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建筑文摘:一种共识单体球蛋白的热力学稳定性和结构域。

Architectural digest: Thermodynamic stability and domain structure of a consensus monomeric globin.

机构信息

T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland.

T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland.

出版信息

Biophys J. 2023 Aug 8;122(15):3117-3132. doi: 10.1016/j.bpj.2023.06.016. Epub 2023 Jun 23.

Abstract

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 a b-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.

摘要

代表一组同源序列共识的人工蛋白质因其热力学稳定性增加和活性保守而受到关注。在这里,我们将共识方法应用于 b 型血红素结合蛋白,以检查可分离辅因子对增强稳定性的贡献以及创建通用血红素环境的化学后果。我们针对第 1 组截断血红蛋白 (TrHb1) 亚家族的蛋白质,因为它们的体积小(约 120 个残基)且易于表征。从大约 300 个代表性序列的精选集合中获得的一级结构产生了富含酸性残基的高度可溶性共识球蛋白 (cGlbN)。光学和 NMR 光谱学表明在预期的位置和两种构象中具有高亲和力的血红素结合。在中性 pH 下,通过一对组氨酸残基实现了近端和远端铁配位,就像在一些天然 TrHb1 中观察到的那样,并且在远端具有不稳定的配位。与研究过的 TrHb1 不同,在血红素结合后,cGlbN 表现出相同程度的二级结构,无论血红素是否与蛋白质结合。变性需要盐酸胍,并表明 apo 和 holoprotein 在两个转变中展开 - 第一个(在中点约 2 M 处发生)在 holoprotein 中向更高的变性剂浓度转移(约 3.7 M),反映了由于血红素结合而导致的稳定性增加,而第二个转变(约 6.2 M)在两种形式中都很常见。因此,共识序列稳定了蛋白质,但暴露了球蛋白结构中两个单独的合作亚结构的存在,在具有典型稳定性的 TrHb1 中被掩盖为一个单一的结构域。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的方式。

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