Isotope-edited ESEEM: A new method for probing copper binding sites in neurodegenerative proteins

The interactions between intrinsically disordered domains in neurodegenerative proteins are often stabilized by the inclusion of physiologic metal ions such as copper or zinc. Characterizing the metal ion coordination environment in such cases is critical for assessing the stability and organization of these relevant protein-protein interactions but is challenging given the lack of regular molecular order of the relevant protein domains. We recently developed a new pulsed electron paramagnetic resonance approach that takes advantage of electron spin echo envelope modulation (ESEEM) with selective ¹⁵N isotope labeling. Histidine side chains with ¹⁴N or ¹⁵N coordinated to Cu²⁺ give distinct, fully resolvable signals in both ESEEM and its two-dimensional companion method HYSCORE. The approach was applied to the cellular prion protein, PrP^C, to investigate how its otherwise neurotoxic, disordered N-terminal domain is regulated through a Cu²⁺ linkage to its globular C-terminal domain. Sortase-mediated ligation created an expressed murine prion protein with segmental ¹⁵N-labeling exclusive to the N-terminal domain. Isotope-edited ESEEM identifies the specific His residues from both domains that participate in this essential regulatory process. Extending this approach to hetero-protein complexes focused on how Cu²⁺ may facilitate the interaction between PrP^C and the Aβ peptide, a dominant component of senile plaques in Alzheimer's disease. Combining ¹⁵N-labeled PrP^C with unlabeled Aβ shows that both proteins simultaneously coordinate Cu²⁺ with Aβ contributing a single His side chain. Collectively, this review highlights isotope-edited ESEEM as an effective method for achieving residue-specific insights into metal coordination within structured and disordered protein complexes.