Total chemical synthesis and X-ray crystal structure of a protein diastereomer: [D-Gln 35]ubiquitin
Total chemical synthesis and X-ray crystal structure of a protein diastereomer: [D-Gln 35]ubiquitin
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DOI：
10.1002/anie.200463040
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发表时间：
2005-01-01
影响因子：
16.6
通讯作者：
Kent, SB
中科院分区：
文献类型：
--
作者：
Bang, D;Makhatadze, GI;Kent, SB
自然界中发现的蛋白质含有由l-氨基酸和甘氨酸组成的多肽链。多肽链上氨基酸的顺序决定了蛋白质分子的折叠三级结构，蛋白质的生物活性取决于这种折叠结构。非天然氨基酸已被用来构建自然界中不存在的蛋白质，以研究氨基酸的化学结构在折叠蛋白质分子的形成和稳定性中的作用特别是，将d-氨基酸残基取代到蛋白质分子中，其对胰岛素与胰岛素受体结合的影响已被探索最近，有人提出用氨基酸替代来增强蛋白质的稳定性[3a]，并通过用d-Ala残基代替Gly残基来探索K+离子通道选择性过滤器中的结构-功能关系。[3b]然而，尽管它们在研究蛋白质结构-功能关系方面具有潜在的重要性，但关于d-氨基酸残基的掺入如何影响决定蛋白质分子折叠、稳定性和功能的局部和全局构象的详细知识却很少。我们开始了解天然蛋白质是如何将d-氨基酸残基纳入其整体结构的。为了明确地探索d-氨基酸掺入对蛋白质分子局部和全局构象的扰动，我们合成并测定了化学工程球形蛋白泛素（76个氨基酸）的x射线晶体结构，该蛋白泛素在α螺旋的C-cap区以达米诺酸残基代替Gly - 35残基。该残基之所以被定位，是因为天然泛素[4]中Gly35残基（φ= 818, f= 58）的构象只允许存在于左手α螺旋中的l-氨基酸或d-氨基酸残基中。在此，我们报告：1)一种有效的全化学合成泛素的策略；2)直接观察拉尼-镍还原法(即l-Cys！l-Ala);3)天然泛素和泛素蛋白非对映体[d-Gln35]泛素的高分辨率晶体结构；4)天然泛素的分子结构与泛素蛋白非对映体具有惊人的相似性。本文还讨论了这些结果的意义。我们的研究重点是利用化学技术研究蛋白质的折叠和稳定性，因此我们开始建立一个有效的全化学合成模型蛋白泛素及其各种类似物我们试图使用最近开发的一罐结扎方法[9]共价组装三个无保护的肽段。然而，泛素分子不具有天然化学连接[10]所需的半胱氨酸残基（泛素的氨基酸序列如图1a所示）。我们注意到，人红细胞泛素有两个丙氨酸残基，位于第28位和第46位，因此适合用作连接位点（Swiss-Prot accession number P62988）因此，我们采用了一种蛋白质脱硫策略[11]，可以在Cys 28和Cys 46上使用天然化学连接，之后半胱氨酸残基转化为天然Ala28和Ala46残基。我们的综合策略如图1b所示。合成天然泛素的数据如图1所示。c端肽和肽-α硫酯采用人工分步Boc化学（Boc= butoxycarbonyl）“原位中和”方案，采用固相肽合成方法制备几百毫克的高纯度肽…
Proteins found in nature contain polypeptide chains made up of l-amino acids and glycine. The sequence of amino acids in the polypeptide chain defines the folded tertiary structure of the protein molecule, and the protein owes its biological activity to that folded structure. Non-natural amino acids have been used to construct proteins that are not found in nature to investigate the role of amino acid chemical structure in the formation and stability of the folded protein molecule.[1] In particular, substitution of d-amino acid residues into the protein molecule has been explored for its effects on the binding of insulin to insulin receptors.[2] More recently, damino acid substitution has been proposed for enhancing protein stability,[3a] and the structure–function relationships in the K+ ion channel selectivity filter have been explored by incorporation of a d-Ala residue in place of a Gly residue.[3b] However, despite their potential importance for investigating protein structure–function relationships, there is little detailed knowledge of how the incorporation of a d-amino acid residue affects the local and global conformations that define the folding, stability, and function of the protein molecule.We set out to understand how a native protein would incorporate a d-amino acid residue into its overall architecture. To definitively explore the perturbation of the local and global conformation in a protein molecule that results from incorporation of d-amino acids, we synthesized and determined the X-ray crystal structure of the chemically engineered globular protein ubiquitin (76 amino acids) with a damino acid residue in place of the Gly 35 residue in the C-cap region of an α helix. This residue was targeted because the conformation of the Gly35 residue (φ= 818, f= 58) in native ubiquitin [4] is only allowed for an l-amino acid in a lefthanded α helix or for a d-amino acid residue. Herein, we report: 1) an efficient strategy for the total chemical synthesis of ubiquitin; 2) the direct observation of the conservation of amino acid configuration after protein desulfurization by Raney-nickel reduction (that is, l-Cys! l-Ala); 3) highresolution crystal structures for native ubiquitin and for the ubiquitin protein diastereomer [d-Gln35] ubiquitin; and 4) a striking similarity between the molecular structures of native ubiquitin and the ubiquitin protein diastereomer. The significance of these results is also discussed. Our research focuses on the investigation of protein folding and stability by using chemical techniques, so we set out to establish an efficient total chemical synthesis of the model protein ubiquitin and its various analogues.[5] We sought to use the recently developed one-pot ligation method [9] to covalently assemble three unprotected peptide segments. However, the ubiquitin molecule does not have the cysteine residues that are needed for native chemical ligation [10](the amino acid sequence of ubiquitin is shown in Scheme 1a). We noted that human erythrocytic ubiquitin has two alanine residues, which are in positions 28 and 46 and so are suitably located for use as ligation sites (Swiss-Prot accession number P62988).[4] Thus, we adopted a proteindesulfurization strategy [11] that enables the use of native chemical ligation at Cys 28 and cys 46, after which the cysteine residues are converted into the native Ala28 and Ala46 residues. Our synthetic strategy is shown in Scheme 1b. Data for the synthesis of native ubiquitin are shown in Figure 1. The C-terminal peptide and the peptide-αthioesters were prepared by solid-phase peptide synthesis by using manual stepwise Boc-chemistry (Boc= butoxycarbonyl)“in situ neutralization” protocols.[12] Several hundred milligrams of high-purity peptide were …