Short-range ENDOR distance measurements between Gd(iii) and trifluoromethyl labels in proteins

The measurement of distances in proteins can be challenging in the 5-20 angstrom range, which is outside those accessible through conventional NMR and EPR methods. Recently it was demonstrated that distances in this range could be measured between a nitroxide as a paramagnetic spin label and a nearby fluorine atom (F-19) as a nuclear spin label using high-field (W-band/3.4 T) ENDOR spectroscopy. Here we show that such measurements can also be performed using a gadolinium ion (Gd3+) as the paramagnetic tag. Gd3+ has two advantages. (i) A greater electronic spin (S = 7/2) and fast electronic spin-lattice (T-1) relaxation, improving sensitivity by allowing data to be collected at lower temperatures. (ii) A narrow EPR signal for the -1/2 <-> 1/2 transition, and therefore no orientation selection artefacts. Signal intensities can be further enhanced by using a trifluoromethyl ((CF3)-F-19) group instead of a single F-19 atom. Using the protein calbindin D-9k with a Ca2+ ion replaced by a Gd3+ ion and a trifluoromethylphenylalanine in position 50, we show that distances up to about 10 angstrom can be readily measured. Longer distances proved more difficult to measure due to variable electronic T-M relaxation rates, which lead to broader Lorentzian ENDOR lineshapes. Gd3+ complexes (Gd3+ tags), which reliably display longer T-M times, allow longer distances to be measured (8-16 angstrom). We also provide preliminary evidence that the intensity of ENDOR signals follows the predicted 1/r(6) dependence, indicating that distances r > 20 angstrom can be measured by this method.

Automated summary

This study demonstrates the use of gadolinium ions as paramagnetic tags for measuring longer distances in proteins. Gadolinium ions have greater electronic spin and faster electronic spin-lattice relaxation compared to nitroxide radicals, enhancing the sensitivity of the measurements. The use of trifluoromethyl groups further enhances signal intensity.