The effect of pressure and guanidine hydrochloride on azurins mutated in the hydrophobic core
The effect of pressure and guanidine hydrochloride on azurins mutated in the hydrophobic core
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DOI：
10.1046/j.1432-1327.1999.00751.x
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发表时间：
1999-10-01
期刊：
影响因子：
--
通讯作者：
Finazzi-Agrò, A
中科院分区：
文献类型：
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作者：
Mei, G;Di Venere, A;Finazzi-Agrò, A
用荧光光谱和圆二色谱研究了盐酸胍对铜绿假单胞菌蓝铜蛋白天青蛋白的去折叠作用。变性转变可以通过简单的两态模型拟合。从天然状态到未折叠状态的总自由能变化为9.4 +/-0.4 kcal. mol（-1），而对于金属耗尽的酶（脱辅基天青蛋白）获得了较低的值（6.4 +/-0.4 kcal. mol（-1）），这表明铜原子起着重要的稳定作用。天青蛋白和脱辅基天青蛋白几乎不受流体静压力高达3000巴。定点诱变已被用来稳定的疏水核心的天青蛋白。特别地，疏水性残基Ile7或Phe110已被丝氨酸取代。盐酸胍解折叠的自由能变化分别为Ile7Ser和Phe110Ser的5.8 +/-0.3 kcal. mol（-1）和4.8 +/-0.3 kcal. mol（-1），表明两种突变体的稳定性远低于野生型蛋白。稳态荧光测量表明，突变的脱辅基蛋白即使在高压下也可以可逆变性。Ile7Ser和Phe110Ser的去折叠体积分别为-24 mL. mol（-1）和-55 mL. mol（-1），表明疏水残基的紧密堆积是蛋白质稳定性的基础.这表明疏水核心的正确组装是折叠过程中最早和最关键的事件之一，对蛋白质的从头设计具有重要意义。
The unfolding of the blue-copper protein azurin from Pseudomonas aeruginosa by guanidine hydrochloride, under nonreducing conditions, has been studied by fluorescence techniques and circular dichroism. The denaturation transition may be fitted by a simple two-state model. The total free energy change from the native to the unfolded state was 9.4 +/- 0.4 kcal.mol(-1), while a lower value (6.4 +/- 0.4 kcal.mol(-1)) was obtained for the metal depleted enzyme (apo-azurin) suggesting that the copper atom plays an important stabilization role. Azurin and apo-azurin were practically unaffected by hydrostatic pressure up to 3000 bar.Site-directed mutagenesis has been used to destabilize the hydrophobic core of azurin. In particular either hydrophobic residue Ile7 or Phe110 has been substituted with a serine. The free energy change of unfolding by guanidinium hydrochloride, resulted to be 5.8 +/- 0.3 kcal.mol(-1) and 4.8 +/- 0.3 kcal.mol(-1) for Ile7Ser and Phe110Ser, respectively, showing that both mutants are much less stable than the wild-type protein. The mutated apoproteins could be reversible denatured even by high pressure, as demonstrated by steady-state fluorescence measurements. The change in volume associated to the pressure-induced unfolding was estimated to be -24 mL.mol(-1) for Ile7Ser and -55 mL.mol(-1) for Phe110Ser.These results show that the tight packing of the hydrophobic residues that characterize the inner structure of azurin is fundamental for the protein stability. This suggests that the proper assembly of the hydrophobic core is one of the earliest and most crucial event in the folding process, bearing important implication for de novo design of proteins.