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PreprintCited by 5Advanced NMR Techniques and ApplicationsProtein Structure and DynamicsLipid Membrane Structure and Behavior

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Evaluation of TOCSY mixing for sensitivity-enhancement in solid-state NMR and application of 4D experiments for side-chain assignments of the full-length 30 kDa membrane protein GlpG

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Öster, Carl, Chevelkov, Veniamin, Lange, Adam·2024·Journal of Biomolecular NMR
DOI 10.26434/chemrxiv-2024-f6qmrPMC PMC11832555

Preprint — published as 10.1007/s10858-024-00454-7 · Paper page

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Abstract

Chemical shift assignments of large membrane proteins by solid-state NMR experiments are challenging. Recent advancements in sensitivity-enhanced pulse sequences, have made it feasible to acquire 1H-detected 4D spectra of these challenging protein samples within reasonable timeframes. However, obtaining unambiguous assignments remains difficult without access to side-chain chemical shifts. Drawing inspiration from sensitivity-enhanced TOCSY experiments in solution NMR, we have explored the potential of 13C-13C TOCSY mixing as a viable option for triple sensitivity-enhanced 4D experiments aimed at side-chain assignments in solid-state NMR. Through simulations and experimental trials, we have identified optimal conditions to achieve uniform transfer efficiency for both transverse components and to minimize undesired cross-transfers. Our experiments, conducted on the 30 kDa membrane protein GlpG embedded in E. coli liposomes, have demonstrated enhanced sensitivity compared to the most effective dipolar and J-coupling-based 13C-13C mixing sequences. Notably, a non-uniformly sampled 4D hCXCANH spectrum with exceptionally high sensitivity was obtained in just a few days using a 600 MHz spectrometer equipped with a 1.3 mm probe operating at a magic angle spinning rate of 55 kHz.

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Figures

Fig. 1 Evaluation of the suitability for sensitivity-enhancement of the DIPSI-3 mixing sequence on MLF. (A) Scheme of 13C- 13C mixing sequences (DIPSI-3, C9481, and hSPEPS). (B) Sensitivity-enhanced 2D hnCACX spectra of 13C15N labelled MLF recorded at 55 kHz MAS on a 600 MHz spectrometer using the different 13C- 13C mixing sequences. Black crosses represent wanted peaks (assignments shown in the spectrum at the top) and red crosses represent mirrored peaks originating from unwanted cross-transfers. (C) Comparison of peak intensities in the spectra resulting from the different mixing sequences. The peak intensities are normalized to the highest intensity (the combined intensity for all peaks of the methionine (Mtot) in the C9 spectrum). The colour coding is the same in panels A, B and C. (D) Bar plot summarizing the simulated transfer efficiencies of DIPSI-3 under the same conditions as in the experiments (16.3 ms mixing). Radio-frequency inhomogeneity is simulated by varying the nutation frequency by ± 10% from the ideal conditions (10 kHz). (E) Example plots representing the full simulations of DIPSI-3 for CαY to CβX (left, (i), CαZ to CβX (middle, (ii) and CαZ to CβY (right, (iii). The grey shaded bars in E represent the values at 16.3 ms mixing, these are indicated in the bar plot in panel D ((i), (ii) and (iii). The colour coding is the same in panels D and E
Fig. 1 Evaluation of the suitability for sensitivity-enhancement of the DIPSI-3 mixing sequence on MLF. (A) Scheme of 13C- 13C mixing sequences (DIPSI-3, C9481, and hSPEPS). (B) Sensitivity-enhanced 2D hnCACX spectra of 13C15N labelled MLF recorded at 55 kHz MAS on a 600 MHz spectrometer using the different 13C- 13C mixing sequences. Black crosses represent wanted peaks (assignments shown in the spectrum at the top) and red crosses represent mirrored peaks originating from unwanted cross-transfers. (C) Comparison of peak intensities in the spectra resulting from the different mixing sequences. The peak intensities are normalized to the highest intensity (the combined intensity for all peaks of the methionine (Mtot) in the C9 spectrum). The colour coding is the same in panels A, B and C. (D) Bar plot summarizing the simulated transfer efficiencies of DIPSI-3 under the same conditions as in the experiments (16.3 ms mixing). Radio-frequency inhomogeneity is simulated by varying the nutation frequency by ± 10% from the ideal conditions (10 kHz). (E) Example plots representing the full simulations of DIPSI-3 for CαY to CβX (left, (i), CαZ to CβX (middle, (ii) and CαZ to CβY (right, (iii). The grey shaded bars in E represent the values at 16.3 ms mixing, these are indicated in the bar plot in panel D ((i), (ii) and (iii). The colour coding is the same in panels D and ECC BY · PubMed Central
Fig. 2 Evaluation of DIPSI-3 mixing on 2H13C15N labelled GlpG. (A) 2D hCC spectra recorded at 55 kHz MAS on a 600 MHz spectrometer using sensitivity-enhanced and standard experiments with different 13C- 13C mixing sequences. Black crosses represent peaks used for comparison. (B) Normalized peak intensities for 8 different peaks that can be identified in all spectra. (C) Schematic drawing of the 13C- 13C mixing sequences used. The same colour coding is used in all panels
Fig. 2 Evaluation of DIPSI-3 mixing on 2H13C15N labelled GlpG. (A) 2D hCC spectra recorded at 55 kHz MAS on a 600 MHz spectrometer using sensitivity-enhanced and standard experiments with different 13C- 13C mixing sequences. Black crosses represent peaks used for comparison. (B) Normalized peak intensities for 8 different peaks that can be identified in all spectra. (C) Schematic drawing of the 13C- 13C mixing sequences used. The same colour coding is used in all panelsCC BY · PubMed Central
Fig. 3 Summary of the side-chain assignments enabled by the triple sensitivity-enhanced 4D experiments. (A) Scheme of how the resonances are visualized in the strip plots with the magnetization transfer pathways indicated (red arrows). (B) Strip plots for the region Q44 to W38, showing the side-chain assignments for residue i in orange (hCxCaNH) and i-1 in blue (hCxCacoNH) connected to the backbone 15N and 1HN resonances of residue i. (C) Summary of the side-chain assignments plotted onto a model of GlpG. Residues with Cα but no side-chain assignments are coloured in dark gray, residues with Cβ assignments are coloured in green and residues with no Cβ assignments but with other carbon side-chains assigned are coloured in orange.(D) Scheme summarizing the side-chain assignments for each residue type found in GlpG (except glycines and prolines). Green circles indicate side-chains that were generally assigned, orange circles represent side-chains that were sometimes assigned and red circles represent side-chains that could not be assigned.
Fig. 3 Summary of the side-chain assignments enabled by the triple sensitivity-enhanced 4D experiments. (A) Scheme of how the resonances are visualized in the strip plots with the magnetization transfer pathways indicated (red arrows). (B) Strip plots for the region Q44 to W38, showing the side-chain assignments for residue i in orange (hCxCaNH) and i-1 in blue (hCxCacoNH) connected to the backbone 15N and 1HN resonances of residue i. (C) Summary of the side-chain assignments plotted onto a model of GlpG. Residues with Cα but no side-chain assignments are coloured in dark gray, residues with Cβ assignments are coloured in green and residues with no Cβ assignments but with other carbon side-chains assigned are coloured in orange.(D) Scheme summarizing the side-chain assignments for each residue type found in GlpG (except glycines and prolines). Green circles indicate side-chains that were generally assigned, orange circles represent side-chains that were sometimes assigned and red circles represent side-chains that could not be assigned.CC BY · PubMed Central

