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.

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.

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.

催化熟练且可超过的从头酶的结构和体内组装。

• 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