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

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)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.



Source: Journal of Biomolecular NMR
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