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Metadata

Authors
Öster, C., Chevelkov, V., & Lange, A.
Affiliations
  • Leibniz-Forschungsinstitut für Molekulare Pharmakologie (DE)
  • Humboldt-Universität zu Berlin (DE)
Corresponding author
  • Öster, Carl
  • Lange, Adam
ORCID
Venue
Journal of Biomolecular NMR
Publisher
American Chemical Society (ACS)
Published
2024-08-16
Volume
79
Issue
1
Pages
25-34
ISSN
0925-2738 (ISSN-L)
DOI
10.26434/chemrxiv-2024-f6qmr
PMC
PMC11832555
OpenAlex
W4406721611
Cited by
5
References
2
Citations by year
2026: 2 · 2025: 3
FWCI
1.26
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biomolecular NMR (publishedVersion, cc-by)
  • ChemRxiv (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • edoc Publication server (Humboldt University of Berlin) (submittedVersion, cc-by)
  • edoc Publication server (Humboldt University of Berlin) (cc-by)
Funders
Deutsche Forschungsgemeinschaft (EXC 2008/1 (UniSysCat) - 390540038)
Topics
  • Physical Sciences › Chemistry › Spectroscopy › Advanced NMR Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Lipid Membrane Structure and Behavior
Keywords
sensitivity enhancement, side-chain assignment, membrane proteins, GlpG, transfer efficiency, dipolar coupling, spin-spin coupling, chemical shift assignment
MeSH terms
Carbon Isotopes, Escherichia coli, Membrane Proteins, Nuclear Magnetic Resonance, Biomolecular, Escherichia coli Proteins

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CoChat. (2026). Citation check: Evaluation of TOCSY mixing for sensitivity-enhancement in solid-state NMR and application of 4D experiments for side-chain assignments of the full-length 30 kDa membrane protein GlpG. https://app.cochat.ai/papers/10.26434/chemrxiv-2024-f6qmr
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Öster, C., Chevelkov, V., & Lange, A. (2024). Evaluation of TOCSY mixing for sensitivity-enhancement in solid-state NMR and application of 4D experiments for side-chain assignments of the full-length 30 kDa membrane protein GlpG. Journal of Biomolecular NMR. https://doi.org/10.26434/chemrxiv-2024-f6qmr

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