13Cα decoupling during direct observation of carbonyl resonances in solution NMR of isotopically enriched proteins

Direct detection of 13C can be advantageous when studying uniformly enriched proteins, in particular for paramagnetic proteins or when hydrogen exchange with solvent is fast. A scheme recently introduced for long-observation-window band-selective homonuclear decoupling in solid state NMR, LOW-BASHD (Struppe et al. in J Magn Reson 236:89–94, 2013) is shown to be effective for 13Cα decoupling during direct 13C′ observation in solution NMR experiments too. For this purpose, adjustment of the decoupling pulse parameters and delays is demonstrated to be important for increasing spectral resolution, to reduce three-spin effects, and to decrease the intensity of decoupling side-bands. LOW-BASHD then yields 13C′ line widths comparable to those obtained with the popular IPAP method, while enhancing sensitivity by ca 35 %. As a practical application of LOW-BASHD decoupling, requiring quantitative intensity measurement over a wide dynamic range, the impact of lipid binding on the 13C′-detected NCO spectrum of the intrinsically disordered protein α-synuclein is compared with that on the 1H-detected 1H–15N HSQC spectrum. Results confirm that synuclein’s “dark state” behavior is not caused by paramagnetic relaxation or rapid hydrogen exchange.