Recent progress in NMR spectroscopy: Toward the study of intrinsically disordered proteins of increasing size and complexity

Abstract

SummaryThanks to recent fast progress, NMR is now in a strategic position to provide unique atomic resolution information on a variety of different biological macromolecules in different conditions (solution, solid state, in-cell). We would like here to present recent developments that enable to focus on intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) of proteins of increasing size and complexity. They have attracted the attention of the scientific community challenging well accepted ideas and concepts and demanding an expansion of the structure function paradigm.

Synergistic Regulation of Coregulator/Nuclear Receptor Interaction by Ligand and DNA

“…Preliminary 2D [ 1 H, 15 N]-HSQC NMR analysis of our SRC-2 RID construct, which contains three LXXLL motifs and a C-terminal extension containing a related LEEIL motif, revealed very poor spectral dispersion and missing peaks that hindered the interaction analysis (Figure 1A). We therefore utilized 13 C-detected NMR methods (Felli and Pierattelli, 2012), which alleviated issues of poor spectral dispersion and rapid exchange of amide protons with solvent that are often problematic with disordered proteins such as SRC-2 RID (Figure S1). 2D [ 13 C, 15 N]-CON (Figure 1B) and [ 13 C, 15 N]-intraCAN (Figure 1C) spectra afforded dispersed spectra that revealed significant changes upon addition of PPARγ-RXRα, including observation of 16 proline residues in SRC-2 RID that cannot be observed in 2D [ 1 H, 15 N]-HSQC NMR data.…”

Detection of disordered regions in globular proteins using 13C‐detected NMR

“…The truncation after residue 487 led to the generation of the N_meninDC, which was used for NMR experiments. The 13 C, 15 N-labeled N_meninDC protein was expressed by growing bacterial cells in isotopically enriched M9 minimal media. The purification was carried out following previously described protocol.…”

“…15 Additional advantages of carbondetected experiments include the observation of resonances corresponding to the backbone Ca and C 0 carbons allowing for the detection of all amino acids, including proline, which is frequently found in disordered regions. 16 The use of 13 20 Here, we demonstrate that 13 C-based NMR techniques provide a very efficient approach to characterize disordered regions within large, globular proteins.…”

Enhancing the resolution of multi-dimensional heteronuclear NMR spectra of intrinsically disordered proteins by homonuclear broadband decoupling

“…(right) Close-up view of the valine methyl region. Whereas the conventional CT 1 H,13 C HSQC (left) shows significant resonance overlapping most of the peaks are well separated in the HD1 H,13 C HSQC experiment and can be unambiguously assigned.…”