Oral Presentation 23rd International Society of Magnetic Resonance Conference 2023

Measuring Ångström-scale distances using high-field Gd3+–19F ENDOR (#32)

Martyna M Judd 1 , Elwy H Abdelkader 2 , Haocheng Qianzhu 2 , Mian Qi 3 , Caitlin Lindsay 1 , Anthony Barancewicz 1 , Jeffrey R Harmer 4 , Thomas Huber 1 , Adeheid Godt 3 , Anton Savitsky 5 , Gottfried Otting 2 , Nick Cox 1
  1. Research School of Chemistry, Australian National University, Canberra, ACT, Australia
  2. ARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, The Australian National University, Canberra, ACT, Australia
  3. Faculty of Chemistry and Center for Molecular Materials, Bielefeld University, Bielefeld, Germany
  4. Centre for Advanced Imaging, The University of Queensland, Brisbane, Queensland, Australia
  5. Faculty of Physics, Technical University Dortmund, Dortmund, Germany

19F-ENDOR has recently emerged as a powerful technique for measuring Ångström-scale distances.1,2 However, extending the upper limit of ENDOR distance detection as well as the versatility of 19F-ENDOR across a range of protein systems requires careful pulse parameter optimisation,  spin label design, and understanding the spin label conformational dynamics.

Our work demonstrates the advantages of achieving these developments by using Gd3+-19F label pairs for ENDOR at W-band (94 GHz).  Most notably, Gd3+-19F ENDOR allows significantly faster and more sensitive detection of distant 19F nuclei compared to other electronic spin labels.3 Gd3+ has high intrinsic EPR intensity (S = 7/2), fast electronic spin-lattice (T1) relaxation, and is free of orientation selection effects, allowing 5-18 Å distances to be rapidly sampled. Signal intensities can be further enhanced by using a trifluoromethyl (C19F3) group instead of a single 19F atom.2,3 Here we discuss two ways in which Gd3+-19F ENDOR enhances the scope of ENDOR measurement in proteins:

1: In metalloproteins, where the Gd3+ position is fixed, we can measure short (<12 Å) distances and constrain the precise ring orientation of both CF3-Phe tags,2 and novel mono- and di-fluorinated aromatic amino acid tags relative to the Gd3+ centre. For both metalloproteins and proteins labelled with synthetic Gd3+ labels, the measured 19F-ENDOR spectra can distinguish between favoured conformations adopted by 19F-labelled aromatic residues to additionally validate and refine rotamer library simulations. Pairing these 19F tagging approaches with synthetic Gd3+ labels may therefore further open up avenues to more accurately characterising nuclear and electronic tag dynamics, enabling a more modular implementation of these spin labels.

2: Longer distaces ~18 Å and distance distributions can be measured using DEER-type Gd3+ labels, tethered to the protein surface, and displaying reliably longer electronic phase-memory (TM) times.2 We find that the integrated 19F-ENDOR intensity in Gd3+-tagged proteins follows the predicted 1/r6 dependence,3 and we are currently assessing how to optimise the label properties and measurement parameters to detect <25 Å distances using this approach.   The development of the 19F-ENDOR integration method into a tool for rapid screening for ligand/drug-target binding in biomolecules will also be discussed.  

  1. A. Meyer, S. Dechert, S. Dey, C. Höbartner and M. Bennati, Angew. Chemie Int. Ed., 2020, 59, 373–379, DOI: 10.1002/anie.201908584.
  2. A. Kehl, M. Hiller, F. Hecker, I. Tkach, S. Dechert, M. Bennati and A. Meyer, J. Magn. Reson., 2021, 333, 107091, DOI: 10.1016/j.jmr.2021.107091.
  3. M. M. Judd, E. H. Abdelkader, M. Qi, J. Harmer, T. Huber, A. Godt, A. Savitsky, G. Otting and N. Cox, Phys. Chem. Chem. Phys., 2022, online, 1–11, DOI: 10.1039/D2CP02889A.
  4. P.-P. Zänker, G. Jeschke and D. Goldfarb, J. Chem. Phys., 2004, 122, 24515, DOI: 10.1063/1.1828435.