A J-modulated protonless NMR experiment characterizes the conformational ensemble of the intrinsically disordered protein WIP

Eva Rozentur-Shkop, Gil Goobes, Jordan H. Chill

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


Intrinsically disordered proteins (IDPs) are multi-conformational polypeptides that lack a single stable three-dimensional structure. It has become increasingly clear that the versatile IDPs play key roles in a multitude of biological processes, and, given their flexible nature, NMR is a leading method to investigate IDP behavior on the molecular level. Here we present an IDP-tailored J-modulated experiment designed to monitor changes in the conformational ensemble characteristic of IDPs by accurately measuring backbone one- and two-bond J(15N,13Cα) couplings. This concept was realized using a unidirectional (H)NCO 13C-detected experiment suitable for poor spectral dispersion and optimized for maximum coverage of amino acid types. To demonstrate the utility of this approach we applied it to the disordered actin-binding N-terminal domain of WASp interacting protein (WIP), a ubiquitous key modulator of cytoskeletal changes in a range of biological systems. One- and two-bond J(15N,13Cα) couplings were acquired for WIP residues 2–65 at various temperatures, and in denaturing and crowding environments. Under native conditions fitted J-couplings identified in the WIP conformational ensemble a propensity for extended conformation at residues 16–23 and 45–60, and a helical tendency at residues 28–42. These findings are consistent with a previous study of the based upon chemical shift and RDC data and confirm that the WIP2–65 conformational ensemble is biased towards the structure assumed by this fragment in its actin-bound form. The effects of environmental changes upon this ensemble were readily apparent in the J-coupling data, which reflected a significant decrease in structural propensity at higher temperatures, in the presence of 8 M urea, and under the influence of a bacterial cell lysate. The latter suggests that crowding can cause protein unfolding through protein–protein interactions that stabilize the unfolded state. We conclude that J-couplings are a useful measureable in characterizing structural ensembles in IDPs, and that the proposed experiment provides a practical method for accurately performing such measurements, once again emphasizing the power of NMR in studying IDP behavior.

Original languageEnglish
Pages (from-to)243-257
Number of pages15
JournalJournal of Biomolecular NMR
Issue number4
StatePublished - 1 Dec 2016

Bibliographical note

Funding Information:
We thank Drs. Hugo Gottlieb and Keren Keinan-Adamsky for spectrometer assistance and Mr. Israel Tabakman for technical assistance. Early contributions of Dr. Renana Gross to this work are greatly appreciated. Financial support by the Heritage Legacy fund (Israel Science Foundation award 491/10) is gratefully acknowledged. Establishment of the 700 MHz spectrometer system in the NMR lab was supported by Fundacion Adar and a Converging Technologies award. JHC acknowledges support of the Christians for Israel Chair for Medical Research.

Publisher Copyright:
© 2016, Springer Science+Business Media Dordrecht.


  • Actin-binding module
  • Crowding effects
  • C′-detected experiments
  • Intrinsically disordered proteins
  • J-modulated experiments
  • WASP-interacting protein


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