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 [Formula: see text] NMR relaxation methodology have been applied to a large number of systems. However, the low dispersion of [Formula: see text] 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 [Formula: see text] relaxation parameters ([Formula: see text], [Formula: see text], NOE, cross-correlated relaxation rates [Formula: see text] and [Formula: see text]) in doubly [Formula: see text],[Formula: see text]-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 [Formula: see text]-subunit of RNA polymerase from Bacillus subtilis. Comparison with data obtained using a sample labeled by [Formula: see text] only showed that the presence of [Formula: see text] has a negligible effect on [Formula: see text], [Formula: see text], and on the cross-relaxation rate (calculated from NOE and [Formula: see text]), and that these relaxation rates can be used to calculate accurate spectral density values. Partially [Formula: see text]-labeled sample was used to test if the observed increase of [Formula: see text] [Formula: see text] in the presence of [Formula: see text] corresponds to the [Formula: see text] dipole-dipole interactions in the [Formula: see text],[Formula: see text]-labeled sample.

Boosting the resolution of low-field $$^{15}\hbox {N}$$ relaxation experiments on intrinsically disordered proteins with triple-resonance NMR

“…The pulse sequence is designed to be suitable for the analysis of the dynamics of IDPs as was shown for the C-terminal domain of the δ subunit of RNA polymerase -a protein with highly repetitive amino acid sequence and characterized by very low diversity of NMR frequencies of backbone nuclei. Relaxation rates measured at low field identify segments with slower motional modes within the disordered region of the δ subunit in agreement with previous studies [Srb et al(2017), Kadeřávek et al(2014)].…”

“…Examples of peak intensity decays are shown in Figure 2 and supplementary information material Figures S1-S3. Longitudinal relaxation rates measured at 0.33 T are shown in Figure 3 together with the previously reported [Srb et al(2017)] longitudinal and transverse relaxation rates acquired at 14.1 T for the same system under the same conditions. All discussed experiments were performed with uniformly 13 C, 15 N labeled samples, so the autorelaxation rates reflect not only the relaxation due to the direct 1 H-15 N DD interaction together with the contribution of the anisotropy of the amide 15 N chemical shielding tensor (CSA), but also the effects of the dipolar interactions between the 15 N and neighboring 13 C nuclei as discussed previously [Srb et al(2017)].…”

“…Longitudinal relaxation rates measured at 0.33 T are shown in Figure 3 together with the previously reported [Srb et al(2017)] longitudinal and transverse relaxation rates acquired at 14.1 T for the same system under the same conditions. All discussed experiments were performed with uniformly 13 C, 15 N labeled samples, so the autorelaxation rates reflect not only the relaxation due to the direct 1 H-15 N DD interaction together with the contribution of the anisotropy of the amide 15 N chemical shielding tensor (CSA), but also the effects of the dipolar interactions between the 15 N and neighboring 13 C nuclei as discussed previously [Srb et al(2017)]. Although 48 out of 68 residues in the negatively charged part of the C-terminal domain are either glutamic or aspartic acids in this part of the sequence, resulting in very poor signal dispersion, we were able to extract quantitative information for 80% of them.…”

“…In contrast to transverse relaxation rates, the longitudinal relaxation rates at low field can not be biased by contributions of a chemical exchange. High-field transverse crosscorrelated cross-relaxation rates (CSA-DD) is another relaxation rate which is not sensitive to contributions of chemical exchange [Abyzov et al(2016), Khan et al(2015), Srb et al(2017), Kadeřávek et al(2014)]. However, unless the analysis is based on both longitudinal and transverse CSA/DD cross-correlated cross relaxation [Kroenke et al(1998)], the analysis combining the auto-relaxation rates and transverse CSA-DD cross-correlated cross-relaxation rates requires a very accurate determination of amplitudes and orientations of amide chemical shielding tensors which may also vary over the sequence of proteins and might be difficult to obtain.…”

“…Previously reported high-resolution relaxometry experiments of protein backbone 15 N amides [Charlier et al(2013), Clarkson et al(2009] are based on signal detection using 2D 15 N-1 H HSQC spectra. Here, the dimensionality of the spectra was extended with the addition of a carbonyl dimension [Srb et al(2017)] and the evolution times in the indirect dimensions are increased using non-uniform sampling (NUS) to keep the experimental time in reasonable limits. Such an HNCO-type experiment provides a sufficient resolution for the quantitative site-specific analysis of the δ subunit of RNA polymerase with extremely low chemical shift dispersion of its intrinsically disordered and highly repetitive C-terminal domain.…”