Pre-Homonuclear Decoupling Enables High-Resolution NMR Analysis of Intrinsically Disordered Proteins in Solution

Probing the atomic details of intrinsically disordered proteins is crucial to understanding their biological function and relation to pathogenesis. Although amide-detected NMR experiments are widely employed in protein studies, 3 J HNHα couplings between amide ( 1 H N ) and alpha ( 1 H α ) protons impose an intrinsic limit on the achievable 1 H N linewidth. Here, we present a homonuclear decoupling method that narrows the α-synuclein 1 H N linewidths to 3–5 Hz. Tightly distributed 1 J CαHα coupling values were employed to generate homogeneous antiphase coherences of 2H α z H N y and 4H α (2) z H α (3) z H N y for nonglycine and glycine residues, respectively, which were combined with their in-phase H N y counterparts to achieve homonuclear decoupling. By reducing the multiplet structure to a singlet, the width of the 1 H N cross-peak was reduced by ∼3-fold in the 2D HSQC and 3D intra-HNCA spectra, and good spectral quality was achieved without the need for postprocessing.