15N detection harnesses the slow relaxation property of nitrogen: Delivering enhanced resolution for intrinsically disordered proteins

Abstract

<p id="d7478795e374">Intrinsically disordered proteins (IDPs) have attracted significant attention due to their roles in crucial cellular processes. NMR is the only technique that allows the study of IDPs at atomic-level resolution. However, narrow chemical shift dispersion, rapid exchange with solvent, and high proline content challenge conventional ¹H-detected experiments. Here, we report the development of a suite of 3D experiments based on ¹⁵N direct detection that harnesses the slow relaxation and the larger chemical shift dispersion of ¹⁵N nuclei for complete backbone assignment of IDPs, including proline residues, which are critical to the study of IDPs. Using this approach, we have assigned the regulatory domain of NFATC2 and have identified a likely mechanism by which 14-3-3 proteins regulate NFAT nuclear translocation. </p><p class="first" id="d7478795e386">Studies over the past decade have highlighted the functional significance of intrinsically disordered proteins (IDPs). Due to conformational heterogeneity and inherent dynamics, structural studies of IDPs have relied mostly on NMR spectroscopy, despite IDPs having characteristics that make them challenging to study using traditional ¹H-detected biomolecular NMR techniques. Here, we develop a suite of 3D ¹⁵N-detected experiments that take advantage of the slower transverse relaxation property of ¹⁵N nuclei, the associated narrower linewidth, and the greater chemical shift dispersion compared with those of ¹H and ¹³C resonances. The six 3D experiments described here start with aliphatic ¹H magnetization to take advantage of its higher initial polarization, and are broadly applicable for backbone assignment of proteins that are disordered, dynamic, or have unfavorable amide proton exchange rates. Using these experiments, backbone resonance assignments were completed for the unstructured regulatory domain (residues 131–294) of the human transcription factor nuclear factor of activated T cells (NFATC2), which includes 28 proline residues located in functionally important serine–proline (SP) repeats. The complete assignment of the NFATC2 regulatory domain enabled us to study phosphorylation of NFAT by kinase PKA and phosphorylation-dependent binding of chaperone protein 14-3-3 to NFAT, providing mechanistic insight on how 14-3-3 regulates NFAT nuclear translocation. </p>