Development of membrane protein structure analysis using heavy atom labeled lipids

• Project/Area Number: 25650051
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Biophysics
• Research Institution: Osaka University
• Principal Investigator: SUGIYAMA SHIGERU 大阪大学, 理学(系)研究科(研究院), 特任准教授 (90615428)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2014: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000); Fiscal Year 2013: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: 構造生物学 / 膜タンパク質結晶 / バイセル / 重原子標識脂質 / 結晶成長 / 重原子誘導体結晶 / 膜脂質 / 異常分散 / 溶液撹拌法 / Ｘ線構造解析 / 脂質 / X線構造解析

Outline of Final Research Achievements

The specific lipids binding to membrane protein play a crucial role in membrane protein organization and function, resulting in a very important to elucidate the detailed mechanism of its specific lipid recognition. Membrane protein is crystallized after solubilization by detergents, although this treatment often disrupts innate structures and functions. Here we focused on crystallization of bacteriorhodopsin (bR) using various lipid bicelles as membrane mimicking systems. Bicells are small bilayer disks that form in lipid / amphiphile mixtures. Eight lipid bicelles were crystallized by mixing bR, resulting successfully in formation of bR-bicelle crystals. X-ray crystal structure analyses indicated that heavy atom labeled lipids are successfully incorporated into the bR-bicelle crystal, successfully interacting with bR. Our bicelle crystallization technique may be applicable for structurally unknown membrane proteins.

Research Products

• [Journal Article] Grease matrix as a versatile carrier of proteins for serial crystallography. (2015)
  • Author(s): Sugahara M, Shimamura T, et al.
  • Journal Title: Nat Methods. Volume: 12 Issue: 1 Pages: 61-63
  • DOI: 10.1038/nmeth.3172
  • NAID: 120005682565
  • Peer Reviewed
• [Journal Article] Crystallization and preliminary crystallographic studies of PotA, a membrane-associated ATPase of the spermidine-preferential uptake system in Thermotoga maritime. (2014)
  • Author(s): Sugiyama, S. et al.
  • Journal Title: Acta Crystallogr. F Struct. Biol. Commun. Volume: 70 Issue: 6 Pages: 738-741
  • DOI: 10.1107/s2053230x14008607
  • Peer Reviewed
• [Presentation] 膜タンパク質研究基盤の構築に向けた合成リン脂質の利用 (2015)
  • Author(s): 1.杉山成, 斎木悠, 島田真典, 垣ノ内啓介, 新山真由美, 花島慎弥, 溝端栄一, 井上豪, 松森信明, 村田道雄
  • Organizer: 第15回蛋白質科学会年会
  • Place of Presentation: あわぎんホール（徳島県、徳島市）
  • Year and Date: 2015-06-24 – 2015-06-26
• [Presentation] 重元素化脂質含有バイセルを用いた膜タンパク質結晶化法の検討 (2015)
  • Author(s): 斎木悠，花島慎弥，村田道雄，松森信明，溝畑栄一，井上豪，川竹悟史，垣之内啓介，杉山成
  • Organizer: 日本化学会 第95春季年会
  • Place of Presentation: 日本大学理工学部船橋キャンパス（千葉県、船橋市）
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] バクテリオロドプシンを機能的に再構成する膜脂質の検討 (2014)
  • Author(s): 川竹悟史、梅川雄一、松岡茂、杉山成、園山正史、村田道雄
  • Organizer: 第14回日本蛋白質科学会年会
  • Place of Presentation: ワークピア横浜（神奈川県、横浜市）
  • Year and Date: 2014-06-25 – 2014-06-27
• [Presentation] Novel fatty acid analogues for the understanding of lipid-protein interactions (2013)
  • Author(s): H. Ishida, S. Sugiyama, M. Hirose, S. Lethu, H. Ano, F. Sato, D. Matsuoka, T. Hara, E. Mizohata, T. Inoue, S. Matsuoka, and M. Murata
  • Organizer: The International Conference on Structural Genomics 2013 (ICSG2013)
  • Place of Presentation: Sapporo