Replacement EPR spectrometer to the multiuser Biomedical EPR Facility at the University of Essex

我们建议用最先进的X波段光谱仪取代埃塞克斯大学生物医学EPR设施中目前的电子顺磁共振(EPR)光谱仪，该光谱仪具有未来升级的能力。EPR光谱仪可以检测在所有氧化/还原反应中形成的分子状态，称为顺磁。这些顺磁状态，其中最著名的是自由基，可以是暂时的(寿命短或长)，也可以是稳定的。酶和蛋白质中顺磁态的形成和消失的过程是非常有趣的，因为一种类型的顺磁中心向另一种类型的顺磁中心转变的顺序事件可以被及时跟踪，从而提供了揭示酶如何工作的分子机制的图景。这些信息对于理解广泛的生物过程很重要。目前的EMX EPR光谱仪(Bruker)已有23年的历史，并出现了严重的间歇性故障。在这些年里，生物医学EPR设施为英国一系列研究小组进行的以蛋白质和酶为重点的研究做出了重大贡献。该机构在蛋白质自由基和金属蛋白质和酶方面开发了高度专业化的专业知识。我们为酶和底物之间快速反应的EPR波谱冷冻样品的内部设备(由BBSRC资助，BB/E02355X/1)允许获得关于顺磁物种在反应时间过程中的出现和转变的有价值的动力学数据。这些数据集可用于计算机生成的动力学模型，以验证有关酶机制的假设。为了保持出版物、补助金和员工培训中记录的设施的高质量产出，我们需要一台类似级别的EPR光谱仪(E500 Bruker)，但与当前模型相比具有实质性的优势，即：a)最新的电子和软件包；b)该系统未来可以升级为以第二工作频率和脉冲方案运行。申请者财团由来自埃塞克斯大学两所学院的11名学者和8名外部合作者组成，他们都在各自的领域拥有良好的国际声誉。这些科学家的研究范围很广，但都需要EPR光谱学作为他们研究的方法。提案中列出了10个项目，这些项目需要并将极大地受益于所要求的仪器的能力：1.探索变铅青链霉菌酶的生物技术潜力2。电子通过和蛋白质之间的转移。热敏智能材料相变温度跃变的动力学研究。金属蛋白的时间分辨EPR和时间分辨晶体结构的集成。酶和蛋白质电子顺磁共振谱的深度神经网络分析6。膜蛋白在聚合物基脂颗粒中的滞留动力学7。细菌亚铁蛋白的新生物化学8。新的血液过氧化物酶9。饮食和心肌组织呼吸中缺铁10。来自链霉菌的新的转运蛋白样膜蛋白埃塞克斯生物医学EPR设施的现有Bruker EMX光谱仪一直是制作高质量出版物和获得赠款的关键工具，在该设施存在期间，资金和研究成果具有明确的上升轨迹。更新换代的光谱仪将确保我们在生物分子电子顺磁共振光谱领域的世界领先地位得以保持。

We propose to replace the current Electron Paramagnetic Resonance (EPR) spectrometer, at the Biomedical EPR Facility at the University of Essex, with the latest state of the art X-band spectrometer with capabilities for future upgrades.EPR spectroscopy allows detection of molecules in the states, called paramagnetic, that are formed in all oxidation/reduction reactions. These paramagnetic states, of which the best known are free radicals, can be transient (with either short or long life time) or stable. The processes of paramagnetic states formation within enzymes and proteins, as well as their disappearance, are of great interest since the sequential events of one type of paramagnetic centre transforming into another can be followed in time, thus providing a picture of the molecular mechanism revealing how an enzyme works. Such information is important for understanding a wide range of biological processes.The current EMX EPR spectrometer (Bruker) is 23 years old and has developed a serious intermittent fault. Throughout these years, the Biomedical EPR Facility has contributed significantly to the research focused on proteins and enzymes conducted by a range of research groups in the UK. The Facility has developed highly specialised expertise in protein-based free radicals and metallo-proteins and enzymes. Our in-house built apparatus for freezing samples for EPR spectroscopy of rapid reactions between enzyme and substrate (funded by BBSRC, BB/E02355X/1), allows valuable kinetic data pertaining to the appearance and transformation of paramagnetic species over a reaction time course to be obtained. Such data sets can be used in computer generated kinetic models to verify hypotheses about the enzyme mechanism.To maintain the high quality output of the Facility documented in publications, grant awards and staff training, we require an EPR spectrometer of a similar class (E500 Bruker) but with substantial advantages compared to the current model, namely: a) up-to-date electronics and software package; b) the system can be upgraded in the future to operate at a second working frequency and a pulsed regime.The consortium of applicants consists of 11 academics from two Schools of the University of Essex and 8 external collaborators, all with excellent international reputations in their fields. The scope of research by these scientists is wide but all require EPR spectroscopy as a method for their research.The proposal features 10 projects that require and will benefit greatly from the capabilities of the requested instrument:1. Exploring the biotechnological potential of Streptomyces lividans enzymes2. Electron transfer through and between proteins3. Kinetics of the phase transition in thermoresponsive smart materials on temperature jump4. Integrated time-resolved EPR and time-resolved crystal structures of metalloproteins5. Deep neural network analysis of the EPR spectra of enzymes and proteins6. Dynamics of membrane proteins sequestered in polymer based lipid particles7. New biochemistry of bacterioferritin8. De novo haem peroxidases9. Iron deficiency in diet and cardiac muscle tissue respiration10. New transporter-like membrane protein from StreptomycesThe existing Bruker EMX spectrometer at the Biomedical EPR Facility at Essex has been the key instrument in producing high quality publications and obtaining grant awards with a clear upward trajectory of funds and research outputs over the period of the Facility existence. The replacement spectrometer would ensure that our world leading position in EPR spectroscopy of biomolecules will be maintained.