Noncovalent‐Interaction‐Promoted Ligation for Protein Labeling
Noncovalent‐Interaction‐Promoted Ligation for Protein Labeling
复制标题
用于蛋白质标记的非共价相互作用促进连接
DOI：
10.1002/cbic.201000007
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发表时间：
2010
期刊：
影响因子：
3.2
通讯作者：
K. Kikuchi
中科院分区：
文献类型：
--
作者：
Y. Hori;Yuka Egashira;Ryosuke Kamiura;K. Kikuchi
用荧光探针、同位素或其他功能化合物对蛋白质进行化学标记可提供通过传统生物化学或分子生物学方法无法获得的有价值的信息。 2] 特别是，表达蛋白连接（EPL）是分析蛋白质功能和结构的强大标记方法，并已广泛应用于目的蛋白（POI）的C端标记。在EPL中，具有C末端硫酯的蛋白质通过硫酯转移作用与具有N末端Cys的肽（或蛋白质）连接，随后形成酰胺键。该技术的关键组成部分是内含肽，它对于在蛋白质 C 末端生成硫酯至关重要。该技术的主要优点是连接反应具有相当大的化学选择性，因此可靠地获得了所需的半合成蛋白质。然而，反应通常效率低下，因此需要高浓度（毫米或亚毫米）的反应物才能有效连接。 [图3,5,6]因此，当反应物在水中的溶解度低时，使用增溶试剂例如盐酸胍或表面活性剂来溶解反应物。 6] 这一限制极大地阻碍了该方法应用于去除增溶剂后不保留其结构和功能的蛋白质。此外，除了少数例外，该技术尚未应用于哺乳动物细胞裂解物或活细胞中的蛋白质标记，因为蛋白质表达水平对于连接反应而言不够高。为了克服这些障碍，我们开发了一种基于非共价相互作用和内含肽介导的连接的新型蛋白质标记方法。该方法通过使用低浓度范围的硫酯蛋白和合成肽探针，可以在细胞裂解液中进行有效且特异的蛋白质标记。为了获得有效的蛋白质标记，将亲和标签引入到反应物中。这确保了反应物的局部浓度会增加。因此，预计连接反应会在低浓度下发生。选择形成反向平行卷曲线圈的亲和标签 Acid-a1 和 Base-a1 是因为这些肽很小（30 个氨基酸）并且相互作用机制众所周知。 9] 在本研究中，将卷曲螺旋的相互作用与内含肽介导的连接结合起来进行蛋白质标记（方案 1）。采用麦芽糖结合蛋白 (MBP) 作为模型 POI，并将 Acid-a1 融合到其 C 末端 (MBP-Acid)。内含肽进一步与MBP-Acid的C端连接，生成融合蛋白MBPAcid-In，其可以在MBP-Acid的C端形成硫酯。作为标记试剂，采用Fmoc固相法制备了N末端含有Cys的荧光素结合的Basea1（F-Base）。还合成了带有 C 末端硫酯的 Acid-a1 (Acid-T)，用于相互作用分析和与 F-Base 的模型连接。首先，测量圆二色性 (CD) 光谱，以确定尽管对肽进行了修饰以及 Acid-a1 与蛋白质结构域融合，但卷曲螺旋相互作用是否仍保留。 F-base 和 Acid-T 均显示出无序二级结构的圆二色光谱特征（图 1 A）。在 Acid-T 中添加 F 碱导致 208 和 222 nm 处的负面棉花效应显着增加。这些结果表明肽彼此相互作用形成α-螺旋结构。与肽相比，在没有 FBase 的情况下 MBP-Acid-In 的 CD 谱表明 MBP-Acid-In 包含有序的二级结构，包括 α-螺旋（图 1 B）。这与之前关于这些蛋白质晶体结构的报道是一致的。图11] MBP-Acid-In在远紫外区域的信号在F-Base存在下增强；这表明无螺旋含量增加。因此，这些结果表明F-碱基方案1。基于非共价卷曲螺旋相互作用和内含肽介导的连接的蛋白质标记原理。线圈用Base-a1和Acid-a1表示； POI : 感兴趣的蛋白质； 6-CF：6-羧基荧光素。
Chemical labeling of proteins with fluorescent probes, isotopes, or other functional compounds provides valuable information that cannot be obtained through conventional biochemistry or molecular biology methods. 2] In particular, expressed protein ligation (EPL) is a powerful labeling method for analyzing protein function and structure, and has been widely applied to C-terminal labeling of proteins of interest (POI). In EPL, proteins with a C-terminal thioester are linked to peptides (or proteins) with an N-terminal Cys by transthioesterification, and an amide bond is subsequently formed. The critical component of this technology is intein, which is essential for generating thioester at the protein’s C terminus. The major advantage of this technique is that the ligation reaction is considerably chemoselective and, thus, a desirable semisynthetic protein is reliably obtained. However, the reaction is generally inefficient and, therefore, a high concentration (mm or sub-mm) of reactants is required for efficient ligation. 3, 5, 6] Consequently, when the solubility of the reactants in water is low, a solubilization reagent, such as guanidine hydrochloride or a surfactant, is used to solubilize the reactants. 