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

• 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
• 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

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）技术是一种光谱学技术