Assembly of Artificial Oxidoreductases

人工氧化还原酶的组装

• 批准号: BB/I014063/1
• 负责人: Ross Anderson
• 金额: $ 37.56万
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI014063%2F1
• 关键词: Assembly; Artificial; Oxidoreductases

Proteins are biological molecules constructed from linear chains of amino acids that adopt complex 3D structures informed by their amino acid sequence. Each protein typically has a unique structure that is indelibly linked to the function it performs in nature. Enzymes are proteins that catalyze the chemical reactions that occur in the cell, examples of which facilitate the capture and storage of chemical energy from respiration and photosynthesis. The design of new artificial proteins and enzymes remains one of the great challenges in biochemistry, testing our fundamental understanding of the nature of protein as a material. Unlocking the exceptionally powerful array of chemistries that natural enzymes perform promises routes to new drugs, therapies and sources of renewable green energy. Most attempts to construct new enzymes have focussed on modifying natural proteins and enzymes to introduce new catalytic function with modest degrees of success. The problems associated with redesigning natural proteins are due to the layers of complexity that nature incorporates through natural selection into a protein's complicated 3D structure. This complexity serves to complicate functional deconstruction of naturally evolved proteins and enzymes, rendering their redesign intrinsically difficult. We believe that this complexity is not a necessary feature of proteins and enzymes. Our method to effectively avoid such complexity is to work with proteins that have been untouched by natural selection. These simple proteins, neoproteins, are small, robust protein scaffolds with generic amino acid sequences that serve as templates onto which natural protein functions can be added. Non-protein components of certain proteins and enzymes, such as the heme molecule of the protein hemoglobin, can be effectively supported in neoproteins and the various functions that these molecules perform in natural proteins can be exploited. An example of how this method can be effectively used is the creation of a heme-binding neoprotein capable of reversibly binding oxygen, a function common to myoglobin, hemoglobin and the recently discovered neuroglobin. Functional elements of engineering are added step-by-step and the requirements to form such a protein are surprisingly few in number. And, as E. coli produces the artificial protein in large quantities, the oxygen-binding neoprotein is exceptionally cheap to produce and easy to alter through standard molecular biology techniques. Since the oxygen bound state in heme proteins is a pre-requisite for a multitude of catalytic processes in natural proteins, we plan to take inspiration from nature to further the development of these proteins into artificial enzymes. We have developed the oxygen-binding neoprotein to include hemes rigidly attached to the protein backbone. This alleviates problems associated with heme loss from previous designs and allows for an unprecedented control of neoprotein properties and function. Since natural oxygen-dependent catalysis requires that oxygen be 'activated' by the controlled addition of electrons, we will explore this reaction in our oxygen binding neoproteins, gaining valuable information about the generation and stability of intermediates capable of powerful oxygenic catalysis. Ultimately, we plan to combine the oxygen binding and electron delivery functions into either a single protein or a combination of associated protein subunits with discrete functions. Much as modular furniture design uses combinations of smaller functionally independent subunits such as legs, drawers, shelves and assembles them to particular specifications, we think an analogous approach can be applied to the construction of new proteins and enzymes whose functions are dictated by the designer. An advantage of this approach is that through the reproduction of enzyme and protein function in artificial proteins a deep fundamental understanding of the workings of their natural counterparts is gained.

