Exploring Chromophore-Binding Pocket: High-Resolution Solid-State 1H-13C Interfacial Correlation NMR Spectra with Windowed PMLG Scheme

High-resolution two-dimensional (2D) H-1-C-13 heteronuclear correlation spectra are recorded for selective observation of interfacial 3-5.5 contacts of the uniformly C-13-labeled phycocyanobilin (PCB) chromophore with its unlabeled binding pocket. The experiment is based on a medium- and long-distance heteronuclear correlation (MELODI-HETCOR) method. For improving H-1 spectral resolution, a windowed phase-modulated Lee-Goldburg (wPMLG) decoupling scheme is applied during the t (1) evolution period. Our approach allows for identification of chromophore-protein interactions, in particular for elucidation of the hydrogen-bonding networks and charge distributions within the chromophore-binding pocket. The resulting pulse sequence is tested on the cyanobacterial (Cph1) phytochrome sensory module (residues 1-514, Cph1 Delta 2) containing uniformly C-13- and N-15-labeled PCB chromophore (u-[C-13,N-15]-PCB-Cph1 Delta 2) at 17.6 T.