Enabling NMR studies of sparsely labelled large proteins by automated assignment

• Project/Area Number: 17K07312
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: Tokyo Metropolitan University
• Principal Investigator: Guentert Peter 首都大学東京, 理学研究科, 客員教授 (20392110)
• Co-Investigator(Kenkyū-buntansha): 池谷 鉄兵 首都大学東京, 理学研究科, 助教 (30457840)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000); Fiscal Year 2019: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: protein NMR / in-cell NMR / resonance assignment / automated assignment / methyl groups / isotope labeling / protein / NMR / 選択的安定同位体標識 / CYANA / 蛋白質構造解析

Outline of Final Research Achievements

Proteins that are large, membrane-bound, or studied in living cells by in-cell NMR can in general not be assigned by the conventional solution NMR method that relies on uniform 13C/15N-labeling because the resonance lines become too broad and overlapping. Interpretable spectra can be restored by sparse labeling of methyl groups although resonance assignments remain difficult to obtain. Here we developed the MethylFLYA method that can assign large, methyl-labeled proteins using NOESY spectra in conjunction with a known 3D structure. MethylFLYA finds assignments by optimizing a mapping between expected peaks based on the protein sequence, and the measured peaks identified by peak picking. The new approach has been applied to large proteins up to 468 kDa size and to proteins in living cells.

Academic Significance and Societal Importance of the Research Achievements

The methods developed in this research project makes new classes of proteins more easily accessible to detailed NMR studies. Previously, NMR resonance assignments for these proteins could only be determined by time-consuming experimental methods such as extensive mutagenesis.

