Milliwatt three- and four-pulse double electron electron resonance for protein structure determination

Area of Science:

• Biophysics
• Spectroscopy
• Structural Biology

Background:

• Electron paramagnetic resonance (EPR) spectroscopy, particularly double electron-electron resonance (DEER), is crucial for protein structure determination.
• Traditional DEER experiments require high-power microwave amplifiers (>300 W) to achieve short pulse lengths for broad spectral excitation.
• Existing methods face limitations in sensitivity and require large sample volumes.

Purpose of the Study:

• To demonstrate the efficacy of a novel self-resonant microhelix for low-power EPR experiments.
• To achieve significantly reduced dead times in 3-pulse DEER experiments.
• To explore the potential for developing cost-effective, benchtop EPR spectrometers.

Main Methods:

• Utilized a self-resonant microhelix with high B1 conversion efficiency and a low Q-value.
• Performed 3-pulse DEER experiments at X-band (9.5 GHz) using less than 1 W of microwave power.
• Tested on molecular ruler and T4 lysozyme samples at concentrations down to 100 μM.

Main Results:

• Achieved dead times as low as 14 ± 2 ns with significantly reduced power (<1 W).
• Experiments were conducted in an active volume 120 times smaller than standard resonators, with only an 11-fold decrease in signal-to-noise ratio.
• Demonstrated superior sensitivity of 3-pulse DEER over 4-pulse DEER with the new microhelix.

Conclusions:

• The self-resonant microhelix enables high-performance DEER experiments at significantly lower power levels.
• Shorter dead times and smaller active volumes facilitate the study of fast-relaxing species and volume-limited samples.
• This technology paves the way for accessible, low-cost benchtop X-band pulse EPR spectrometers, broadening community adoption.