Anderson J L Ross, Armstrong Craig T, Kodali Goutham, Lichtenstein Bruce R, Watkins Daniel W, Mancini Joshua A, Boyle Aimee L, Farid Tammer A, Crump Matthew P, Moser Christopher C, Dutton P Leslie
School of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK.
The Johnson Research Foundation, Dept. of Biochemistry and Biophysics, University of Pennsylvania, PA19104-6059, USA.
Chem Sci. 2014 Feb 1;5(2):507-514. doi: 10.1039/C3SC52019F. Epub 2013 Oct 31.
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 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 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 .
成功利用人造蛋白质推动合成生物学发展，既需要在活细胞环境中制造出具有功能的人工蛋白质，还需要这些蛋白质能够在该环境中与其他蛋白质和底物进行有效相互作用。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原始、未经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。然而，我们在此表明，我们能够有效地与将血红素共价结合到天然细胞色素c中的天然细胞机制相结合，以产生一种人工c型细胞色素模型。此外，这种c型细胞色素模型设计有一个可置换的组氨酸血红素配体，该配体打开后允许功能性氧结合，这是从氧储存和运输到催化羟基化等更复杂功能中的主要事件。为了利用在单个模型框架内自由结合各种氧化还原辅因子所带来的功能范围，这种c型细胞色素模型设计有第二个非血红素C四吡咯结合位点，能够构建一个基本的电子传递链，并且当血红素C铁被锌取代以形成锌卟啉时，可得到一种光激活的人工氧化还原蛋白。我们在此描述的工作代表了蛋白质设计的一项重大进展，为基于新型c型血红素的氧化还原酶设计提供了一个强大的平台，同时也是一个同样重要的原理验证，即配备辅因子的人造蛋白质可以在活细胞中表达，为构建功能有用的人造蛋白质铺平了道路。
