Relaxation-based distance measurements between a nitroxide and a lanthanide spin label

Area of Science:

• Biophysical Chemistry
• Structural Biology
• Spectroscopy

Background:

• Electron paramagnetic resonance (EPR) techniques enable distance measurements between labels on biomacromolecules, crucial for structural studies of non-crystallizable or large systems.
• Current EPR distance measurement methods face limitations in sensitivity and accessible distance range.
• Theoretical studies suggest enhancing longitudinal relaxation of nitroxide labels using lanthanide complexes at cryogenic temperatures could overcome these limitations.

Purpose of the Study:

• To extend the distance range and sensitivity of EPR-based distance measurements in structural biology.
• To develop and validate a method for determining lanthanide complex relaxivity without direct lanthanide relaxation rate measurements or model compounds.
• To measure distances in complex biomacromolecular systems unsuitable for traditional structural biology techniques.

Main Methods:

• Utilized electron paramagnetic resonance (EPR) spectroscopy with nitroxide and lanthanide (specifically dysprosium-DOTA complex) labels.
• Measured the enhancement of longitudinal relaxation of the nitroxide label induced by the lanthanide complex at cryogenic temperatures.
• Determined lanthanide complex relaxivity by analyzing the dependence of relaxation enhancement on temperature or concentration in frozen solutions, avoiding direct lanthanide relaxation measurements.

Main Results:

• Successfully determined the relaxivity of the dysprosium-DOTA complex using two independent calibration techniques (temperature and concentration dependence), yielding satisfying agreement.
• Measured a distance of approximately 2.7 nm in a nitroxide-spacer-lanthanide model compound, consistent with its modeled structure.
• Identified error sources for both calibration techniques and examined their impact.

Conclusions:

• The developed EPR-based method, utilizing lanthanide complexes at cryogenic temperatures, effectively extends distance measurement capabilities for biomacromolecules.
• The approach allows for accurate relaxivity determination and distance measurements without relying on direct lanthanide relaxation rates or well-defined model compounds.
• Theoretical analysis suggests further extending the upper distance limit requires measurements at lower magnetic fields and temperatures.