Articles | Volume 5, issue 1
https://doi.org/10.5194/mr-5-33-2024
© Author(s) 2024. This work is distributed under
the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
https://doi.org/10.5194/mr-5-33-2024
© Author(s) 2024. This work is distributed under
the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
Deuteration of proteins boosted by cell lysates: high-resolution amide and Hα magic-angle-spinning (MAS) NMR without the reprotonation bottleneck
Federico Napoli
Institute of Science and Technology Austria (ISTA), Am Campus 1, 3400 Klosterneuburg, Austria
Jia-Ying Guan
Univ. Grenoble Alpes, CNRS, CEA, IBS, 38000 Grenoble, France
Charles-Adrien Arnaud
Univ. Grenoble Alpes, CNRS, CEA, IBS, 38000 Grenoble, France
Pavel Macek
Univ. Grenoble Alpes, CNRS, CEA, IBS, 38000 Grenoble, France
Hugo Fraga
Univ. Grenoble Alpes, CNRS, CEA, IBS, 38000 Grenoble, France
Cécile Breyton
Univ. Grenoble Alpes, CNRS, CEA, IBS, 38000 Grenoble, France
Institute of Science and Technology Austria (ISTA), Am Campus 1, 3400 Klosterneuburg, Austria
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Lucky N. Kapoor, Natalia Ruzickova, Predrag Živadinović, Valentin Leitner, Maria Anna Sisak, Cecelia Mweka, Jeroen Dobbelaere, Georgios Katsaros, and Paul Schanda
Magn. Reson. Discuss., https://doi.org/10.5194/mr-2025-9, https://doi.org/10.5194/mr-2025-9, 2025
Preprint under review for MR
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By reviewing attendee lists of ten MR (magnetic resonance) meetings over the last year, we estimate the climate footprint of conferences and explore possibilities to reduce it. This manuscript will facilitate discussions about possible actions the community may take.
Kathrin Aebischer, Lea Marie Becker, Paul Schanda, and Matthias Ernst
Magn. Reson., 5, 69–86, https://doi.org/10.5194/mr-5-69-2024, https://doi.org/10.5194/mr-5-69-2024, 2024
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To characterize the amplitude of dynamic processes in molecules, anisotropic parameters can be measured using solid-state NMR. However, the timescales of motion that lead to such a scaling of the anisotropic interactions are not clear. Using numerical simulations in small spin systems, we could show that mostly the magnitude of the anisotropic interaction determines the range of timescales detected by the scaled anisotropic interaction, and experimental parameters play a very minor role.
Alicia Vallet, Adrien Favier, Bernhard Brutscher, and Paul Schanda
Magn. Reson., 1, 331–345, https://doi.org/10.5194/mr-1-331-2020, https://doi.org/10.5194/mr-1-331-2020, 2020
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We introduce ssNMRlib, a library of pulse sequences and jython scripts for user-friendly setup and acquisition of solids-state NMR experiments. ssNMRlib facilitates all steps of data acquisition, including calibration of various pulse-sequence parameters and semi-automatic setup of even complex high-dimensional experiments, using an intuitive graphical user interface, launched directly within Bruker's Topspin acquisition program.
Related subject area
Field: Solid-state NMR | Topic: (Bio)Chemistry
Analysis of the electronic structure of the primary electron donor of photosystem I of Spirodela oligorrhiza by photochemically induced dynamic nuclear polarization (photo-CIDNP) solid-state nuclear magnetic resonance (NMR)
Geertje J. Janssen, Patrick Eschenbach, Patrick Kurle, Bela E. Bode, Johannes Neugebauer, Huub J. M. de Groot, Jörg Matysik, and Alia Alia
Magn. Reson., 1, 261–274, https://doi.org/10.5194/mr-1-261-2020, https://doi.org/10.5194/mr-1-261-2020, 2020
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Natural photosynthetic reaction centers (RCs) are built up by two parallel branches of cofactors. While photosystem II and purple bacterial RCs selectively use one branch for light-driven electron transfer, photosystem I, as also shown here, is using both branches. Comparing NMR chemical shifts, we shown that the two donor cofactors in photosystem I are similarly distinguished to those in purple bacterial RCs (Schulten et al., 2002; Biochemistry 41, 8708). Alternative reasons are discussed.
