Impact of two-bond ¹⁵N–¹⁵N scalar couplings on ¹⁵N transverse relaxation measurements for arginine side chains of proteins

NMR relaxation of arginine (Arg) ¹⁵N_ε nuclei is useful for studying side-chain dynamics of proteins. In this work, we studied the impact of two geminal ¹⁵N–¹⁵N scalar couplings on measurements of transverse relaxation rates (R₂) for Arg side-chain ¹⁵N_ε nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal ¹⁵N–¹⁵N couplings (²J_NN) of the ¹⁵N_ε nuclei and found that the magnitudes of the ²J_NN coupling constants were virtually uniform with an average of 1.2 Hz. Our simulations, assuming ideal 180° rotations for all ¹⁵N nuclei, suggested that the two ²J_NN couplings of this magnitude could in principle cause significant modulation in signal intensities during the Carr–Purcell-Meiboom–Gill (CPMG) scheme for Arg ¹⁵N_εR₂ measurements. However, our experimental data show that the expected modulation via two ²J_NN couplings vanishes during the ¹⁵N CPMG scheme. This quenching of J modulation can be explained by the mechanism described in Dittmer and Bodenhausen (Chemphyschem 7:831–836, 2006). This effect allows for accurate measurements of R₂ relaxation rates for Arg side-chain ¹⁵N_ε nuclei despite the presence of two ²J_NN couplings. Although the so-called recoupling conditions may cause overestimate of R₂ rates for very mobile Arg side chains, such conditions can readily be avoided through appropriate experimental settings.