Copper stabilizes azurin by decreasing the unfolding rate.
Copper stabilizes azurin by decreasing the unfolding rate.
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铜通过降低解折叠速率来稳定天青蛋白。
DOI：
10.1006/abbi.2000.2369
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发表时间：
2001
期刊：
影响因子：
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通讯作者：
Wittung-Stafshede,P
中科院分区：
文献类型：
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作者：
Pozdnyakova,I;Guidry,J;Wittung-Stafshede,P
辅助因子通常稳定蛋白质的天然状态，然而，它们对折叠动力学的影响仍然知之甚少。在这里，我们报告了azurin，一种具有ß-桶结构的蓝铜蛋白，辅助因子在很大程度上稳定了天然蛋白（(GU) 23 kJ/mol）。热力学稳定性的提高是由于展开速度的降低，而折叠动力学不受影响。这表明蓝蛋白中铜位点的形成对折叠没有速率限制。大量的研究表明，蛋白质折叠的动力学和复杂程度各不相同。一个关键的发现是，虽然许多大蛋白质填充折叠中间体，但较小的蛋白质通常在没有动力学中间体的情况下直接折叠到天然状态（1-3）。对于这种小蛋白质，诸如序列、大小、稳定性和拓扑结构等参数可能在不同程度上影响蛋白质的折叠速率。最近的一项研究发现，一大批不相关的小蛋白质的拓扑结构和折叠速度之间存在统计学上显著的相关性(4)。研究表明，主要具有局部相互作用的蛋白质（如α-螺旋）具有快速的折叠转变，而具有更复杂拓扑结构的蛋白质（如ß-sheets）通常折叠得更慢。许多蛋白质需要辅因子的结合来执行其生物活性，这些分子在其同源辅因子存在的细胞环境中折叠。辅助因子已被证明可以稳定许多蛋白质的天然状态（5-8）。然而，由于动力学折叠研究经常是在没有潜在的复杂配体的情况下进行的，所以辅助因子影响折叠途径的方式仍然知之甚少。为了解决金属辅助因子在ß-桶蛋白折叠中的作用，我们比较了全息和载脂蛋白的折叠过程。铜绿假单胞菌azurin是一个小的（128个残基）蓝铜蛋白，具有ß-barrel结构，协调一个氧化还原活性铜[Cu (II) 2/Cu (I)对]（9-11）。拓扑结构为希腊密钥（图1，插入）；许多蛋白质中的结构基序。铜由两个组氨酸咪唑、一个半胱氨酸硫酸盐和两个较弱的轴向配体（蛋氨酸的硫和甘氨酸的羰基）配位。铜-半胱氨酸键的高共价性质赋予了Cu (II) -azurin独特的光谱特性：最重要的是，在625 nm处有强烈的吸收。一个包含azurin c端部分的小肽，包括三个铜配体，被证明可以特异性地结合Cu （II）并获得结构；因此，这段多肽被认为是折叠的起始位点（12）。Azurin可以采用折叠结构，不存在金属辅因子。载脂蛋白和全脂蛋白的晶体结构表明，在有金属和没有金属的情况下，它们的整体三维结构是相同的（9,11）。在化学变性剂盐酸胍（GuHCl）诱导下，Cu (II) -azurin展开后，铜最初仍然结合在多肽上，其配位包括半胱氨酸、一个组氨酸和一个第三配体（13-15）。然而，展开反应只是部分可逆的：半胱氨酸硫醇和变性态的Cu （II）之间发生缓慢的氧化还原反应（13）。这个缓慢的不可逆过程可以用一个线性时间函数来近似，从第一个阶段中减去它，得到每个变性条件下可逆展开步骤的速率和幅度（13）。根据这些数据，构建了Cu (II) -azurin的平衡展开曲线（再现于图1）。Cu （II）的展开动力学和平衡展开转变。
Cofactors often stabilize the native state of proteins, however, their effects on folding dynamics remain poorly understood. Here we report that in the case of azurin, a blue-copper protein with a ß-barrel structure, the cofactor largely stabilizes the native protein ((GU) 23 kJ/mol). The increase in thermodynamic stability is specifically due to a decrease in the unfolding speed, whereas the folding kinetics are not affected. This indicates that formation of the copper-site in azurin is not rate-limiting for folding. A considerable body of work has demonstrated that proteins fold with widely differing kinetics and with mechanisms of varying complexity. A key finding is that while many large proteins populate folding intermediates, smaller proteins often fold directly to the native state without kinetic intermediates (1–3). For such small proteins, parameters such as sequence, size, stability, and topology may to various extents affect the protein-folding rates. A recent study identified a statistically significant correlation between the topology and folding speed for a large set of small, unrelated proteins (4). It was shown that proteins with mainly local interactions (such as α-helices) have rapid folding transitions, whereas proteins with more complex topologies (such as ß-sheets) usually fold more slowly. Many proteins require the binding of cofactors to perform their biological activity, and these molecules fold in a cellular environment where their cognate cofactors are present. Cofactors have been shown to stabilize the native state of many proteins (5–8). However, since kinetic-folding studies are frequently conducted in the absence of potentially complicating ligands, the manner in which cofactors affect the folding pathway remains poorly understood. To address the role of a metal cofactor in folding of a ß-barrel protein, we compare the folding processes of holo-and apo-azurin. Pseudomonas aeruginosa azurin is a small (128-residue) blue-copper protein with a ß-barrel structure that coordinates a redox-active copper [Cu (II) 2/Cu (I) pair](9–11). The topology is that of a Greek key (Fig. 1, inset); a structural motif found in many proteins. The copper is coordinated by two histidine imidazoles, one cysteine thiolate, and two weaker axial (sulfur of methionine and the carbonyl of glycine) ligands. The highly covalent nature of the copper–cysteine bond gives Cu (II)–azurin unique spectroscopic properties: most importantly, an intense absorption at 625 nm. A small peptide comprising the C-terminal part of azurin, including three copper ligands, was shown to specifically bind Cu (II) and acquire structure; therefore, this stretch of the polypeptide was proposed to act as an initiation site for folding (12). Azurin can adopt a folded structure without the presence of metal cofactor. Crystal structures of apo-and holo-azurin have shown that the overall three-dimensional structure is identical with and without metal (9, 11). Upon Cu (II)–azurin unfolding, induced by chemical denaturant guanidine hydrochloride (GuHCl), the copper initially remains bound to the polypeptide with a coordination including the cysteine, one histidine, and a third ligand (13–15). However, the unfolding reaction is only partly reversible: a slow redox reaction takes place between the cysteine thiol and Cu (II) in the denatured state (13). This slow irreversible process could be approximated by a linear time function, which was subtracted from the first phase to yield the rate and amplitude of the reversible unfolding step at each denaturant condition (13). From this data the equilibrium-unfolding curve for Cu (II)–azurin was constructed (reproduced in Fig. 1). Both the unfolding kinetics and the equilibriumunfolding transition for Cu (II …