From carbon nanotubes to zinc porphyrins: engineering proteins to interface with non- biological molecular systems.

从碳纳米管到锌卟啉：工程蛋白质与非生物分子系统的接口。

• 批准号: 1642423
• 金额: --
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1642423
• 关键词: carbon; nanotubes; zinc; porphyrins; engineering

Bacterial resistance to antibiotics is one of the most significant crises in modern healthcare. The most widely utilised class of antibiotics (and therapeutics in general)are the beta-lactams, which include ampicillin, amoxicillin and methicillin. By far the main mechanism bacteria use to confer beta-lactam resistance is the production of beta-lactamases that hydrolyses the pseudo-peptide bond in the beta-lactam ring critical for its antibacterial activity. The aim of this project is to generate a high-information content miniaturised sensor with the capacity to rapidly detect the presence and type of beta-lactamase to allow more effective clinical use of current beta-lactams. To achieve this engineered beta-lactamase inhibitory protein (BLIP) will be selectively interfaced with a graphene field-effect transistor so that protein-protein interactions are transduced into a change in resistance of graphene-bridged microelectrodes through both bulk binding effects and electrostatic surface interactions. This system has the potential to sense events connected to single protein molecules. BLIP is the ideal sensor as it binds a wide variety of clinically relevant beta-lactamases with very distinct surface-facing electrostatics and interaction properties, which will potentially offer discrete electronic signatures. A key requirement is defined and directed interfacing of BLIP with graphene to maximise communication between the two components. This will be achieved using a synthetic biology approach to design in silico residue positions for the recombinant incorporation of non-native chemical handles (e.g. phenyl azide chemistry) that will allow high precision and optimal interfacing of protein with graphene. Detailed single molecule analysis (AFM/STM) of the protein-graphene interface will lead to the microfabrication of the protein-graphene-electrode biosensing platform and begin monitoring and assessing binding events through changes in conductivity. First rotation project (Jones, Cardiff). The student will initiate the first round of BLIP variant design in silico, introduce the selected mutations, and using a reprogrammed genetic code approach incorporate the new reactive handles using non-natural amino acid at the defined positions. The student will produce and functionally test the selected BLIP variants to ascertain the effect of the mutations. Second rotation project (Cardiff and Exeter Physics). The student will begin optimising approaches for directly assembling the BLIP variants on graphene. Two approaches will be taken: (1) direct covalent linkage through the photogeneration of reactive nitrene radicals; (2) Click addition of ?-stacking adducts to the BLIP. The student will analyse the BLIP-graphene interface using single molecule imaging approaches (AFM-STM). Functional studies will also begin through imaging the formation of BLIP-beta-lactamase complexes on grapheme.

细菌对抗生素的耐药性是现代医疗保健中最严重的危机之一。最广泛使用的抗生素类(以及一般的治疗药物)是β-内酰胺类药物，它包括氨苄西林、阿莫西林和甲氧西林。到目前为止，细菌用来传递对β-内酰胺类药物耐药性的主要机制是产生β-内酰胺酶，这种酶能分解对其抗菌活性至关重要的β-内酰胺环中的伪肽键。该项目的目的是产生一种高信息含量的微型传感器，能够快速检测β-内酰胺酶的存在和类型，以便更有效地临床使用当前的β-内酰胺类药物。为了实现这一点，将有选择地将β-内酰胺酶抑制蛋白(BIP)与石墨烯场效应管连接，从而通过体结合效应和静电表面相互作用将蛋白质-蛋白质相互作用转化为石墨烯桥微电极的电阻变化。该系统有可能感知与单个蛋白质分子有关的事件。BLIP是理想的传感器，因为它结合了多种临床相关的β-内酰胺酶，具有非常独特的面向表面的静电和相互作用特性，这将可能提供离散的电子签名。定义了BLIP与石墨烯的直接接口以使两个组件之间的通信最大化，这是一个关键要求。这将使用合成生物学方法在硅基残基位置进行设计，以便重组并入非天然化学手柄(例如苯叠氮化物化学)，从而实现蛋白质与石墨烯的高精度和最佳接口。对蛋白质-石墨烯界面的详细单分子分析(AFM/STM)将导致蛋白质-石墨烯-电极生物传感平台的微制造，并开始通过电导率的变化来监测和评估结合事件。第一个轮换项目(琼斯，加的夫)。学生将在电子计算机中启动第一轮blip变体设计，引入选定的突变，并使用重新编程的遗传密码方法在定义的位置使用非天然氨基酸合并新的反应手柄。学生将产生并从功能上测试选定的blip变体，以确定这些突变的效果。第二轮换项目(加的夫和埃克塞特物理学)。这名学生将开始优化直接在石墨烯上组装blip变体的方法。将采取两种方法：(1)通过光产生反应性的硝酸根直接共价键；(2)点击加成？-堆积加合物到斑点。学生将使用单分子成像方法(AFM-STM)分析斑点-石墨烯界面。功能研究还将开始，通过成像在石墨烯上形成斑点-β-内酰胺酶复合体。

项目成果

Switching protein metalloporphyrin binding specificity by design from iron to fluorogenic zinc.

通过设计将蛋白质金属卟啉结合特异性从铁切换到荧光锌。

• DOI: 10.1039/d0cc00596g
• 发表时间: 2020
• 期刊: Chemical communications (Cambridge, England)
• 作者: Bowen BJ

Switching metalloporphyrin binding specificity of a b -type cytochrome to fluorogenic zinc by design

通过设计将b型细胞色素的金属卟啉结合特异性切换为荧光锌

• DOI: 10.1101/832923
• 发表时间: 2019
• 作者: Bowen B

Site-specific Protein Photochemical Covalent Attachment to Carbon Nanotube Side Walls and its Electronic Impact on Single Molecule Function.

• DOI: 10.1021/acs.bioconjchem.9b00719
• 发表时间: 2019-11
• 期刊: Bioconjugate chemistry
• 影响因子: 4.7
• 作者: Suzanne Thomas;David Jamieson;R. Gwyther;B. Bowen;A. Beachey;H. Worthy;J. Macdonald;M. Elliott;Oliver Kieran Castell;Darran Dafydd Jones