NMR methodology


NMR spectroscopy is ideally suited to investigate Intrinsically disordered proteins (IDPs) or disordered protein regions (IDRs). As a technique, it is very sensitive to changes in molecular dynamics, and the inherent disorder incurs favourable spectroscopic properties that allow the design of very informative experiments. However, IDPs also present unique challenges to NMR spectroscopy. Compared to their globular counterparts, IDPs display significantly lower chemical shift dispersion, leading – even for small proteins – to very crowded NMR spectra, impairing extraction of valuable structural information. Moreover, the fast interconversion between conformers makes data interpretation not straightforward. We are interested in developing new approaches that harvest a maximum of spectral information that can be translated into structural information, such as residual dipolar couplings (RDCs). Central to this is the use of ‘pure shift’ techniques, whereby the signals are homodecoupled so that multiplets in the 1H spectrum of typically 10-50 Hz wide reduce to singlets of only a few Hz wide, improving resolution by an order of magnitude. Such methods have proven very valuable for small molecules, including the measurement of 1H-1H scalar couplings and RDCs (Sinnaeve et al. Angew. Chem. Int. Ed. 2015; Anal. Chem. 2018; Angew. Chem. Int. Ed. 2020), and also hold potential for IDPs.
We are particularly interested in investigating the structure-functional relationships of proline-rich motifs (PRMs) and oligoproline stretches, which are found abundantly in IDPs. They are key to protein-protein interactions (involving for instance SH3 domains), and mediate biological processes such as cell signaling or neurodegenerative diseases. Examples of PRMs are found in the proteins Huntingtin (implicated in Huntington disease), tau (implicated in Alzheimer disease), and NS5A (a protein of the Hepatitis C Virus). The unique features of proline — the lack of an amide proton, its aliphatic five-membered ring, and the cis/trans peptide bond isomerization — make PRMs particularly challenging for NMR study. Besides the use of homodecoupling techniques, we are interested in the use of fluorinated prolines to investigate the role of individual proline conformations and exploit 19F NMR for interaction studies.

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Tau_1

(A) 1H-1H TOCSY spectrum at 600 MHz, correlating the Hα spectral region with the aliphatic region of the shown proline-rich peptide sequence derived from the proline-rich domain of tau. Clearly, multiplet overlap limits the resolution. (B) F1-PSYCHE TOCSY spectrum of the same region, introducing pure shift resolution in both dimensions. All side-chain protons can now be resolved. (C) 1D 1H, 1D pure shift and PSYCHEDELIC spectra (Sinnaeve et al. Angew. Chem. Int. Ed. 2015) of estradiol to measure individual 1H-1H J-couplings at pure shift resolution, allowing measurement of couplings as simple doublets in crowded spectral regions. (D) 1D 1H, 1D pure shift and PSYCHEDELIC experiment on (+)-isopinocampheol dissolved in poly(γ-benzyl)-D-glutamate/CD2Cl2 to extract individual 1H-1H RDCs (Sinnaeve et al. Angew. Chem. Int. Ed. 2020).