Issue 41, 2022

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

Abstract

The measurement of distances in proteins can be challenging in the 5–20 Å 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 (19F) 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 (T1) relaxation, improving sensitivity by allowing data to be collected at lower temperatures. (ii) A narrow EPR signal for the −½ ↔ ½ transition, and therefore no orientation selection artefacts. Signal intensities can be further enhanced by using a trifluoromethyl (C19F3) group instead of a single 19F atom. Using the protein calbindin D9k with a Ca2+ ion replaced by a Gd3+ ion and a trifluoromethylphenylalanine in position 50, we show that distances up to about 10 Å can be readily measured. Longer distances proved more difficult to measure due to variable electronic TM relaxation rates, which lead to broader Lorentzian ENDOR lineshapes. Gd3+ complexes (Gd3+ tags), which reliably display longer TM times, allow longer distances to be measured (8–16 Å). We also provide preliminary evidence that the intensity of ENDOR signals follows the predicted 1/r6 dependence, indicating that distances r > 20 Å can be measured by this method.

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

Supplementary files

Article information

Article type
Paper
Submitted
25 Jun 2022
Accepted
13 Sep 2022
First published
13 Sep 2022

Phys. Chem. Chem. Phys., 2022,24, 25214-25226

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

M. Judd, E. H. Abdelkader, M. Qi, J. R. Harmer, T. Huber, A. Godt, A. Savitsky, G. Otting and N. Cox, Phys. Chem. Chem. Phys., 2022, 24, 25214 DOI: 10.1039/D2CP02889A

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