蛋白质是由氨基酸的线性链构建的生物分子，这些氨基酸采用了由其氨基酸序列告知的复杂的3D结构。每种蛋白质通常都有一个独特的结构，该结构与其在自然界中执行的功能无关紧要。酶是催化细胞中发生的化学反应的蛋白质，其示例有助于从呼吸和光合作用中捕获和储存化学能。新的人工蛋白质和酶的设计仍然是生物化学中的重大挑战之一，它测试了我们对蛋白质本质作为一种材料的基本理解。解锁天然酶的异常强大的化学阵列，可以承诺提供新药物，疗法和可再生绿色能源的途径。大多数构建新酶的尝试都集中在修饰天然蛋白质和酶以适度的成功程度引入新的催化功能。与重新设计自然蛋白有关的问题是由于自然通过自然选择纳入蛋白质复杂的3D结构的复杂性层。这种复杂性使自然进化的蛋白质和酶的功能解构复杂化，从而使它们的内在困难重新设计。我们认为，这种复杂性不是蛋白质和酶的必要特征。我们有效避免这种复杂性的方法是处理自然选择未受的蛋白质。这些简单的蛋白质（Neoperins）是小型，可靠的蛋白质支架，具有通用氨基酸序列，可作为模板，可以添加天然蛋白质功能。某些蛋白质和酶的非蛋白质成分，例如蛋白质血红蛋白的血红素分子，可以在Neoprotein中得到有效支持，并且可以利用这些分子在天然蛋白中执行的各种功能。该方法如何有效使用的一个例子是创建能够可逆结合氧的血红素结合蛋白，这是肌红蛋白，血红蛋白和最近发现的神经蛋白的共同功能。逐步添加工程功能元素，形成这种蛋白质的要求很少。而且，由于大肠杆菌大量生产人造蛋白质，因此结合的氧蛋白质非常便宜，并且可以通过标准分子生物学技术易于改变。由于血红素蛋白中的氧结合态是自然蛋白质中多种催化过程的先决条件，因此我们计划从自然中汲取灵感，以进一步发展这些蛋白质为人造酶。我们已经开发出氧结合毒素，包括刚性附着在蛋白质主链上的血液。这减轻了与先前设计的血红素损失相关的问题，并允许对新蛋白质特性和功能进行前所未有的控制。由于天然氧依赖性催化要求通过受控的电子添加氧气“激活”氧气，因此我们将在我们的氧结合毒素中探索这种反应，从而获得有关能够有强大氧催化的中间体的产生和稳定性的有价值的信息。最终，我们计划将氧结合和电子递送功能结合到单个蛋白质或相关蛋白亚基与离散功能的组合中。就像模块化家具设计使用较小功能独立亚基的组合，例如腿部，抽屉，架子和将它们组装成特定的规格，我们认为可以将类似方法应用于构建新蛋白质和功能的新蛋白质和酶，其功能由设计师决定。这种方法的一个优点是，通过复制人工蛋白质中的酶和蛋白质功能，就获得了对自然对应物的起作用的深层理解。

项目成果

Arginine side chain interactions and the role of arginine as a gating charge carrier in voltage sensitive ion channels.

• DOI: 10.1038/srep21759
• 发表时间: 2016-02-22
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Armstrong CT;Mason PE;Anderson JL;Dempsey CE
• 通讯作者: Dempsey CE

Construction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme.

• DOI: 10.1038/s41467-017-00541-4
• 发表时间: 2017-08-25
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Watkins DW;Jenkins JMX;Grayson KJ;Wood N;Steventon JW;Le Vay KK;Goodwin MI;Mullen AS;Bailey HJ;Crump MP;MacMillan F;Mulholland AJ;Cameron G;Sessions RB;Mann S;Anderson JLR
• 通讯作者: Anderson JLR

A suite of de novo c-type cytochromes for functional oxidoreductase engineering.

一套用于功能性氧化还原酶工程的从头 C 型细胞色素。

• DOI: 10.1016/j.bbabio.2015.11.003
• 发表时间: 2016
• 期刊: Biochimica et biophysica acta
• 作者: Watkins DW

Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette.

• DOI: 10.1039/c3sc52019f
• 发表时间: 2014-02-01
• 期刊: Chemical science
• 影响因子: 8.4
• 作者: Anderson JLR;Armstrong CT;Kodali G;Lichtenstein BR;Watkins DW;Mancini JA;Boyle AL;Farid TA;Crump MP;Moser CC;Dutton PL
• 通讯作者: Dutton PL

Expression and In Vivo Loading of De Novo Proteins with Tetrapyrrole Cofactors.

使用四吡咯辅因子表达和体内装载 De Novo 蛋白质。

• DOI: 10.1007/978-1-0716-1826-4_8
• 发表时间: 2022
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Curnow P