intact disulfide bond, indicating that the disulfide bond is important for giving azurin its stable structure. The C3A/C26I mutant is more stable and the SB mutant is less stable than C3A/C26A, both in terms of folding energy and thermal denaturation. The folding intermediate of the wild-type Zn2+-azurin is not observed for the disulfide-deficient C3A/C26A mutant. The rate of unfolding for the C3A/C26A mutant is similar to that of the wild-type protein, suggesting that the site of the mutation is not involved in an early unfolding reaction.