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首页> 外文期刊>Journal of Molecular Biology >Stabilisation of a (betaalpha)8-barrel protein designed from identical half barrels.
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Stabilisation of a (betaalpha)8-barrel protein designed from identical half barrels.

机译：从相同的半桶设计的（betaalpha）8桶蛋白的稳定性。

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It has been suggested that the common (betaalpha)(8)-barrel enzyme fold has evolved by the duplication and fusion of identical (betaalpha)(4)-half barrels, followed by the optimisation of their interface. In our attempts to reconstruct these events in vitro we have previously linked in tandem two copies of the C-terminal half barrel HisF-C of imidazole glycerol phosphate synthase from Thermotoga maritima and subsequently reconstituted in the fusion construct HisF-CC a salt bridge cluster present in wild-type HisF. The resulting recombinant protein HisF-C*C, which was produced in an insoluble form and unfolded with low cooperativity at moderate urea concentrations has now been stabilised and solubilised by a combination of random mutagenesis and selection in vivo. For this purpose, Escherichia coli cells were transformed with a plasmid-based gene library encoding HisF-C*C variants fused to chloramphenicol acetyltransferase (CAT). Stable and soluble variants were identified by the survival of host cells on solid medium containing high concentrations of the antibiotic. The selected HisF-C*C proteins, which were characterised in vitro in the absence of CAT, contained eight different amino acid substitutions. One of the exchanges (Y143C) stabilised HisF-C*C by the formation of an intermolecular disulfide bond. Three of the substitutions (G245R, V248M, L250Q) were located in the long loop connecting the two HisF-C copies, whose subsequent truncation from 13 to 5 residues yielded the stabilised variant HisF-C*C Delta. From the remaining substitutions, Y143H and V234M were most beneficial, and molecular dynamics simulations suggest that they strengthen the interactions between the half barrels by establishing a hydrogen-bonding network and an extensive hydrophobic cluster, respectively. By combining the loop deletion of HisF-C*C Delta with the Y143H and V234M substitutions, the variant HisF-C**C was generated. Recombinant HisF-C**C is produced in soluble form, forms a pure monomer with its tryptophan residues shielded from solvent and unfolds with similar cooperativity as HisF. Our results show that, starting from two identical and fused half barrels, few amino acid exchanges are sufficient to generate a highly stable and compact (betaalpha)(8)-barrel protein with wild-type like structural properties.
机译：已经提出，通过重复和融合相同的（betaα）（4）-半桶，并对其界面进行优化，共同的beta-（8）-桶酶折叠得到了发展。在我们尝试体外重建这些事件的过程中，我们先前串联了两个副本，分别来自海栖嗜热菌的咪唑甘油磷酸合酶的C末端半桶HisF-C，随后在融合构建体HisF-CC中重建了一个盐桥簇在野生型HisF中。产生的重组蛋白HisF-C * C，以不溶形式产生，在中等尿素浓度下以低协同性展开，现已通过随机诱变和体内选择相结合的方法得以稳定和溶解。为此目的，用基于质粒的基因文库转化大肠杆菌细胞，该基因文库编码与氯霉素乙酰基转移酶（CAT）融合的HisF-C * C变体。通过宿主细胞在含有高浓度抗生素的固体培养基上的存活来鉴定稳定和可溶的变体。所选的HisF-C * C蛋白在没有CAT的情况下进行了体外表征，包含8个不同的氨基酸取代。其中一种交换（Y143C）通过形成分子间二硫键来稳定HisF-C * C。三个取代（G245R，V248M，L250Q）位于连接两个HisF-C拷贝的长环中，其随后的13至5个残基截短产生了稳定的变体HisF-C * C Delta。从其余的取代中，Y143H和V234M是最有益的，并且分子动力学模拟表明它们分别通过建立氢键网络和广泛的疏水簇来增强半桶之间的相互作用。通过结合HisF-C * C Delta的环缺失与Y143H和V234M取代，产生了变体HisF-C ** C。重组HisF-C ** C以可溶形式产生，形成纯色单体，其色氨酸残基被溶剂屏蔽，并且以与HisF相似的协同性展开。我们的结果表明，从两个相同且融合的半桶开始，很少的氨基酸交换足以生成具有野生型结构特性的高度稳定且紧凑的β-（8）-桶蛋白。

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