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

摘要

NMR relaxation of arginine (Arg) ¹⁵Nε 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 ¹⁵Nε nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal ¹⁵N-¹⁵N couplings (²JNN) of the ¹⁵Nε nuclei and found that the magnitudes of the ²JNN 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 ²JNN couplings of this magnitude could in principle cause significant modulation in signal intensities during the Carr-Purcell-Meiboom-Gill (CPMG) scheme for Arg ¹⁵Nε R2 measurements. However, our experimental data show that the expected modulation via two ²JNN couplings vanishes during the ¹⁵N CPMG scheme. This quenching of J modulation can be explained by the mechanism described in ChemPhysChem 7, 831–836. This effect allows for accurate measurements of R2 relaxation rates for Arg side-chain ¹⁵Nε nuclei despite the presence of two ²JNN 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.