Distance measurements on orthogonal Cu(II) spin labels and natural Cu(II) centers in proteins and peptides by 19F ENDOR spectroscopy

• Applicant: Dr. Andreas Meyer
• Subject Area: Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 575891254

19F Electron nuclear double resonance (ENDOR) is a spectroscopic method to determine biomolecular distances in the 1 - 2 nanometer range. The physical principle is measuring the distance dependent, magnetic dipolar coupling between a 19F nuclear spin and the electron spin of a paramagnetic center. In typical applications both the 19F nucleus as well as the paramagnetic center are introduced as spin labels into a biological macromolecule. Nitroxide radicals are without any doubt the most popular spin labels, even though they feature some disadvantageous properties, most importantly a flexible linker (length scale ca. 0.7 nanometers) as attachment to the biomolecule. This flexible linker leads to major uncertainties when interpreting the measured distances. The core idea of my proposed project is establishing Cu(II) spin centers for 19F ENDOR measurements. These allow circumventing the problems described for nitroxide labels and offer additional advantages. At the same time, Cu(II) also features challenging spectroscopic properties, for example fast magnetic relaxation. It is unclear to which extent 19F ENDOR with Cu(II) centers is feasible. Therefore, the starting point of the project is devoted to systematically developing and optimizing experimental procedures using rigid Cu(II) spin labels in a model protein. Preliminary results for this part have already been obtained and confirm the feasibility of the proposed experiments. The protocols developed in the model protein are subsequently used to investigate the structure of a peptide hormone/Cu(II) complex, for which a partly disordered structure is expected. The partial disorder prevents using X-ray crystallography and cryo-EM in this medium-sized system. Since the peptide contains a natural Cu(II) binding site, any additional flexibility by introducing a spin label is completely circumvented here. The last part of the project investigates the suitability of the 19F/Cu(II) pair in the frame work of orthogonal labelling strategies, which are practically not used at all in ENDOR. For this, a model system which contains additional nitroxide radicals is proposed. This allows selective measurement of pairwise distances (Cu/19F, Cu/nitroxide, nitroxide/19F) as well as establishing three-spin-correlations. Such a network of information is far more valuable than any singular pair distance obtained using current protocols.

• DFG Programme: Research Grants