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

项目总结/摘要：电子顺磁共振（EPR）是一种光谱技术， 测量未成对电子的能量吸收，并用于监测与局部电子的相互作用。 分子环境这些未成对电子可以在酶的催化过程中自然产生 或使用定点自旋标记进行工程化。详细研究这些顺磁状态对于 了解蛋白质结构与功能的关系，不成对电子与配位球的关系， 蛋白质的二级结构在这份提案中，我重点介绍了X的三项技术和方法发展， 频段（标称9.5 GHz），将显着提高EPR光谱的生物医学研究 社区我将（i）提高自共振微螺旋的EPR灵敏度，用于蛋白质单晶EPR， 小到中等大小（0.1-3 nl）晶体，（ii）建立真正的自由感应衰减检测EPR体积有限 冷冻样品（85 nl），和（iii）开发一种新的谐振器，自谐振微螺旋，先进的时间- 域连续波（CW）实验与微流体（500 nl）样品处理。第一，增强 一项关键的使能技术，即自谐振微螺旋，将使EPR的灵敏度提高一个数量级 由于应用了创新的匹配电路和低温低噪声放大器，为了提高采用 这个原型，我将实现一个更标准的蛋白质晶体处理工作流程，包括计算机- 可控测角仪该原型将被设计成易于集成到商业X波段脉冲 光谱仪由于自谐振微螺旋具有测量的谐振器效率参数 3.2 mT/W1/2，其大于市售谐振器的5倍， 典型的80 ns脉冲减少了3个数量级（从45 W到仅43 mW）。降低入射功率， 采用机载低温低噪声三端口传输线耦合方案 放大器将建立小于5 ns的谐振器死区时间。通过利用这些特征，我可以开发一个 新的光谱仪原型为真正的自由感应衰变检测EPR，这将大大提高EPR 并且允许目前不可能的先进脉冲方法 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- 共振微螺旋，这将增加浓度的灵敏度CW EPR的70倍相比， 微螺旋，将微流体EPR转化为药物发现的可行工具。自谐振 微螺旋使一个新的初期绝热通道实验，率先在这里，监测T1的变化 和T2随未成对电子微环境的变化而变化。 将CW和绝热快速扫描灵敏度提高一个数量级的样本采集方法 相同的测量时间。总之，这些关键的使能技术将进一步采用纳米EPR， 其中在X波段对小于500 nl的体积受限样品进行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.