Identifying the copper coordination environment between interacting neurodegenerative proteins: A new approach using pulsed EPR with 14N/15N isotopic labeling
Year: 2025
Type: article
Abstract: The trafficking and aggregation of neurodegenerative proteins often involves the interaction between intrinsically disordered domains, stabilized by the inclusion of physiologic metal ions such as copper or zinc. Characterizing the metal ion coordination environment is critical for assessing the stability and organization of these relevant protein-protein interactions but is challenging given the lack of regular molecular order or global structure. The cellular prion protein (PrP C ) binds both monomers and aggregates of the Alzheimer’s amyloid-beta peptide (Aβ), promoting interactions of relevance to Aβ internalization across the cellular plasma membrane and aberrant signaling in neurodegenerative disease, respectively. Both proteins bind Cu 2+ with high affinity, suggesting the existence of a ternary complex with copper bridging between the two proteins through His coordination. In this work, we describe a novel approach utilizing multiple EPR experiments to characterize the simultaneous Cu 2+ coordination of PrP C and Aβ. Uniformly 15 N-labeled PrP C is used in conjunction with natural abundance 14 N Aβ, the combination of which leads to distinct energy manifolds for paramagnetic Cu 2+ and resolved by the pulsed EPR experiments ESEEM and HYSCORE. We develop acquisition parameters to simultaneously optimize 14 N (I = 1) and 15 N (I = ½) pulsed EPR signals and we also advance the theory of ESEEM and HYSCORE to quantitatively describe multiple 15 N imidazole coordination. Together, these findings provide a detailed view of how Cu 2+ bridges between the two proteins in this complex, along with a global strategy for assessing the copper environment with other interacting neurodegenerative proteins.
Source: Journal of Biological Chemistry
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,430
OpenAlex ID: W4407493191