The authors develop an MspA nanopore single-molecule assay that resolves three reproducible current states of monomeric α‑synuclein, maps them to α‑helical and random‑coil subdomains, and generates a biophysical parameter matrix to profile structural heterogeneity of variants.

By directly sensing monomeric α‑synuclein conformations at single‑molecule resolution, this method can reveal early misfolding mechanisms, enable screening for compounds that stabilize nonpathogenic conformers, and serve as a potential biomarker platform for Parkinson's therapeutic discovery.

Parkinson's disease (PD) is closely associated with the misfolding of α-synuclein (αSyn) proteins, which remains obscure due to the long-standing challenge to elucidate monomeric αSyn structure. Here, we present a novel single-molecule technique to efficiently analyze αSyn monomer with Mycobacterium smegmatis porin A (MspA) nanopores. The nanopore signals elicited by αSyn consist of three distinct, reproducible current levels. Notably, through systematic measurement of αSyn fragments, reference peptide with α-helical structure and molecular dynamics simulation study, we demonstrate that these current levels correspond to α-helical and random-coil structures within the protein. The α-helical structures are successfully assigned to the specific αSyn subdomains. A biophysical parameter figure matrix derived from the αSyn signals is further generated and readily applied to profile the structural heterogeneity of monomeric αSyn variants. These results establish that MspA nanopores can directly sense the formation of α-helical structure in individual αSyn monomer, which advances our capability to investigate this highly flexible pathological protein and may contribute to elucidating its misfolding mechanism.