Hyperthermostable recombinant human heteropolymer ferritin derived from a novel plasmid design
Hyperthermostable recombinant human heteropolymer ferritin derived from a novel plasmid design
复制标题
DOI：
10.1002/pro.4543
复制
发表时间：
2022-12
期刊：
影响因子：
8
通讯作者：
A. Srivastava;Lucas J. Scalcione;P. Arosio;Fadi Bou-Abdallah
中科院分区：
文献类型：
--
作者：
A. Srivastava;Lucas J. Scalcione;P. Arosio;Fadi Bou-Abdallah
哺乳动物铁蛋白主要是由2种结构相似但功能和遗传上不同的亚基类型组成的杂聚物，称为H（重）和L（轻）。这两个亚基以不同的H和L比例共同组装，形成24-mer壳状蛋白纳米笼，其中数千个铁原子可以在中空腔体内矿化。在这里，我们使用差示扫描量热法（DSC）来研究铁蛋白的稳定性，并了解H和L亚基的各种组合如何赋予蛋白质结构-功能关系的各个方面。使用最近工程化的质粒设计，能够合成具有特定H与L亚基比率的复杂铁蛋白纳米结构，我们表明，与其H-铁蛋白同源物（Tm = 93 ± 1° C）相比，均聚物L和杂聚物富含L的铁蛋白具有显着的超热稳定性（Tm = 115 ± 1° C）。我们的数据揭示了蛋白质的热稳定性和铁蛋白壳上存在的L亚基的数量之间的显着的线性相关性。观察到强烈且出乎意料的铁诱导的蛋白质热不稳定效应（ΔTm高达20°C）。据我们所知，这是第一次报告重组人均聚物和异聚物铁蛋白，其表现出令人惊讶的高解离温度，在所有已知的铁蛋白物种中最高，包括许多已知的超嗜热蛋白和酶。我们的L和富含L的铁蛋白的这种极端热稳定性可能在生物技术应用中具有巨大的潜力。
Mammalian ferritins are predominantly heteropolymeric species consisting of 2 structurally similar, but functionally and genetically distinct subunit types, called H (Heavy) and L (Light). The two subunits co‐assemble in different H and L ratios to form 24‐mer shell‐like protein nanocages where thousands of iron atoms can be mineralized inside a hollow cavity. Here, we use differential scanning calorimetry (DSC) to study ferritin stability and understand how various combinations of H and L subunits confer aspects of protein structure–function relationships. Using a recently engineered plasmid design that enables the synthesis of complex ferritin nanostructures with specific H to L subunit ratios, we show that homopolymer L and heteropolymer L‐rich ferritins have a remarkable hyperthermostability (Tm = 115 ± 1°C) compared to their H‐ferritin homologues (Tm = 93 ± 1°C). Our data reveal a significant linear correlation between protein thermal stability and the number of L subunits present on the ferritin shell. A strong and unexpected iron‐induced protein thermal destabilization effect (ΔTm up to 20°C) is observed. To our knowledge, this is the first report of recombinant human homo‐ and hetero‐polymer ferritins that exhibit surprisingly high dissociation temperatures, the highest among all known ferritin species, including many known hyperthermophilic proteins and enzymes. This extreme thermostability of our L and L‐rich ferritins may have great potential for biotechnological applications.