Rational Design of a Cu(II) Spin Label Improves the Sensitivity of Distance Measurements

The measurement of distance constraints in biomolecules using Cu(II) spin labels at Q-band EPR is complicated due to the selective excitation of only some orientations of the label. Here, we introduce a new Cu(II) spin label, in which the divalent metal is coordinated equatorially by four nitrogen atoms. Such chelation reduces the <i>g</i> anisotropy of the Cu(II) spin, which reduces the spectral width. We show that the complex coordinates to target labeling sites on a protein and provides narrow distance distributions on proteins. DEER data acquired across different fields at Q-band show the same period of modulation, which suggests proper orientational averaging. Thus, a single measurement at the magnetic field corresponding to the maximum intensity is sufficient to measure the distance distribution. Although the label does not bind stoichiometrically, we observe a 2.3-fold improvement in sensitivity in half the data acquisition time compared to those of existing Cu(II) spin labels.