Calibration ruler for CW-EPR distance measurement using diradical molecule of rigid structure.

Many experimental factors and uncontrollable factors may introduce errors in the distance measurement by continuous wave electron paramagnetic resonance. To deal with this problem, several C60 nitroxide diradical adducts with rigid structure and definite molecular dimension were used as distance calibration rulers. Based on the improvement of distance calculation program via adding simulation programs of experimental spectra and dipolar broadening function, respectively, the distance calibration method was developed under different conditions such as different solvent, solution concentration, measuring temperature, and microwave power. As a result, stable distance calibration rulers were established within the range of 8-13 Å. The distance calibration effect was evaluated resulting in a corresponding distance measurement precision of 0.84 Å. The results suggested that the influence of non-dipolar spectral broadening factors could be overcome, and the established experimental and calculation methods were suitable to a wide range of situations. The developed method will ensure more accurate and objective distance measurement in biomacromolecular analysis.