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