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

利用电子顺磁共振研究酶结构/功能关系的新兴技术

基本信息

批准号：
10630488
负责人：
Jason W Sidabras
金额：
$ 31.12万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-09 至 2028-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10630488
关键词：
Adopted Adoption Amplifiers Automobile Driving Biological Biology Biomedical Research Characteristics Communities Complement Complex Computers Coupling Crystallography Data Collection Development Directed Molecular Evolution Drug Design Electron Spin Resonance Spectroscopy Electrons Emerging Technologies Engineering Environment Enzymes Ferredoxin Fluorescence Fluorescence Resonance Energy Transfer Freezing Frequencies Geometry Goals Hemoglobin Measures Metalloproteins Methodology Methods Microfluidics Molecular Monitor Muramidase Noise Oxidation-Reduction Performance Physiologic pulse Process Protein Analysis Proteins Publishing Rapid screening Research Research Personnel Sampling Scanning Scheme Secondary Protein Structure Signal Transduction Site Spin Labels Structure Structure-Activity Relationship System Techniques Technology Time absorption aqueous clinical diagnostics cryogenics design drug discovery experimental study high throughput screening improved innovation instrumentation magnetic field method development microwave electromagnetic radiation nano nanolitre novel protein complex protein structure function prototype structural biology technology development temporal measurement tool transmission process usability

项目摘要

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.

项目摘要/摘要：电子顺磁共振 (EPR) 是一种光谱技术，测量不成对电子的能量吸收，并用于监测与局部电子的相互作用这些不成对的电子可以在酶的催化过程中自然发生。或使用定点自旋标记进行工程化，详细研究这些顺磁态对于实现这一点至关重要。了解不成对电子与配位层的蛋白质结构-功能关系以及在这个提案中，我重点关注 X- 的三个技术和方法开发。频段（标称 9.5 GHz）将显着改善生物医学研究的 EPR 光谱我将 (i) 增强自共振微螺旋对蛋白质单晶 EPR 的 EPR 敏感性。小到中型 (0.1–3 nl) 晶体，(ii) 建立真正的自由感应衰变检测 EPR 的体积限制冷冻样品（85 nl），以及（iii）开发一种新的谐振器，即自谐振微螺旋，用于先进的时间- 使用微流体（500 nl）样品处理的域连续波（CW）实验首先，增强。自谐振微螺旋这一关键技术将使 EPR 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，以提高采用率。在这个原型中，我将实施一个更标准的蛋白质晶体处理工作流程，包括计算机- 该原型设计可轻松集成到商用 X 波段脉冲中。因为自谐振微螺旋的测量谐振器效率参数为 3.2 mT/W1/2，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 减少到仅 43 mW）。使用板载低温低噪声实施创新的 3 端口传输线耦合方案放大器将建立一个小于 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原型可实现真正的自由感应衰变检测 EPR，这将显着提高 EPR 生物样品的信号强度，并允许采用目前不可能的先进脉冲方法最后，我将介绍一种新型微谐振器，即自谐振器。共振微螺旋，与传统方法相比，CW EPR 的浓度灵敏度提高了 70 倍借助微螺旋，将微流控 EPR 转变为药物发现的可行工具。 microspiral 实现了此处首创的新的初期绝热通道实验，该实验可监测 T1 的变化和 T2 随着不成对电子微环境的变化该实验得到了新的支持。将连续波和绝热快速扫描灵敏度提高一个数量级的样本采集方法总的来说，这些关键的支持技术将进一步推动纳米 EPR 的采用，在 X 波段对小于 500 nl 的体积限制样品进行 EPR 实验变得可行。