Cited articles
Agarwal, V., Penzel, S., Szekely, K., Cadalbert, R., Testori, E., Oss, A., Past, J., Samoson, A., Ernst, M., Böckmann, A., and Meier, B. H.: De novo 3D structure determination from sub-milligram protein samples by solid-state 100 kHz MAS NMR spectroscopy, Angew. Chem. Int. Edit., 53, 12253–12256, 2014. a
Anderson, E.: Growth requirements of virus-resistant mutants of Escherichia coli strain “B”, P. Natl. Acad. Sci. USA, 32, 120–128, 1946. a
Andreas, L. B., Stanek, J., Le Marchand, T., Bertarello, A., Paepe, D. C.-D., Lalli, D., Krejčíková, M., Doyen, C., Öster, C., Knott, B., Wegner, S., Engelke, F., Felli, I. C., Pierattelli, R., Dixon, N. E., Emsley, L., Herrmann, T., and Pintacuda, G.: Protein residue linking in a single spectrum for magic-angle spinning NMR assignment, J. Biomol. NMR, 62, 253–261, 2015. a
Andreas, L. B., Jaudzems, K., Stanek, J., Lalli, D., Bertarello, A., Le Marchand, T., Cala-De Paepe, D., Kotelovica, S., Akopjana, I., Knott, B., Wegner, S., Engelke, F., Lesage, A., Emsley, L., Tars, K., Herrmann, T., and Pintacuda, G.: Structure of fully protonated proteins by proton-detected magic-angle spinning NMR, P. Natl. Acad. Sci. USA, 113, 9187–9192, 2016. a
Arnaud, C.-A., Effantin, G., Vivès, C., Engilberge, S., Bacia, M., Boulanger, P., Girard, E., Schoehn, G., and Breyton, C.: Bacteriophage T5 tail tube structure suggests a trigger mechanism for Siphoviridae DNA ejection, Nat. Commun., 8, 1953, https://doi.org/10.1038/s41467-017-02049-3, 2017. a
Aucharova, H., Klein, A., Medina, S., Söldner, B., Vasa, S. K., and Linser, R.: Protein deuteration via algal amino acids to overcome proton back-exchange for fast- MAS solid-state NMR of large proteins, Chem. Commun., 60, 3083–3086, https://doi.org/10.1039/D4CC00213J, 2024. a, b
Barbet-Massin, E., Pell, A. J., Retel, J. S., Andreas, L. B., Jaudzems, K., Franks, W. T., Nieuwkoop, A. J., Hiller, M., Higman, V., Guerry, P., Bertarello, A., Knight, M. J., Felletti, M., Le Marchand, T., Kotelovica, S., Akopjana, I., Tars, K., Stoppini, M., Bellotti, V., Bolognesi, M., Ricagno, S., Chou, J. J., Griffin, R. G., Oschkinat, H., Lesage, A., Emsley, L., Herrmann, T., and Pintacuda, G.: Rapid proton-detected NMR assignment for proteins with fast magic angle spinning, J. Am. Chem. Soc., 136, 12489–12497, 2014. a
Böckmann, A., Gardiennet, C., Verel, R., Hunkeler, A., Loquet, A., Pintacuda, G., Emsley, L., Meier, B. H., and Lesage, A.: Characterization of different water pools in solid-state NMR protein samples, J. Biomol. NMR, 45, 319–327, 2009. a
Bonaccorsi, M., Le Marchand, T., and Pintacuda, G.: Protein structural dynamics by magic-angle spinning NMR, Curr. Opin. Struc. Biol., 70, 34–43, 2021. a
Bougault, C., Ayala, I., Vollmer, W., Simorre, J.-P., and Schanda, P.: Studying intact bacterial peptidoglycan by proton-detected NMR spectroscopy at 100 kHz MAS frequency, J. Struct. Biol., 206, 66–72, 2019. a
Chevelkov, V., Rehbein, K., Diehl, A., and Reif, B.: Ultrahigh resolution in proton solid-state NMR spectroscopy at high levels of deuteration, Angew. Chem. Int. Edit., 45, 3878–3881, 2006. a
Chevelkov, V., Fink, U., and Reif, B.: Quantitative analysis of backbone motion in proteins using MAS solid-state NMR spectroscopy, J. Biomol. NMR, 45, 197–206, 2009. a
