Site-Dependent Structural Effects of R1 Spin Labeling in an Aβ42 Protofilament Characterized by Molecular Dynamics Simulations

Abstract Site-directed spin labeling coupled with electron paramagnetic resonance (EPR) is used to characterize structural features in amyloid fibrils, but whether the nitroxide spin label R1 perturbs the structure it reports remains unclear. Molecular dynamics simulations compared wild-type Aβ42 with ten R1-labeled variants in a five-layer protofilament derived from the type I human-brain fibril structure. We quantified nitroxide proximity, regional interlayer packing, backbone hydrogen bonding, R1–protein contacts, and R1 side-chain rotamer sampling. Simulated nitroxide contacts reproduced the experimental distinction between sites with strong and weak spin–spin interactions, although some sites deviated from the trend. Regional interlayer organization and backbone hydrogen bonding were preserved relative to wild-type. However, structural responses were site-dependent and sometimes extended beyond the labeling site. The 32R1 system produced the largest rearrangement of the C-terminal tail. Comparison with the corresponding unlabeled cysteine control showed smaller perturbation, indicating that the response at position 32 arose primarily from the R1 label rather than the cysteine substitution itself. Thus, R1 generally preserved the protofilament architecture and reflected the local packing environment while producing label-specific structural adjustments. These results support R1 as a reporter of local packing in Aβ42 fibrils and demonstrate the value of site-resolved comparisons for identifying label-induced effects.