Short-range distance measurements in proteins using gadolinium complexes as paramagnetic tags and trifluoromethyl as a nuclear label

Abstract

The measurement of short (5-20 Å) distances in proteins can be challenging, outside the range of conventional methods. Recently it was demonstrated that distances in this range could be measured between an organic paramagnetic spin label (nitroxide radical) and a nearby fluorine atom (19F) using high field (W-band / 3.4 T) ENDOR spectroscopy. Here we show that the same measurements can be performed using paramagnetic Gadolinium (Gd3+). This species has two advantages: i) a higher electronic spin (S = 7/2) and fast electronic spin-lattice (T1) relaxation, allowing spectra to be collected at lower temperatures, with both improving sensitivity; and ii) a narrow EPR signal which leads to the suppression of orientation selection artefacts as seen earlier for DEER spectroscopy. Signal intensities can be further enhanced by replacing a single fluorine with a methyl tri-fluoro (CF3) group. We show that replacing divalent metals such as Ca2+ with a Gd3+ ion allows short distances (0-10 Å) to be measured in proteins - but likely exclude longer distances due to variable electronic TM relation rates, leading to broader Lorentzian ENDOR lineshapes. Larger Gd3+ chelate complexes (Gd3+ tags), which display intrinsically slower, robust TM rates, allow longer distances to be measured (8-16 Å). We also provide preliminary evidence that the intensity of ENDOR signals follow the predicted 1/r6 dependence, potentially allowing longer distances (>20 Å) to be measured. Show more