Felix, J., Weinhäupl, K., Chipot, C., Dehez, F., Hessel, A., Gauto, D. F., Morlot, C., Abian, O., Gutsche, I., Velazquez-Campoy, A., Schanda, P., and Fraga, H.: Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors, Sci. Adv., 5, eaaw3818, https://doi.org/10.1126/sciadv.aaw3818 , 2019. a
Fraga, H., Arnaud, C.-A., Gauto, D. F., Audin, M., Kurauskas, V., Macek, P., Krichel, C., Guan, J.-Y., Boisbouvier, J., Sprangers, R., Breyton, C., and Schanda, P.: Solid-State NMR H-N-(C)-H and H-N-C-C 3D/4D Correlation Experiments for Resonance Assignment of Large Proteins, Chem. Phys. Chem., 18, 2697–2703, 2017. a
Gardner, K. H. and Kay, L. E.: The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins, Annu. Rev. Bioph. Biom., 27, 357–406, 1998. a
Gardner, K. H., Zhang, X., Gehring, K., and Kay, L. E.: Solution NMR Studies of a 42 kDa Escherichia Coli Maltose Binding Protein/β-Cyclodextrin Complex: Chemical Shift Assignments and Analysis, J. Am. Chem. Soc., 120, 11738–11748, 1998. a
Gauto, D. F., Estrozi, L. F., Schwieters, C. D., Effantin, G., Macek, P., Sounier, R., Sivertsen, A. C., Schmidt, E., Kerfah, R., Mas, G., Colletier, J.-P., Güntert, P., Favier, A., Schoehn, G., Schanda, P., and Boisbouvier, J.: Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex, Nat. Commun., 10, 1–12, 2019a. a, b, c, d, e, f
Gauto, D. F., Macek, P., Barducci, A., Fraga, H., Hessel, A., Terauchi, T., Gajan, D., Miyanoiri, Y., Boisbouvier, J., Lichtenecker, R., Kainosho, M., and Schanda, P.: Aromatic Ring Dynamics, Thermal Activation, and Transient Conformations of a 468 kDa Enzyme by Specific 1H–13C Labeling and Fast Magic-Angle Spinning NMR, J. Am. Chem. Soc., 141, 11183–11195, 2019b. a
Gauto, D. F., Macek, P., Malinverni, D., Fraga, H., Paloni, M., Sučec, I., Hessel, A., Bustamante, J. P., Barducci, A., and Schanda, P.: Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR, Nat. Commun., 13, 1927, https://doi.org/10.1038/s41467-022-29423-0, 2022. a
Good, D. B., Wang, S., Ward, M. E., Struppe, J., Brown, L. S., Lewandowski, J. R., and Ladizhansky, V.: Conformational dynamics of a seven transmembrane helical protein Anabaena Sensory Rhodopsin probed by solid-state NMR, J. Am. Chem. Soc., 136, 2833–2842, 2014. a
Huber, M., Hiller, S., Schanda, P., Ernst, M., Böckmann, A., Verel, R., and Meier, B. H.: A Proton-Detected 4D Solid-State NMR Experiment for Protein Structure Determination, Chem. Phys. Chem., 12, 915–918, 2011. a
Imbert, L., Lenoir-Capello, R., Crublet, E., Vallet, A., Awad, R., Ayala, I., Juillan-Binard, C., Mayerhofer, H., Kerfah, R., Gans, P., Miclet, E., and Boisbouvier, J.: In Vitro Production of Perdeuterated Proteins in H2O for Biomolecular NMR Studies, in: Meth. Mol. Biol., vol. 2199, 127–149, Humana, 2021. a
Jain, M. G., Lalli, D., Stanek, J., Gowda, C., Prakash, S., Schwarzer, T. S., Schubeis, T., Castiglione, K., Andreas, L. B., Madhu, P. K., Pintacuda, G., and Agarwal, V.: Selective 1H-1H Distance Restraints in Fully Protonated Proteins by Very Fast Magic-Angle Spinning Solid-State NMR, J. Phys. Chem. Lett., 8, 2399–2405, 2017. a
Klein, A., Rovó, P., Sakhrani, V. V., Wang, Y., Holmes, J. B., Liu, V., Skowronek, P., Kukuk, L., Vasa, S. K., Güntert, P., Mueller, L. J., and Linser, R.: Atomic-resolution chemical characterization of (2x) 72-kDa tryptophan synthase via four-and five-dimensional 1H-detected solid-state NMR, P. Natl. Acad. Sci. USA, 119, e2114690119, https://doi.org/10.1073/pnas.2114690119, 2022. a
