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Proc Natl Acad Sci U S A. 2016 Aug 16;113(33):9187-92. doi: 10.1073/pnas.1602248113. Epub 2016 Aug 3.

Structure of fully protonated proteins by proton-detected magic-angle spinning NMR.

Author information

1
Centre de Résonance Magnétique Nucléaire à Très Hauts Champs, Institut des Sciences Analytiques (UMR 5280 - CNRS, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1), Université de Lyon, 69100 Villeurbanne, France;
2
Biomedical Research and Study Centre, LV-1067 Riga, Latvia;
3
Bruker Biospin, 76287 Rheinstetten, Germany;
4
Centre de Résonance Magnétique Nucléaire à Très Hauts Champs, Institut des Sciences Analytiques (UMR 5280 - CNRS, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1), Université de Lyon, 69100 Villeurbanne, France; Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
5
Centre de Résonance Magnétique Nucléaire à Très Hauts Champs, Institut des Sciences Analytiques (UMR 5280 - CNRS, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1), Université de Lyon, 69100 Villeurbanne, France; Guido.Pintacuda@ens-lyon.fr.

Abstract

Protein structure determination by proton-detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of signals from side chains is extremely limited. Here, we show in two fully protonated proteins that, at 100-kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of (1)H-(1)H proximities that define a protein structure. The high data quality allowed the correct identification of internuclear distance restraints encoded in 3D spectra with automated data analysis, resulting in accurate, unbiased, and fast structure determination. Additionally, we find that narrower proton resonance lines, longer coherence lifetimes, and improved magnetization transfer offset the reduced sample size at 100-kHz spinning and above. Less than 2 weeks of experiment time and a single 0.5-mg sample was sufficient for the acquisition of all data necessary for backbone and side-chain resonance assignment and unsupervised structure determination. We expect the technique to pave the way for atomic-resolution structure analysis applicable to a wide range of proteins.

KEYWORDS:

NMR spectroscopy; magic-angle spinning; protein structures; proton detection; viral nucleocapsids

PMID:
27489348
PMCID:
PMC4995937
DOI:
10.1073/pnas.1602248113
[Indexed for MEDLINE]
Free PMC Article

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