Communication Dans Un Congrès Année : 2024

Fast access to protein dynamics using ultra-selective 15N-1H spectroscopy

Résumé

Spectral congestion in 2D protein NMR spectra is a ubiquitous problem. The usual strategy to overcome this issue is by adding further dimensions to the experiment or increase the number of time-increments in the existing indirect dimensions, but this results in longer experimental times. This problems is exacerbated in zz-exchange experiments, where additional exchange cross peaks are present, and especially in relaxation measurement experiments, where multiple experiments have to be acquired in order to sufficiently sample the signal decay. Especially for intrinsically disordered proteins, which often feature very congested spectra and limited sample life times, the required experimental time becomes impractically long. An alternative approach is to use selective experiments that target particular 1H-15N correlations, allowing the use of simple 1D experiments as a readout. Selective Hartmann-Hahn coherence transfer has been previously proposed for this purpose, but the selectivity of these experiments was limited (1,2). Here, we propose a scheme that provides clean 1D spectra with an improved selectivity of 15-20 Hz in 15N and 1H with only a modest cost in sensitivity. Our approach uses an alternative scheme for selective polarization transfer that, to the best or knowledge, has been neglected until now (1). This is combined with an improved purge element based on 15N chemical shift offset (2). We will first demonstrate these principles by quantifying the exchange rates between two slowly exchanging conformers of the SH3GL3 protein. The forms display very similar chemical shifts in the 1H-15N HSQC, but the new selective zz exchange experiment successfully resolved the cross and diagonal peaks. Next, we introduce new selective R1, R2 and hetNOE experiments to quantify the dynamics along a 16-residue polyglutamine stretch within a huntingin exon 1 protein fragment, the causative agent for Huntington’s disease. The severely overlapped polyQ signals until now precluded such relaxation measurements. We demonstrate, for the first time, the gradual increase in conformational dynamics from the N- to C-terminal ends of the polyQ using NMR relaxation and without requiring site-specific isotopic labelling schemes. Our novel pulse sequences provides access to slow, intermediate or fast protein dynamics in a fraction of the experimental time required using non-selective (pseudo-3D) experiments (3), and is a substantial improvement compared to previously proposed selective Hartmann-Hahn schemes (4). The new experiments provide sufficiently selective and clean spectra at acceptable sensitivity, and will be very valuable for any biomacromolecule featuring highly crowded heteronuclear 2D spectra. References 1.Pelupessy, P. and Chiarparin, E., Concepts Magn. Reson. 2000, 12, 103-124. 2.Korzhnev, D. M., Orekhov, V. Y. and Kay, L. E., J. Am. Chem. Soc. 2005, 127, 713-721. 3.Farrow, N. A., Zhang, O., Forman-Kay, J. D. and Kay, L. E., J. Biomol NMR 1994, 5, 727-734. 4.Nishizawa, M., Walinda, E., Morimoto, D. and Sugase, K., J. Biomol. NMR 2020, 74, 205-211.

Mots clés

Fichier non déposé

Dates et versions

hal-05017163 , version 1 (02-04-2025)

Identifiants

  • HAL Id : hal-05017163 , version 1

Citer

Wiktor Adamski, François-Xavier Cantrelle, Géraldine Levy, Davy Sinnaeve. Fast access to protein dynamics using ultra-selective 15N-1H spectroscopy. 20th edition of the Young Belgian Magnetic Resonance Scientists symposium - YBMRS 2024, Nov 2024, Blankenberge, Belgium. ⟨hal-05017163⟩
68 Consultations
0 Téléchargements

Partager

  • More