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, ³J_HNHα couplings between amide (¹H^N) and alpha (¹H^α) protons impose an intrinsic limit on the achievable ¹H^N linewidth. Here, we present a homonuclear decoupling method that narrows the α-synuclein ¹H^N linewidths to 3-5 Hz. Tightly distributed ¹J_CαHα coupling values were employed to generate homogeneous antiphase coherences of 2H^αH^N and 4H^α(2)H^α(3)H^N for nonglycine and glycine residues, respectively, which were combined with their in-phase H^N counterparts to achieve homonuclear decoupling. By reducing the multiplet structure to a singlet, the width of the ¹H^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.