title: A J-modulated protonless NMR experiment characterizes the conformational ensemble of the intrinsically disordered protein WIP.
abstract: 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)NCO13C-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 WIP2-65conformational 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.
authors.affiliation: Department of Chemistry, Bar Ilan University, 52900, Ramat Gan, Israel.
authors.forename: Eva
authors.initials: E
authors.lastname: Rozentur-Shkop
authors.affiliation: Department of Chemistry, Bar Ilan University, 52900, Ramat Gan, Israel.
authors.forename: Gil
authors.initials: G
authors.lastname: Goobes
authors.affiliation: Department of Chemistry, Bar Ilan University, 52900, Ramat Gan, Israel. Jordan.Chill@biu.ac.il.
authors.forename: Jordan H
authors.identifier: 0000-0002-9518-824X
authors.initials: JH
authors.lastname: Chill
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D064267:Intrinsically Disordered Proteins / Q000737:chemistry* / Q000378:metabolism; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins; D025801:Ubiquitin / Q000737:chemistry
chemical_list: D000199:Actins; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D064267:Intrinsically Disordered Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; C109903:WIPF1 protein, human
keywords: 13C′-detected experiments; Actin-binding module; Crowding effects; Intrinsically disordered proteins; J-modulated experiments; WASP-interacting protein