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

ABSTRACT: 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.

SUBMITTER: Chhabra S

PROVIDER: S-EPMC5828609 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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

Chhabra Sandeep S Fischer Patrick P Takeuchi Koh K Dubey Abhinav A Ziarek Joshua J JJ Boeszoermenyi Andras A Mathieu Daniel D Bermel Wolfgang W Davey Norman E NE Wagner Gerhard G Arthanari Haribabu H

Proceedings of the National Academy of Sciences of the United States of America 20180205 8

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 r ...[more]

PMID: 29432148

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Project description:Backbone (15N) NMR relaxation is one of the main sources of information on dynamics of disordered proteins. Yet, we do not know very well what drives 15N relaxation in such systems, i.e., how different forms of motion contribute to the measurable relaxation rates. To address this problem, we have investigated, both experimentally and via molecular dynamics simulations, the dynamics of a 26-residue peptide imitating the N-terminal portion of the histone protein H4. One part of the peptide was found to be fully flexible, whereas the other part features some transient structure (a hairpin stabilized by hydrogen bonds). The following motional modes proved relevant for 15N relaxation. 1) Sub-picosecond librations attenuate relaxation rates according to S2 ?0.85-0.90. 2) Axial peptide-plane fluctuations along a stretch of the peptide chain contribute to relaxation-active dynamics on a fast timescale (from tens to hundreds of picoseconds). 3) ?/? backbone jumps contribute to relaxation-active dynamics on both fast (from tens to hundreds of picoseconds) and slow (from hundreds of picoseconds to a nanosecond) timescales. The major contribution is from polyproline II (PPII) ? ? transitions in the Ramachandran space; in the case of glycine residues, the major contribution is from PPII ? (?) ? rPPII transitions, in which rPPII is the mirror-image (right-handed) version of the PPII geometry, whereas ? geometry plays the role of an intermediate state. 4) Reorientational motion of certain (sufficiently long-lived) elements of transient structure, i.e., rotational tumbling, contributes to slow relaxation-active dynamics on ?1-ns timescale (however, it is difficult to isolate this contribution). In conclusion, recent advances in the area of force-field development have made it possible to obtain viable Molecular Dynamics models of protein disorder. After careful validation against the experimental relaxation data, these models can provide a valuable insight into mechanistic origins of spin relaxation in disordered peptides and proteins.

Dataset Information

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

Publications

<sup>15</sup>N detection harnesses the slow relaxation property of nitrogen: Delivering enhanced resolution for intrinsically disordered proteins.

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