Research Products

• [Int'l Joint Research] Goethe University Frankfurt am Main(Germany)
• [Int'l Joint Research] ETH Zurich(Switzerland)
• [Journal Article] Automated assignment of methyl NMR spectra from large proteins (2020)
  • Author(s): Pritisanac, I., Alderson, T. R., Guentert, P.
  • Journal Title: Progress in NMR Spectroscopy Volume: 118-119 Pages: 54-73
  • DOI: 10.1016/j.pnmrs.2020.04.001
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Automated backbone NMR resonance assignment of large proteins using redundant linking from a single simultaneous acquisition (2020)
  • Author(s): Stanek, J., Schubeis, T., Paluch, P., Guentert, P., Andreas, L. B., Pintacuda, G.
  • Journal Title: Journal of the American Chemical Society Volume: 142 Issue: 12 Pages: 5793-5799
  • DOI: 10.1021/jacs.0c00251
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Backbone and methyl assignment of bacteriorhodopsin incorporated into nanodiscs (2020)
  • Author(s): Kooijman, L., Ansorge, P., Schuster, M., Baumann, C., Loehr, F., Jurt, S., Guentert, P., Zerbe, O.
  • Journal Title: Journal of Biomolecular NMR Volume: 74 Issue: 1 Pages: 45-60
  • DOI: 10.1007/s10858-019-00289-7
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Automatic structure-based NMR methyl resonance assignment in large proteins (2019)
  • Author(s): Pritisanac, I., Wuerz, J. M., Alderson, T. R., Guentert, P.
  • Journal Title: Nature Communications Volume: 10 Issue: 1 Pages: 4922-4922
  • DOI: 10.1038/s41467-019-12837-8
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex (2019)
  • Author(s): 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., Guentert, P., Favier, A., Schoehn, G., Schanda, P., Boisbouvier, J.
  • Journal Title: Nature Communications Volume: 10 Issue: 1 Pages: 2697-2697
  • DOI: 10.1038/s41467-019-10490-9
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Protein Structure Determination in Living Cells. (2019)
  • Author(s): Teppei Ikeya, Peter Guentert, Yutaka Ito
  • Journal Title: international journal of molecular sciences Volume: 20 Issue: 10 Pages: 2442-2442
  • DOI: 10.3390/ijms20102442
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] High resolution protein 3D structure determination in living eukaryotic cells (2019)
  • Author(s): Tanaka, T., Ikeya, T., Kamoshida, H., Suemoto, Y., Mishima, M., Shirakawa, M., Guentert, P., Ito, Y.
  • Journal Title: Angewandte Chemie International Edition Volume: in press Issue: 22 Pages: 1-7
  • DOI: 10.1002/anie.201900840
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Global response of diacylglycerol kinase towards substrate binding observed by 2D and 3D MAS NMR (2019)
  • Author(s): Moebius, K., Kazemi, S., Guentert, P., Jakob, A., Heckel, A., Becker-Baldus, J., Glaubitz, C.
  • Journal Title: Scientific Reporrts Volume: 9 Issue: 1 Pages: 3995-3995
  • DOI: 10.1038/s41598-019-40264-8
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Noise peak filtering in multi-dimensional NMR spectra using convolutional neural networks (2018)
  • Author(s): Kobayashi, N., Hattori, Y., Nagata, T., Shinya, S., Guentert, P., Kojima, C., Fujiwara, T.
  • Journal Title: Bioinformatics Volume: 34 Issue: 24 Pages: 4300-4301
  • DOI: 10.1093/bioinformatics/bty581
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Fully automated assignment of methyl resonances of a 36 kDa protein dimer from sparse NOESY data (2018)
  • Author(s): Pritisanac, I., Wuerz, J. M., Guentert, P.
  • Journal Title: Journal of Physics: Conference Series Volume: 1036 Pages: 012008-012008
  • DOI: 10.1088/1742-6596/1036/1/012008
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Extending the applicability of exact nuclear Overhauser enhancements to large proteins and RNA (2018)
  • Author(s): Nichols, P. J., Born, A., Henen, M. A., Strotz, D., Celestine, C., Riek, R., Guentert, P., Voegeli, B.
  • Journal Title: ChemBioChem Volume: 19 Issue: 16 Pages: 1695-1701
  • DOI: 10.1002/cbic.201800237
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Fully automated assignment of methyl resonances of large proteins from sparse NOESY data (2018)
  • Author(s): Pritisanac, I., Wuerz, J. M. & Guentert, P.
  • Journal Title: Journal of Physics Conference Series Volume: printing in progress
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] NMR-based automated protein structure determination (2017)
  • Author(s): Wuerz, J. M., Kazemi, S., Schmidt, E., Bagaria, A. & Guentert, P.
  • Journal Title: Arch. Biochem. Biophys. Volume: 628 Pages: 24-32
  • DOI: 10.1016/j.abb.2017.02.011
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Solution structure of discoidal high-density lipoprotein particles with a shortened apolipoprotein A-I (2017)
  • Author(s): Bibow, S., Polyhach, Y., Eichmann, C., Chi, C. N., Kowal, J., Albiez, S., McLeod, R. A., Stahlberg, H., Jeschke, G., Guentert, P. & Riek, R.
  • Journal Title: Nat. Struct. Mol. Biol. Volume: 24 Issue: 2 Pages: 187-193
  • DOI: 10.1038/nsmb.3345
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Solution structure of the first RNA recognition motif domain of human spliceosomal protein SF3b49 and its mode of interaction with a SF3b145 fragment (2017)
  • Author(s): Kuwasako, K.
  • Journal Title: Protein Science Volume: 26 Issue: 2 Pages: 280-290
  • DOI: 10.1002/pro.3080
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Peak picking multidimensional NMR spectra with the contour geometry based algorithm CYPICK (2017)
  • Author(s): Wuerz, J. & Guentert, P.
  • Journal Title: J. Biomol. NMR Volume: 67 Issue: 1 Pages: 63-76
  • DOI: 10.1007/s10858-016-0084-3
  • Peer Reviewed / Int'l Joint Research
• [Presentation] Structure calculation and automated assignment with CYANA (2020)
  • Author(s): Peter GUENTERT
  • Organizer: 4th G-NMR School, Goettingen, Germany
  • Invited
• [Presentation] Automatic methyl assignment in large proteins (2019)
  • Author(s): Peter GUENTERT
  • Organizer: The 12th Australian and New Zealand Society for Magnetic Resonance (ANZMAG) Conference
  • Int'l Joint Research / Invited
• [Presentation] NMR structure calculation (2019)
  • Author(s): Peter GUENTERT
  • Organizer: EMBO Practical Course on Structure Determination of Biological Macromolecules by Solution NMR, Munich, Germany
  • Int'l Joint Research / Invited
• [Presentation] Structure-based methyl resonance assignment and multi-state eNOE analysis with CYANA (2018)
  • Author(s): Peter GUENTERT
  • Organizer: 28th International Conference on Magnetic Resonance in Biological Systems (ICMRBS 2018)
  • Int'l Joint Research / Invited
• [Presentation] Fully automated assignment of methyl resonances of a 36 kDa protein dimer from sparse NOESY data (2017)
  • Author(s): Iva Pritisanac, Julia Wuerz & Peter Guentert
  • Organizer: International Meeting on “High-Dimensional Data-Driven Science” (HD3-2017), Kyoto, Japan
  • Invited
• [Presentation] Automated biomolecular NMR spectrum analysis for protein structure and dynamics (2017)
  • Author(s): Peter Guentert
  • Organizer: Euromar 2017, Warsaw, Poland
  • Int'l Joint Research / Invited
• [Presentation] Automated NMR spectrum analysis for protein structure and dynamics (2017)
  • Author(s): Peter Guentert
  • Organizer: Gordon Research Seminar “Frontiers of NMR in Life Sciences”, Newry ME, USA
  • Int'l Joint Research / Invited
• [Presentation] NMR structure calculation (2017)
  • Author(s): Peter Guentert
  • Organizer: EMBO Practical Course on Structure Determination of Biological Macromolecules by Solution NMR, Basel, Switzerland
  • Int'l Joint Research / Invited
• [Book] Modern Magnetic Resonance 2nd Ed. (Ed. G. A. Webb) (2017)
  • Author(s): Kazemi, S., Wuerz, J. M., Schmidt, E., Bagaria, A. & Guentert, P.
  • Total Pages: 18
  • Publisher: Springer
  • ISBN: 9783319282756
• [Remarks] CYANA Home Page
  • URL: http://www.cyana.org/