Lamley, J. M., Iuga, D., Öster, C., Sass, H.-J., Rogowski, M., Oss, A., Past, J., Reinhold, A., Grzesiek, S., Samoson, A., and Lewandowski, J. R.: Solid-state NMR of a protein in a precipitated complex with a full-length antibody, J. Am. Chem. Soc., 136, 16800–16806, 2014. a
Lewandowski, J. R.: Advances in Solid-State Relaxation Methodology for Probing Site-Specific Protein Dynamics, Acc. Chem. Res., 46, 2018–2027, 2013. a
Lewandowski, J. R., Dumez, J. N., Akbey, U., Lange, S., Emsley, L., and Oschkinat, H.: Enhanced Resolution and Coherence Lifetimes in the Solid-state NMR Spectroscopy of Perdeuterated Proteins under Ultrafast Magic-angle Spinning, J. Phys. Chem. Lett., 2, 2205–2211, 2011. a
Linser, R., Bardiaux, B., Higman, V., Fink, U., and Reif, B.: Structure Calculation from Unambiguous Long-Range Amide and Methyl 1 H− 1 H Distance Restraints for a Microcrystalline Protein with MAS Solid-State NMR Spectroscopy, J. Am. Chem. Soc., 133, 5905–5912, 2011a. a
Linser, R., Bardiaux, B., Higman, V., Fink, U., and Reif, B.: Structure Calculation from Unambiguous Long-Range Amide and Methyl 1H-1H Distance Restraints for a Microcrystalline Protein with MAS Solid-State NMR Spectroscopy, J. Am. Chem. Soc., 133, 5905–5912, 2011b. a
Linser, R., Sarkar, R., Krushelnitzky, A., Mainz, A., and Reif, B.: Dynamics in the solid-state: perspectives for the investigation of amyloid aggregates, membrane proteins and soluble protein complexes, J. Biomol. NMR, 59, 1–14, 2014. a
Najbauer, E. E., Tekwani Movellan, K., Giller, K., Benz, R., Becker, S., Griesinger, C., and Andreas, L. B.: Structure and gating behavior of the human integral membrane protein VDAC1 in a lipid bilayer, J. Am. Chem. Soc., 144, 2953–2967, 2022. a
Napoli, F. and Schanda, P.: Amide and α-hydrogens extension to the Solid-state NMR assignment of P. horikoshii TET2, BioMagResBank Entry 52400 [data set], https://doi.org/10.13018/BMR52400, 2024. a
Napoli, F., Becker, L. M., and Schanda, P.: Protein dynamics detected by magic-angle spinning relaxation dispersion NMR, Curr. Opin. Struct. Biol., 82, 102660, https://doi.org/10.1016/j.sbi.2023.102660, 2023. a
Nieuwkoop, A. J., Franks, W. T., Rehbein, K., Diehl, A., Akbey, U., Engelke, F., Emsley, L., Pintacuda, G., and Oschkinat, H.: Sensitivity and resolution of proton detected spectra of a deuterated protein at 40 and 60 kHz magic-angle-spinning, J. Biomol. NMR, 61, 161–171, 2015. a
Pervushin, K., Riek, R., Wider, G., and Wüthrich, K.: Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution, P. Natl. Acad. Sci. USA, 94, 12366–12371, 1997. a
Pervushin, K., Riek, R., Wider, G., and Wüthrich, K.: Transverse relaxation-optimized spectroscopy (TROSY) for NMR studies of aromatic spin systems in 13C-labeled proteins, J. Am. Chem. Soc., 120, 6394–6400, 1998. a
Reif, B.: Deuteration for high-resolution detection of protons in protein magic angle spinning (MAS) solid-state NMR, Chem. Rev., 122, 10019–10035, 2021. a
Retel, J. S., Nieuwkoop, A. J., Hiller, M., Higman, V. A., Barbet-Massin, E., Stanek, J., Andreas, L. B., Franks, W. T., Van Rossum, B.-J., Vinothkumar, K. R., Handel, L., de Palma, G. G., Bardiaux, B., Pintacuda, G., Emsley, L., Kühlbrandt, and Oschkinat, H.: Structure of outer membrane protein G in lipid bilayers, Nat. Commun., 8, 2073, https://doi.org/10.1038/s41467-017-02228-2, 2017. a
Schmidt, E. and Güntert, P.: A New Algorithm for Reliable and General NMR Resonance Assignment, J. Am. Chem. Soc., 134, 12817–12829, 2012. a
