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

Abstract

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.