Constructing catalytically proficient and functionally diverse enzymes from simple, de novo designed proteins

从简单、从头设计的蛋白质构建催化能力强且功能多样的酶

• 批准号: 2278910
• 金额: --
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2278910
• 关键词: Constructing; catalytically; proficient; functionally; diverse

Bespoke protein catalysts compatible with the natural biomolecular components of living cells are keyto realising the ambitious goals of synthetic biology and to the provision of cheap, green catalysts forindustrial biotechnology. To this end, the manmade protein maquettes designed in our lab haveproved particularly adaptable to a tractable de novo enzyme design process, as highlighted throughour successful design of a hyperthermostable and catalytically proficient de novo peroxidase, recentlypublished in Nature Communications. Since then, we have atomistically designed a new series ofheme-containing de novo proteins that allow for precision engineering of the protein structure andactive site. We have done so using a powerful combination of Rosetta protein design and MolecularDynamics simulations, allowing for both computational design and rapid structural assessment insilicon prior to protein expression biophysical characterisation.The aims of this project are to implement a powerful synthesis of computational and experimentalmethods in the de novo design of functional, catalytically active heme-containing proteins andenzymes. These proteins will be designed with particular emphasis on addressing challengingchemistries pertinent to industry, including catalytic monooxygenation and carbene transfer activities.Initially, they will be designed and assessed using computational methods (e.g. Rosetta protein designsuite & Molecular Dynamics software), and proteins that are subsequently selected for expression andpurification will be subjected to a comprehensive biophysical analysis (e.g. circular dichroism, EPR andNMR spectroscopies, redox potentiometry, X-ray crystallography). Catalytic activity will bedetermined through a variety of steady-state and pre-steady-state kinetic methods (e.g. plate readerassays, stopped-flow spectrophotometry), and products will be indentured using a variety oftechniques (e.g. NMR spectroscopy, HPLC, LC-MS). QM/MM calculations will be employed to examinethe catalytic cycles in detail, informing future protein designs. Directed evolution will also beemployed to improve and hone nascent activity.

与活细胞的天然生物分子成分兼容的定制蛋白质催化剂是实现合成生物学雄心勃勃的目标和为工业生物技术提供廉价、绿色催化剂的关键。为此，我们实验室设计的人造蛋白质模型已经证明特别适合于易处理的从头酶设计过程，正如我们最近发表在《自然通讯》上的成功设计的耐热和催化熟练的从头酶过氧化物酶所强调的那样。从那时起，我们已经原子设计了一系列新的含血红素的从头蛋白质，允许对蛋白质结构和活性部位进行精确工程。我们使用Rosetta蛋白质设计和分子动力学模拟的强大组合来做到这一点，允许在蛋白质表达生物物理特征之前在硅中进行计算设计和快速结构评估。这个项目的目的是在功能、催化活性的含血红素的蛋白质和酶的从头设计中实现计算和实验方法的强大综合。这些蛋白质的设计将特别注重解决与工业相关的化学挑战，包括催化单氧合和卡宾转移活性。最初，它们将使用计算方法(例如Rosetta蛋白质设计套件和分子动力学软件)进行设计和评估，随后选择用于表达和纯化的蛋白质将经过全面的生物物理分析(例如圆二色谱、EPR和核磁共振光谱、氧化还原电位法、X射线结晶学)。催化活性将通过各种稳态和稳态前动力学方法(例如平板读数法、停流分光光度法)来测定，产品将使用各种技术(例如核磁共振光谱、高效液相色谱、LC-MS)来确定。QM/MM计算将被用来详细检查催化循环，为未来的蛋白质设计提供信息。定向进化也将被用于改善和磨练新生的活动。