Ultra-fast NMR measurement to analyze dynamics of Sox2 in cells

• Project/Area Number: 25650056
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Biophysics
• Research Institution: Suntory Foundation for Life Sciences
• Principal Investigator: SUGASE Kenji 公益財団法人サントリー生命科学財団, その他部局等, 主席研究員 (00300822)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2014: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2013: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
• Keywords: in-cell NMR / タンパク質 / 揺らぎ / 高速測定 / 高速NMR法

Outline of Final Research Achievements

We developed methods that enable rapid measurement of protein dynamics in cells using NMR. Specifically, we found that (i) a 2%-DMSO treatment of cells drastically enhanced the efficiency of protein incorporation into cells using the cell-penetrating peptide, (ii) presaturation of the water signal is useful for rapid NMR measurements to detect flexible regions in a protein, and (iii) the non-uniform sampling (NUS) method was utilized to reduce the total amount of experimental time and the SIFT algorithm, which we have been developing a computer program to reconstruct NUS data, was further developed. As a result, these newly developed methods enabled us to measure an NMR spectrum more rapidly by approximately one-third than the conventional measurement.

Research Products

• [Journal Article] Solid-state NMR spectra of lipid-anchored proteins under magic angle spinning. (2014)
  • Author(s): Nomura, K., Harada, E., Sugase, K., Shimamoto, K.
  • Journal Title: J. Phys. Chem. B. Volume: 118 Issue: 9 Pages: 2405-2413
  • DOI: 10.1021/jp4124106
  • Peer Reviewed
• [Journal Article] Quantitative analysis of location- and sequence-dependent deamination by APOBEC3G using real-time NMR spectroscopy (2014)
  • Author(s): Furukawa, A., Sugase, K., Morishita, R., Nagata, T., Kodaki, T., Takaori-Kondo, A., Ryo, A., Katahira, M.
  • Journal Title: Angew. Chem. Int. Ed. Engl. Volume: 53 Issue: 9 Pages: 2349-2352
  • DOI: 10.1002/anie.201309940
  • NAID: 120005647511
  • Peer Reviewed / Open Access
• [Journal Article] Solution structure of the Q41N variant of ubiquitin as a model for the alternatively folded N2 state of ubiquitin (2013)
  • Author(s): Soichiro Kitazawa, Tomoshi Kameda, Maho Yagi-Utsumi, Kenji Sugase, Nicola J. Baxter, Koichi Kato, Michael P. Williamson, Ryo Kitahara.
  • Journal Title: Biochemistry Volume: 52 Issue: 11 Pages: 1874-1885
  • DOI: 10.1021/bi301420m
  • Peer Reviewed
• [Journal Article] Fast and accurate fitting of relaxation dispersion data using the flexible software package GLOVE (2013)
  • Author(s): Sugase, K., Konuma, T., Lansing, J.C., Wright, P.E.
  • Journal Title: J. Biomol. NMR Volume: 56 Issue: 3 Pages: 275-283
  • DOI: 10.1007/s10858-013-9747-5
  • Peer Reviewed
• [Presentation] Structural dynamics of Sox2 upon binding to DNA (2014)
  • Author(s): Kenji Sugase
  • Organizer: ICMRBS XXVI
  • Place of Presentation: Texas, USA
  • Year and Date: 2014-08-24 – 2014-08-29
  • Invited
• [Presentation] HLA-HIV由来抗原ペプチド複合体-水のダイナミクスと抗原提示能 (2014)
  • Author(s): 菅瀬謙治
  • Organizer: 関西創薬化学コミュニティ
  • Place of Presentation: 新大阪
  • Invited
• [Presentation] 揺らぎが制御するヘム分解酵素反応の遠位効果 (2013)
  • Author(s): 菅瀬謙治
  • Organizer: 第14回若手NMR研究会
  • Place of Presentation: 京都
  • Invited
• [Presentation] Mechanism of the distal regulation of substrate binding through intrinsic fluctuation in an enzyme (2013)
  • Author(s): Kenji Sugase
  • Organizer: The 10th Korea-Japan Bilateral Symposium on Biological NMR
  • Place of Presentation: ソウル、韓国
  • Invited
• [Presentation] Analysis of protein dynamics in living human cells (2013)
  • Author(s): Kenji Sugase
  • Organizer: 第36回日本分子生物学会年会
  • Place of Presentation: 神戸