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

与活细胞的天然生物分子成分相容的定制蛋白质催化剂是实现合成生物学宏伟目标的关键，也是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被证明特别适用于可处理的从头酶设计过程，正如我们成功设计的超热稳定和催化熟练的从头过氧化物酶所强调的那样，最近发表在《自然通讯》上。从那时起，我们原子地设计了一系列新的含血红素的从头蛋白，允许对蛋白质结构和活性位点进行精确工程。我们使用了Rosetta蛋白设计和分子动力学模拟的强大组合，允许在蛋白质表达生物物理表征之前进行硅的计算设计和快速结构评估。这个项目的目的是实现一个强大的综合计算和实验方法在从头设计的功能，催化活性血红素蛋白和酶。这些蛋白质的设计将特别强调解决与工业相关的具有挑战性的化学问题，包括催化单氧作用和碳转移活性。最初，它们将使用计算方法（例如Rosetta protein designsuite & Molecular Dynamics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进行全面的生物物理分析（例如圆二色性，EPR和nmr光谱，氧化还原电位测定法，x射线晶体学）。催化活性将通过各种稳态和预稳态动力学方法（如平板阅读器测定，停止流动分光光度法）来确定，产品将使用各种技术（如核磁共振光谱，HPLC, LC-MS）来测定。QM/MM计算将用于详细检查催化循环，为未来的蛋白质设计提供信息。定向进化还将用于改进和磨练新生活动。

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.