2017 Fiscal Year Research-status Report
高分子量蛋白質のNMR構造解析を目指したスパース選択標識とNMR自動解析法の開発
| Project/Area Number |
17K07312
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| Research Institution | Tokyo Metropolitan University |
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Principal Investigator |
PETER GUENTERT 首都大学東京, 理工学研究科, 客員教授 (20392110)
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| Co-Investigator(Kenkyū-buntansha) |
池谷 鉄兵 首都大学東京, 理工学研究科, 助教 (30457840)
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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Keywords | protein / NMR / in-cell NMR / resonance assignment / automated assignment / methyl groups / isotope labeling |
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| Outline of Annual 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. Sparse labeling, in particular of methyl groups is an approach to restore interpretable spectra by drastically reducing relaxation rates and the number of signals. However, it remains difficult to establish resonance assignments, even if the three-dimensional structure of the protein is already known. In this project we develop computational methods to assign the spectra of sparsely labeled difficult proteins by structure-based automated assignment. On the basis of our existing FLYA algorithm for the assignment of uniformly labeled proteins, a new automated assignment method has been developed that can assign large proteins using methyl group labeling or other sparse labeling techniques, and NOESY spectra in conjunction with a known three-dimensional structure. The FLYA algorithm finds assignments by optimizing a mapping between the expected peaks, which one anticipates to see based on the protein sequence, and the measured peaks, which have been identified by peak picking. The general design of the algorithm makes it possible to exploit peak lists from virtually any type of NMR spectrum. In particular, we could show that FLYA is able to assign proteins using as input exclusively NOESY spectra. The new approach has been applied to proteins large proteins up to 360 kDa size and to proteins in living cells.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
On the basis of the existing FLYA algorithm for the assignment of uniformly labeled proteins, we have developed a new automated assignment method that can assign large proteins using methyl group labeling or other sparse labeling techniques, and NOESY spectra in conjunction with a known three-dimensional structure. The new approach was successfully applied to the 36 kDa ATCase-r2 protein dimer from sparse NOESY data. Applications to other proteins up to 360 kDa size are under way. The automated assignment method is also being applied to in-cell NMR in order to support the first structure determinations of proteins directly in living eukaryotic cells.
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| Strategy for Future Research Activity |
For the final year of the project we plan to publish a full account of our methyl-FLYA method for structure-based assignment of large, specifically methyl-labeled proteins. We will perform automated assignment for five proteins with sizes between 27 and 360 kDa, for which experimental NOESY spectra are available. The first structure determinations of proteins directly in living eukaryotic cells, which use the automated assignment method, shall be completed and published.
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| Causes of Carryover |
研究成果発表のための学会参加費と論文等の出版にかかると経費が，予定よりもかからなかったため．次年度は，初年度の成果を発表する機会が増えるため，残った分の経費は次年度の成果発表の経費として使用予定．
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Research Products
(13 results)
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[Journal Article] Solution structure of discoidal high-density lipoprotein particles with a shortened apolipoprotein A-I2017
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.
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Journal Title
Nat. Struct. Mol. Biol.
Volume: 24
Pages: 187-193
DOI
Peer Reviewed / Int'l Joint Research
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[Journal Article] Solution structure of the first RNA recognition motif domain of human spliceosomal protein SF3b49 and its mode of interaction with a SF3b145 fragment2017
Author(s)
Kuwasako, K., Nameki, N., Tsuda, K., Takahashi, M., Sato, A., Tochio, N., Inoue, M., Terada, T., Kigawa, T., Kobayashi, N., Shirouzu, M., Ito, T., Sakamoto, T., Wakamatsu, K., Guentert, P., Takahashi, S., Yokoyama, S. & Muto, Y.
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Journal Title
Protein Sci.
Volume: 26
Pages: 280-291
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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[Presentation] NMR structure calculation2017
Author(s)
Peter Guentert
Organizer
EMBO Practical Course on Structure Determination of Biological Macromolecules by Solution NMR, Basel, Switzerland
Int'l Joint Research / Invited
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