Sensitive proton-detected solid-state NMR spectroscopy of large proteins with selective CH<sub>3</sub>labelling: application to the 50S ribosome subunit

Kurauskas, Vilius; Crublet, Elodie; Macek, Pavel; Kerfah, Rime; Gauto, Diego F.; Boisbouvier, Jérôme; Schanda, Paul

doi:10.1039/c6cc04484k

Cited by 31 publications

(34 citation statements)

References 22 publications

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“…Typical amide 1 H line widths are of the order of 50-70 Hz, narrower than line widths obtained on 2D-crystals of β-barrel membrane proteins, [26] or on a sample of liposome-embedded proteorhodopsin (a protein that yields highly resolved 13 C-detected spectra) [27]. 1 H line widths of methyls, obtained with CH 3 -labelling [28] of Ile-δ1 sites are as low as ~30-40 Hz (Figure 3c). Notwithstanding these fairly favorable line widths, the large number of residues (more than 300) leads to severe resonance overlap, hampering assignment, and making it difficult to evaluate the completeness of these spectra.…”

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confidence: 99%

Proton‐Detected Solid‐State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs

et al. 2017

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The structure, dynamics and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane, and reconstituted in a suitable membrane-mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co-polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes, forming nanosize discoidal proteolipid particles, also referred to as native nanodiscs. Here we show, for the first time, that high-resolution solid-state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified with the 2 x 34 kDa-large bacterial cation diffusion facilitator CzcD from Cupriavidus metallidurans CH34. Keywords membrane protein; nanodiscs; styrene maleic acid; NMR spectroscopy; solid-state NMR Integral membrane proteins play essential roles in numerous cellular processes. The properties of the surrounding lipid environment are crucial to their structure, dynamics and function. However, structural and biophysical characterizations by most biophysical techniques generally require extraction and purification from the native membrane. Major concerns when working with detergent-solubilized membrane proteins are related to the fact that functionally and structurally important lipids are often stripped away, and that the properties of the detergent micelle differ significantly from the planar lipid bilayer environment. An increasing number of cases of distortions of membrane protein structures in detergents have been reported.[1,2] These known problems with detergents have triggered the development of alternative non-micellar systems, such as amphiphilic polymers (amphipols), bicelles, or nanolipoprotein particles, i.e. a lipid patch surrounded by a membrane-scaffold protein. [3][4][5] A particularly interesting recent approach is the use of the beate.bersch@ibs.fr, paul.schanda@ibs.fr. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts styrene/maleic acid co-polymer (SMA), which is able to self-insert into the lipid bilayer, and to extract membrane proteins directly from the membrane, forming nanosize disc-shaped particles. [5][6][7] SMA has been successfully used to solubilize membrane proteins from reconstituted proteoliposomes [8,9], and also from native cellular membranes, forming socalled native nanodiscs. [7,[10][11][12][13][14] The stability and activity of proteins in SMA-bounded nanodiscs have been shown to be significantly higher than in detergent micelles in several instances, and some cases were reported where the stability was even higher than in the native membrane [15]. These nanodiscs have been used in functional tests, ligand binding studies, electron-microscopy, EPR and other spectroscopic characterizations (reviewed in...

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Proton‐Detected Solid‐State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs

et al. 2017

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“…At the same time, intra-and inter-methyl 1 H, 1 H dipolar interactions induce dipolar broadening that counters the advantage. A comparison of deuterated samples, with selectively labelled CH 3 and CHD 2 methyls at Ile and Val has been carried out previously by Schanda and coworkers for the sedimented 468 kDa dodecameric aminopeptidase TET2 (Kurauskas et al 2016). The authors concluded that at > 60 kHz MAS and 14.1 T (600 MHz for 1 H) magnetic field the two schemes (CH3 and CHD2) yield comparable linewidths, with the CH 3 labelling providing improved sensitivity.…”

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confidence: 91%

MAS dependent sensitivity of different isotopomers in selectively methyl protonated protein samples in solid state NMR

et al. 2019

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Sensitivity and resolution together determine the quality of NMR spectra in biological solids. For high-resolution structure determination with solid-state NMR, protondetection emerged as an attractive strategy in the last few years. Recent progress in probe technology has extended the range of available MAS frequencies up to above 100 kHz, enabling the detection of resolved resonances from sidechain protons, which are important reporters of structure. Here we characterise the interplay between MAS frequency in the newly available range of 70-110 kHz and proton content on the spectral quality obtainable on a 1GHz spectrometer for methyl resonances. Variable degrees of proton densities are tested on microcrystalline samples of the α-spectrin SH3 domain with selectively protonated methyl isotopomers (CH 3 , CH 2 D, CHD 2) in a perdeuterated matrix. The experimental results are supported by simulations that allow the prediction of the sensitivity outside this experimental frequency window. Our results facilitate the selection of the appropriate labelling scheme at a given MAS rotation frequency.

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“…In this work, we use methyl labeling—which has proven itself as an indispensable tool in solution NMR of large proteins,—to simplify spectral data and facilitate proton‐detection ,. The methodology presented here allows for the collection of long‐distance restraints with 3D proton‐detected ssNMR experiments between the following: 1.)…”

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Protein−Protein Interfaces Probed by Methyl Labeling and Proton‐Detected Solid‐State NMR Spectroscopy

et al. 2018

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Proton detection and fast magic‐angle spinning have advanced biological solid‐state NMR, allowing for the backbone assignment of complex protein assemblies with high sensitivity and resolution. However, so far no method has been proposed to detect intermolecular interfaces in these assemblies by proton detection. Herein, we introduce a concept based on methyl labeling that allows for the assignment of these moieties and for the study of protein−protein interfaces at atomic resolution.

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Sensitive proton-detected solid-state NMR spectroscopy of large proteins with selective CH₃labelling: application to the 50S ribosome subunit

Cited by 31 publications

References 22 publications

Proton‐Detected Solid‐State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs

Proton‐Detected Solid‐State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs

MAS dependent sensitivity of different isotopomers in selectively methyl protonated protein samples in solid state NMR

Protein−Protein Interfaces Probed by Methyl Labeling and Proton‐Detected Solid‐State NMR Spectroscopy

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Sensitive proton-detected solid-state NMR spectroscopy of large proteins with selective CH3labelling: application to the 50S ribosome subunit

Cited by 31 publications

References 22 publications

Proton‐Detected Solid‐State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs

Proton‐Detected Solid‐State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs

MAS dependent sensitivity of different isotopomers in selectively methyl protonated protein samples in solid state NMR

Protein−Protein Interfaces Probed by Methyl Labeling and Proton‐Detected Solid‐State NMR Spectroscopy

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Sensitive proton-detected solid-state NMR spectroscopy of large proteins with selective CH₃labelling: application to the 50S ribosome subunit