Architectural digest: Thermodynamic stability and domain structure of a consensus monomeric globin
Architectural digest: Thermodynamic stability and domain structure of a consensus monomeric globin
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DOI：
10.1016/j.bpj.2023.06.016
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发表时间：
2023-08-08
影响因子：
3.4
通讯作者：
Lecomte，Juliette T. J.
中科院分区：
文献类型：
--
作者：
Grundman，Jaime E. Martinez;Johnson，Eric A.;Lecomte，Juliette T. J.
人工合成的蛋白质具有较高的热力学稳定性和保守的生物活性，因此受到人们的广泛关注。在这里，我们应用的共识的方法，ab型血红素结合蛋白，检查的贡献，一个可解离的辅因子，以增强稳定性和化学后果，创造一个通用的血红素环境。我们针对第1组截短血红蛋白（TrHb 1）亚家族的蛋白质，因为它们的小尺寸（约120个残基）和易于表征。一级结构，来自一组精选的1000个代表性序列，产生了一个高度可溶的共识珠蛋白（cGlbN）富含酸性残基。光学和NMR光谱显示高亲和力血红素结合在预期的网站和两个方向。在中性pH值下，近端和远端铁协调实现了一对组氨酸残基，在一些天然TrHb 1 s中观察到的，并与不稳定的连接的远端侧。与研究的TrHb 1 s相反，TrHb 1 s在血红素结合后会发生额外的折叠，cGlbN显示出相同程度的二级结构，无论血红素是否与蛋白质相关。变性需要盐酸胍，并表明载脂蛋白和holoprotein展开在两个过渡-第一个（发生与中点的10.2 M）被转移到更高的变性剂浓度的holoprotein（10.3.7 M），并反映由于血红素结合稳定，而第二个过渡（10.6.2 M）是共同的两种形式。因此，共有序列稳定的蛋白质，但暴露存在两个单独的合作亚结构域内的珠蛋白架构，作为一个单一的结构域在TrHb 1的典型稳定性掩盖。结果表明，在人工血红素蛋白中可以控制特定的化学或热力学特征。
Artificial proteins representing the consensus of a set of homologous sequences have attracted attention for their increased thermodynamic stability and conserved activity. Here, we applied the consensus approach to ab-type heme-binding protein to inspect the contribution of a dissociable cofactor to enhanced stability and the chemical consequences of creating a generic heme environment. We targeted the group 1 truncated hemoglobin (TrHb1) subfamily of proteins for their small size (∼120 residues) and ease of characterization. The primary structure, derived from a curated set of ∼300 representative sequences, yielded a highly soluble consensus globin (cGlbN) enriched in acidic residues. Optical and NMR spectroscopies revealed high-affinity heme binding in the expected site and in two orientations. At neutral pH, proximal and distal iron coordination was achieved with a pair of histidine residues, as observed in some natural TrHb1s, and with labile ligation on the distal side. As opposed to studied TrHb1s, which undergo additional folding upon heme binding, cGlbN displayed the same extent of secondary structure whether the heme was associated with the protein or not. Denaturation required guanidine hydrochloride and showed that apo- and holoprotein unfolded in two transitions—the first (occurring with a midpoint of ∼2 M) was shifted to higher denaturant concentration in the holoprotein (∼3.7 M) and reflected stabilization due to heme binding, while the second transition (∼6.2 M) was common to both forms. Thus, the consensus sequence stabilized the protein but exposed the existence of two separately cooperative subdomains within the globin architecture, masked as one single domain in TrHb1s with typical stabilities. The results suggested ways in which specific chemical or thermodynamic features may be controlled in artificial heme proteins.