| Project/Area Number |
16570102
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Kwansei Gakuin University |
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Principal Investigator |
YAMAGUCHI Hiroshi Kwansei Gakuin University, School of Science and Technology, Associate Professor, 理工学部, 助教授 (10252719)
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| Co-Investigator(Kenkyū-buntansha) |
KINOSHITA Tsutomu Kwansei Gakuin University, School of Science and Technology, Professor, 理工学部, 教授 (30161532)
SATO Mamoru Yokohama City University, International Graduate School of Arts and Sciences, Professor, 大学院・国際総合科学研究科, 教授 (60170784)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2006: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2004: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Notch signal / Expression / Purification / Crystallization / Solution Structure / SAX / シグナル伝達系 / 高次構造解析 / 結晶化条件検索 |
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| Research Abstract |
The Notch signaling pathway is known as a signal transduction system which functions between neighboring cells, and is widely conserved among metazoan. Notch intra cellar domain (NICD), suppressor of hairless (Su(H)) and mastermind (Mam) are Notch signal component proteins. We focused on these proteins from Xenopus laevis in order to investigate for structure and function relationship.
We tried to over express these proteins and to purify, however whole of these proteins were not able to purify, although these proteins were over expressed by using E. coli. The signal molecule, NICD, is multi domain protein, and have an ankyrin repeats domain (X-ANK) and a Ram domain (X-RAM) which contribute to interacting with Mam and Su(H), respectively. X-ANK and X-RAM-ANK regions were prepared in high purity.
In order to obtain the structural information in the solution, CD spectra, dynamic light scattering (DLS) of X-ANK and X-RAM-ANK were observed. As a result of CD spectra, α-helices contents are almost same in both proteins. The results of DLS show molecular size of X-ANK is mono-dispersed and that of X-RAM-ANK is widely dispersed. These results suggest that X-RAM domain is difficult to have a tight structure. The detailed information of X-ANK in the solution was obtained by small angle X-ray scattering experiment. The radius of gyration and the maximum size of ANK were 40 Å and 114 Å, respectively. In addition, a dummy atom model was obtained by ab initio method and was well fitted by two Drosophila ANK models. These results show that ANK exists as a dimer in the solution.
The initial crystallization conditions of X-ANK were obtained by sparse matrix method. The crystals were obtained by vapor diffusion method using PEG400 and salts as precipitating reagents.
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