6] This limitation significantly hinders the application of this method to proteins that do not retain their structure and function after removal of the solubilizing reagent. In addition, barring a few exceptions, this technique has not been applied to protein labeling in mammalian cell lysate or living cells because the protein expression level is not high enough for the ligation reaction. To overcome these obstacles, we developed a novel protein labeling method based on noncovalent interaction and intein-mediated ligation. This method allows efficient and specific protein labeling in cell lysate by using a low concentration range of thioester protein and synthetic peptide probe. In order to attain efficient protein labeling, affinity tags were introduced into the reactants. This ensured that the local concentrations of the reactants would increase. As a result, it is expected that the ligation reaction would occur at low concentration. The affinity tags, Acid-a1 and Base-a1, which form an antiparallel coiled coil, were chosen because these peptides are small (30 amino acids) and the interaction mechanism is well-known. 9] In the present research, interaction of the coiled coil was combined with the intein-mediated ligation for protein labeling (Scheme 1). Maltose binding protein (MBP) was employed as a model POI and Acid-a1 was fused to its C terminus (MBP-Acid). The intein was further connected to the C terminus of MBP-Acid to generate a fusion protein MBPAcid-In, which could form a thioester in the C terminus of MBP-Acid. As a labeling reagent, fluorescein-conjugated Basea1 (F-Base), which contains Cys at its N terminus, was prepared with Fmoc solid phase method. Acid-a1 with C-terminal thioester (Acid-T) was also synthesized for interaction analysis and model ligation with F-Base. First, circular dichroism (CD) spectra were measured to determine whether coiled-coil interaction was retained despite the modification of the peptides and the fusion of Acid-a1 with protein domains. Both F-base and Acid-T showed a CD spectrum characteristic of disordered secondary structures (Figure 1 A). The addition of F-base to Acid-T resulted in a dramatic increase in negative Cotton effects at 208 and 222 nm. These results indicate that the peptides interact with each other to form a-helical structures. In contrast with the peptides, the CD spectrum of MBP-Acid-In in the absence of FBase showed that MBP-Acid-In contains ordered secondary structures, including a-helices (Figure 1 B). This is consistent with previous reports on the crystal structures of those proteins. 11] The signal of MBP-Acid-In in the far-UV region intensified in the presence of F-Base; this demonstrates that the ahelix content increased. Thus, these results suggest that F-base Scheme 1. Principle of protein labeling based on noncovalent coiled-coil interaction and intein-mediated ligation. Coiled coils are represented by Base-a1 and Acid-a1; POI : protein of interest; 6-CF: 6-carboxyfluorescein.
影响因子：
15
作者：
Pellois, JP;Hahn, ME;Muir, TW
通讯作者：
Muir, TW
影响因子：
4
作者：
Gallagher, Sarah S.;Sable, Julia E.;Sheetz, Michael P.;Cornish, Virginia W.
通讯作者：
Cornish, Virginia W.