Schubeis, T., Le Marchand, T., Daday, C., Kopec, W., Tekwani Movellan, K., Stanek, J., Schwarzer, T. S., Castiglione, K., de Groot, B. L., Pintacuda, G., and Andreas L. B.: A β-barrel for oil transport through lipid membranes: Dynamic NMR structures of AlkL, P. Natl. Acad. Sci. USA, 117, 21014–21021, 2020. a
Shaka, A. J., Keeler, J., and Freeman, R.: Evaluation of a new broad-band decoupling sequence – WALTZ-16, J. Magn. Reson., 53, 313–340, 1983. a
Shcherbakov, A. A., Mandala, V. S., and Hong, M.: High-Sensitivity Detection of Nanometer 1H-19F Distances for Protein Structure Determination by 1H-Detected Fast MAS NMR, J. Phys. Chem. B, 123, 4387–4391, 2019. a
Shen, Y. and Bax, A.: Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks, J. Biomol. NMR, 56, 227–241, 2013. a
Singh, H., Vasa, S. K., Jangra, H., Rovó, P., Päslack, C., Das, C. K., Zipse, H., Schäfer, L. V., and Linser, R.: Fast Microsecond Dynamics of the Protein–Water Network in the Active Site of Human Carbonic Anhydrase II Studied by Solid-State NMR Spectroscopy, J. Am. Chem. Soc., 141, 19276–19288, 2019. a
Stanek, J., Schubeis, T., Paluch, P., Güntert, P., Andreas, L. B., and Pintacuda, G.: Automated Backbone NMR Resonance Assignment of Large Proteins Using Redundant Linking from a Single Simultaneous Acquisition, J. Am. Chem. Soc. Soc., 142, 5793–5799, 2020. a
Tekwani, K., Eszter, M., Supriya, E. N., Michele, P., Karin, S., Stefan, G., and Andreas, L. B.: Alpha protons as NMR probes in deuterated proteins, J. Biomol. NMR, 73, 81–91, 2019. a
Tugarinov, V., Hwang, P. M., Ollerenshaw, J. E., and Kay, L. E.: Cross-correlated relaxation enhanced 1H-13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes, J. Am. Chem. Soc., 125, 10420–10428, 2003. a
Vallet, A., Favier, A., Brutscher, B., and Schanda, P.: ssNMRlib: a comprehensive library and tool box for acquisition of solid-state nuclear magnetic resonance experiments on Bruker spectrometers, Magn. Reson., 1, 331–345, https://doi.org/10.5194/mr-1-331-2020, 2020. a, b
Vasa, S. K., Rovó, P., and Linser, R.: Protons as versatile reporters in solid-state NMR spectroscopy, Acc. Chem. Res., 51, 1386–1395, 2018. a
Vranken, W. F., Boucher, W., Stevens, T. J., Fogh, R. H., Pajon, A., Llinas, M., Ulrich, E. L., Markley, J. L., Ionides, J., and Laue, E. D.: The CCPN data model for NMR spectroscopy: development of a software pipeline, Proteins, 59, 687–96, 2005. a
Xiang, S., Grohe, K., Rovó, P., Vasa, S. K., Giller, K., Becker, S., and Linser, R.: Sequential backbone assignment based on dipolar amide-to-amide correlation experiments, J. Biomol. NMR, 62, 303–311, https://doi.org/10.1007/s10858-015-9945-4, 2015. a
Xuncheng, S., Loh, C.-T., Ruhu, Q., and Otting, G.: Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O, J. Biomol. NMR, 50, 35–42, https://doi.org/10.1007/s10858-011-9477-5, 2011. a
Short summary
Protons (1H) are useful reporters of protein structure and dynamics in solid-state NMR. However, 1H abundance is detrimental to the resolution of NMR spectra. Substituting 1H by deuterons has been an efficient strategy to improve spectral quality, but when the crucial backbone amide sites are not protonated, much information is loss. We propose a method to completely protonate the amide sites, while maintaining high-resolution information, which partially also extends to backbone alpha-1H.
Protons (1H) are useful reporters of protein structure and dynamics in solid-state NMR. However,...