Triple resonance 15 N NMR relaxation experiments for studies of intrinsically disordered proteins

Description of protein dynamics is known to be essential in understanding their function. Studies based on a well established \(^{15}\hbox {N}\) NMR relaxation methodology have been applied to a large number of systems. However, the low dispersion of \(^{1}\hbox {H}\) chemical shifts very often observed within intrinsically disordered proteins complicates utilization of standard 2D HN correlated spectra because a limited number of amino acids can be characterized. Here we present a suite of triple resonance HNCO-type NMR experiments for measurements of five \(^{15}\hbox {N}\) relaxation parameters (\(R_1\), \(R_2\), NOE, cross-correlated relaxation rates \(\Gamma _x\) and \(\Gamma _z\)) in doubly \(^{13}\hbox {C}\),\(^{15}\hbox {N}\)-labeled proteins. We show that the third spectral dimension combined with non-uniform sampling provides relaxation rates for almost all residues of a protein with extremely poor chemical shift dispersion, the C terminal domain of \(\delta\)-subunit of RNA polymerase from Bacillus subtilis. Comparison with data obtained using a sample labeled by \(^{15}\hbox {N}\) only showed that the presence of \(^{13}\hbox {C}\) has a negligible effect on \(\Gamma _x\), \(\Gamma _z\), and on the cross-relaxation rate (calculated from NOE and \(R_1\)), and that these relaxation rates can be used to calculate accurate spectral density values. Partially \(^{13}\hbox {C}\)-labeled sample was used to test if the observed increase of \(^{15}\hbox {N}\) \(R_1\) in the presence of \(^{13}\hbox {C}\) corresponds to the \(^{15}\hbox {N}-^{13}\hbox {C}\) dipole–dipole interactions in the \(^{13}\hbox {C}\),\(^{15}\hbox {N}\)-labeled sample.