Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette.
Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette.
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DOI：
10.1039/c3sc52019f
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发表时间：
2014-02-01
期刊：
影响因子：
8.4
通讯作者：
Dutton PL
中科院分区：
文献类型：
--
作者：
Anderson JLR;Armstrong CT;Kodali G;Lichtenstein BR;Watkins DW;Mancini JA;Boyle AL;Farid TA;Crump MP;Moser CC;Dutton PL
成功使用人造蛋白质来推进合成生物学需要在生活环境中制造功能性人工蛋白质，以及这些蛋白质与该环境中的其他蛋白质和底物有效相互作用的能力。通过模型方法制造的蛋白质将复杂的氧化还原酶功能整合到进化上幼稚的、非计算设计的蛋白质结构中，其序列与任何天然蛋白质完全无关。尽管如此，我们在这里表明，我们可以有效地接口与天然细胞机械，共价结合血红素到天然细胞色素c在体内产生人工c型细胞色素模型。此外，这种C型细胞色素模型设计有可置换的组氨酸血红素配体，其打开以允许功能性氧结合，这是从氧储存和运输到催化羟基化的更复杂功能中的主要事件。为了利用来自在单个模型框架内结合各种氧化还原辅因子的自由的功能范围，该c型细胞色素模型被设计为具有第二个非血红素C四吡咯结合位点，使得能够构建基本电子传递链，并且当血红素C铁被锌取代时，产生Zn卟啉，一种光活化的人工氧化还原蛋白。我们在这里描述的工作代表了从头蛋白质设计的一个重大进展，为新的c-型血红素为基础的氧化还原酶设计提供了一个强大的平台，并提供了一个同样重要的原理证明，即辅因子配备的人造蛋白质可以在活细胞中表达，为在体内构建功能有用的人造蛋白质铺平了道路。
The successful use of man-made proteins to advance synthetic biology requires both the fabrication of functional artificial proteins in a living environment, and the ability of these proteins to interact productively with other proteins and substrates in that environment. Proteins made by the maquette method integrate sophisticated oxidoreductase function into evolutionarily naive, non-computationally designed protein constructs with sequences that are entirely unrelated to any natural protein. Nevertheless, we show here that we can efficiently interface with the natural cellular machinery that covalently incorporates heme into natural cytochromes c to produce in vivo an artificial c-type cytochrome maquette. Furthermore, this c-type cytochrome maquette is designed with a displaceable histidine heme ligand that opens to allow functional oxygen binding, the primary event in more sophisticated functions ranging from oxygen storage and transport to catalytic hydroxylation. To exploit the range of functions that comes from the freedom to bind a variety of redox cofactors within a single maquette framework, this c-type cytochrome maquette is designed with a second, non-heme C, tetrapyrrole binding site, enabling the construction of an elementary electron transport chain, and when the heme C iron is replaced with zinc to create a Zn porphyrin, a light-activatable artificial redox protein. The work we describe here represents a major advance in de novo protein design, offering a robust platform for new c-type heme based oxidoreductase designs and an equally important proof-of-principle that cofactor-equipped man-made proteins can be expressed in living cells, paving the way for constructing functionally useful man-made proteins in vivo.