Removal of slow-pulsing artifacts in in-phase ¹⁵N relaxation dispersion experiments using broadband ¹H decoupling

Understanding of the molecular mechanisms of protein function requires detailed insight into the conformational landscape accessible to the protein. Conformational changes can be crucial for biological processes, such as ligand binding, protein folding, and catalysis. NMR spectroscopy is exquisitely sensitive to such dynamic changes in protein conformations. In particular, Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments are a powerful tool to investigate protein dynamics on a millisecond time scale. CPMG experiments that probe the chemical shift modulation of ¹⁵N in-phase magnetization are particularly attractive, due to their high sensitivity. These experiments require high power ¹H decoupling during the CPMG period to keep the ¹⁵N magnetization in-phase. Recently, an improved version of the in-phase ¹⁵N-CPMG experiment was introduced, offering greater ease of use by employing a single ¹H decoupling power for all CPMG pulsing rates. In these experiments however, incomplete decoupling of off-resonance amide ¹H spins introduces an artefactual dispersion of relaxation rates, the so-called slow-pulsing artifact. Here, we analyze the slow-pulsing artifact in detail and demonstrate that it can be suppressed through the use of composite pulse decoupling (CPD). We report the performances of various CPD schemes and show that CPD decoupling based on the 90_x-240_y-90_x element results in high-quality dispersion curves free of artifacts, even for amides with high ¹H offset.