In the field of Alzheimer’s disease (AD) and related Tauopathies, the heightened post-translational modifications (PTMs), especially phosphorylation, of Tau proteins stand as prominent pathological indicators. Phosphorylation can alter its electrostatic properties and/or structural conformation, which in turn affect the protein's bioactivity, seeding potential, and aggregation behavior. This has sparked significant interest in investigating phosphorylation of Tau within the scope of these diseases. Developing methods to efficiently, stably read full sequences, and fingerprint phosphorylation—or even pinpoint their locations—is crucial for understanding how these modifications influence the pathological process at the single-molecule level. Motivated by PTM mapping of patient-derived samples, we employ a well-known mutant of a biological nanopore, Mycobacterium smegmatis porin A, mutant 2 (M2-MspA) to sense recombinant Tau protein and full-length non-phosphorylated and phosphorylated variants. The first question we address is whether wild-type Tau protein can translocate through MspA nanopore. This is inherently challenging for native proteins, as their non-uniform charge distribution and structural heterogeneity typically hinder efficient capture and translocation even in denatured conditions. We find that Tau's intrinsic structural disorder, flexible and acidic terminal regions, coupled with the strong electro-osmotic flow (EOF) generated by the MspA nanopore in a guanidinium chloride (GdmCl) environment, collectively facilitates successful capture and translocation. We will present data on the transport dynamics of tau and its variants, as well as strategies for PTM detection.
Chen et al. (Sun,) studied this question.