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

> **NIH NIH R01** · MEDICAL COLLEGE OF WISCONSIN · 2024 · $327,600

## Abstract

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...

## Key facts

- **NIH application ID:** 10863995
- **Project number:** 5R01GM149568-02
- **Recipient organization:** MEDICAL COLLEGE OF WISCONSIN
- **Principal Investigator:** Jason W Sidabras
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $327,600
- **Award type:** 5
- **Project period:** 2023-06-09 → 2028-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10863995

## Citation

> US National Institutes of Health, RePORTER application 10863995, Emergent Technology for Studying the Structure/Function Relationship of Enzymes Using Electron Paramagnetic Resonance (5R01GM149568-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10863995. Licensed CC0.

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