Intrinsically disordered proteins (IDPs) play an important role in cell signaling, and NMR is well-suited to study conformational ensembles and dynamics of IDPs. However, the intrinsic flexibility of IDPs often results in severe spectral overlap, which hampers accurate NMR data analysis. By labeling the longitudinal spin order of an α proton (i.e., Hα z ) on multiple quantum coherences of backbone nuclei (e.g., N y C′ x Cα y ), we were able to apply pre-homonuclear decoupling (PHD) to transverse relaxation-optimized spectroscopy (TROSY). The proposed scheme provides ultrahigh resolution in both amide proton and nitrogen dimensions, as illustrated in the analysis of Tau and alpha-synuclein (α-Syn) proteins. The PHD-TROSY readout enabled complete backbone resonance assignment of α-Syn using a single 3D HNCA experiment performed on a 600 MHz NMR spectrometer.

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