Emergent Technology for Studying the Structure/Function Relationship of Enzymes Using Electron Paramagnetic Resonance

项目摘要/摘要：电子顺磁共振(EPR)是一种光谱技术， 测量未配对电子对能量的吸收，并用于监测与局域电子的相互作用 分子环境。这些不成对的电子可以自然地出现在酶的催化过程中。 或者使用定点定向自旋标记进行工程设计。详细研究这些顺磁状态对于 了解蛋白质中未配对电子与配位球的结构-功能关系 蛋白质的二级结构。在这份提案中，我重点介绍了X- 频段(名义为9.5 GHz)，将显著改进生物医学研究的EPR波谱 社区。我将(I)提高蛋白质单晶EPR的自共振微螺旋的EPR灵敏度 小到中型(0.1-3nL)晶体，(Ii)建立真正的自由感应衰变检测到的体积有限的EPR 冷冻样品(85毫升)，和(Iii)开发一种新的谐振器，自共振微螺旋，用于先进的时间- 微流控(500 NL)样品处理的区域连续波(CW)实验。首先，增强了 一项关键的使能技术--自共振微螺旋将使EPR的灵敏度提高一个数量级 由于采用了创新的匹配电路和低温低噪声放大器。要促进采用 在这个原型中，我将实现一个更标准的蛋白质晶体处理工作流程，包括一台计算机- 受控测角仪。原型的设计将很容易集成到商业X波段脉冲中 分光计。因为自谐振微螺旋具有测量的谐振器效率参数 3.2 mT/w1/2，这是商业谐振器的5倍以上，所需的功率为 典型的80 ns脉冲减少了3个数量级(从45W减少到仅43 mW)。降低入射功率和 采用车载低温低噪声的新型三端口传输线耦合方案 放大器将建立一个死时间小于5 ns的谐振器。通过利用这些特性，我可以开发一个 新的光谱仪样机探测到了真自由感应衰变的EPR，这将极大地提高EPR 生物样品的信号强度，并允许先进的脉冲方法，这是目前不可能的 采用商用X波段EPR光谱仪设计。最后，我将介绍一种新的微谐振器--自谐振器 谐振式微螺旋，将使连续波EPR的浓度灵敏度提高70倍 利用微螺旋，将微流控EPR转化为一种可行的药物发现工具。自我共鸣 微螺旋使一种新的初始绝热通道实验成为可能，该实验是在这里开创的，它监测T1的变化 T2随未成对电子微环境的变化而变化。这项实验得到了新的 将连续波和绝热快速扫描灵敏度提高一个数量级的样品采集方法 对于相同的测量时间。总体而言，这些关键使能技术将进一步推动纳米EPR的采用， 其中，在X波段对小于500nL的体积有限的样品进行EPR实验变得可行。

PROJECT SUMMARY/ABSTRACT: Electron paramagnetic resonance (EPR) is a spectroscopic technique that measures the absorption of energy by unpaired electrons and is used to monitor interactions with the local molecular environment. These unpaired electrons can naturally occur during the catalytic process of an enzyme or be engineered using site-directed spin labeling. Studying these paramagnetic states in detail is critical for understanding the protein structure–function relationship of the unpaired electrons to coordination sphere and secondary structures of the protein. In this proposal, I focus on three technical and method developments at X- band (nominally 9.5 GHz) that will significantly improve EPR spectroscopy for the biomedical research community. I will (i) enhance the EPR sensitivity of the self-resonant microhelix for protein single-crystal EPR of small to medium-sized (0.1–3 nl) crystals, (ii) establish true free induction decay detected EPR for volume-limited frozen samples (85 nl), and (iii) develop a new resonator, the self-resonant microspiral, for advanced time- domain continuous-wave (CW) experiments with microfluidic (500 nl) sample handling. First, enhancements to a key enabling technology, the self-resonant microhelix, will improve EPR sensitivity by an order of magnitude due to application of an innovative matching circuit and cryogenic low-noise amplifier. To improve adoption of this prototype, I will implement a more standard workflow for protein crystal handling, including a computer- controlled goniometer. The prototype will be designed to easily integrate into a commercial X-band pulse spectrometer. Because the self-resonant microhelix has a measured resonator efficiency parameter of 3.2 mT/W1/2, which is greater than 5 times that of commercially available resonators, the power required for a typical 80 ns pulse is reduced by 3 orders of magnitude (from 45 W to just 43 mW). Reduced incident power and implementation of an innovative 3-port transmission line coupling scheme with an onboard cryogenic low-noise amplifier will establish a resonator deadtime less than 5 ns. By leveraging these characteristics, I can develop a new spectrometer prototype for true free induction decay detected EPR, which will drastically improve the EPR signal intensity of biological samples and allow for advanced pulse methodologies that are currently not possible with commercial X-band EPR spectrometer design. Finally, I will introduce a new micro-resonator, the self- resonant microspiral, which will increase the concentration sensitivity for CW EPR by a factor of 70 compared with the microhelix, transforming microfluidic EPR into a viable tool for drug discovery. The self-resonant microspiral enables a new incipient adiabatic passage experiment, pioneered here, that monitors changes of T1 and T2 with changes of the microenvironment of the unpaired electron. This experiment is supported by new sample acquisition methodology that increases CW and adiabatic rapid scan sensitivity by an order of magnitude for the same measurement time. In total, these key enabling technologies will further the adoption of nano-EPR, where performing EPR experiments on volume-limited samples less than 500 nl at X-band becomes practical.