Kinetic control of one-pot trans-splicing reactions by using a wild-type and designed split intein.
Kinetic control of one-pot trans-splicing reactions by using a wild-type and designed split intein.
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DOI：
10.1002/anie.201102909
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发表时间：
2011-07-11
影响因子：
16.6
通讯作者：
Muir, Tom W.
中科院分区：
文献类型：
--
作者：
Shah, Neel H.;Vila-Perello, Miquel;Muir, Tom W.
库仑力在促进蛋白质-蛋白质相互作用中起着重要作用。已经证明，两个相互作用的蛋白质之间的强静电势与快速的结合速度和强烈的结合亲和力相关。[1]实际上，这一原理已被用来增强工程蛋白质界面的结合性质。[2]然而，很少有研究集中于利用分子间离子对来调节蛋白质-蛋白质相互作用的专一性。[3-5]自然分裂的整型是一个潜在的有趣的静电驱动专一性工程系统，因为形成催化活性结构需要两个带相反电荷的原基的结合。在其内源环境中，分裂内含子催化必要基因片段的蛋白质反式剪接(PTS，方案1)，因此面临进化压力，需要快速与高保真结合，并在催化后缓慢解离，以防止无效复合体的重新形成。由于PTS具有连接反式侧翼蛋白序列(外显子)的能力，因此分裂内含子在许多生物技术应用中得到了广泛的应用。[6]PTS的应用包括用于核磁共振光谱的蛋白质的节段性同位素标记，[7]蛋白质固定化，[8]用外源探针标记蛋白质，[9]蛋白质和肽的环化，[10]和蛋白质功能的控制。自然分裂内含子的序列比对显示高度保守的电荷分离，酸性残基集中在N-内含子上的特定位置，碱性残基保守在C-内含子上(N-末端和C-末端原始体，图1a)。[13]此外，对内含子家族的生物信息学序列分析表明，这种电荷分离在自然分裂内含子中比在完整内含子中更普遍(图1c)。有趣的是，当保守的带电残基被映射到由发菜野生型DNAE内含子(NpuWT)的核磁共振光谱确定的结构上时，发现许多参与了分子间离子对和离子三联体(图1b)。
Coulombic forces play an important role in facilitating protein–protein interactions. It has been demonstrated that a strong electrostatic potential between two interacting proteins correlates with a fast rate of association and a strong binding affinity.[1] Indeed, this principle has been exploited to enhance the binding properties of engineered protein interfaces.[2] Nonetheless, little research has focused on utilizing intermolecular ion pairs to modulate specificity in protein–protein interactions.[3–5] Naturally split inteins are a potentially interesting system for engineering electrostatically driven specificity, as the formation of a catalytically competent structure requires the association of two oppositely charged protomers. In their endogenous environment, split inteins catalyze protein transsplicing (PTS, Scheme 1) of essential gene fragments, and thus are under evolutionary pressure to associate rapidly with high fidelity and dissociate slowly post-catalysis to prevent the re-formation of unproductive complexes. Out of their native context, split inteins have seen widespread use in a number of biotechnological applications, as a result of their capacity to ligate flanking protein sequences (exteins) in trans.[6] Applications of PTS include segmental isotopic labeling of proteins for NMR spectroscopy,[7] protein immobilization,[8] the labeling of proteins with extrinsic probes,[9] protein and peptide cyclization,[10] and control of protein function.[11, 12]Despite the utility of PTS, little is known about what drives efficient association of split intein fragments. Sequence alignments of naturally split inteins show highly conserved charge segregation, with acidic residues concentrated at specific positions on the N-intein and basic residues conserved on the C-intein (N-and C-terminal protomers, Figure 1a).[13] Furthermore, a bioinformatic sequence analysis of the intein family indicates that this charge segregation is significantly more prevalent in naturally split inteins than intact ones (Figure 1c). Interestingly, when the conserved charged residues are mapped onto the structure determined by NMR spectrocopy of the wild-type DnaE intein from Nostoc punctiforme (NpuWT), many are found to be participating in intermolecular ion pairs and ion triads (Figure 1b).[14]
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