| Project/Area Number |
17510186
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Living organism molecular science
|
|---|
| Research Institution | Quantum Beam Science Directorate, Japan Atomic Energy Agency |
|---|
Principal Investigator |
KUROKI Ryota Quantum Beam Science Directorate, Japan Atomic Energy Agency, Principal Scientist, 量子ビーム応用研究部門, 研究主幹 (30391246)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
TAMADA Taro Quantum Beam Science Directorate, Japan Atomic Energy Agency, Senior Researcher, 量子ビーム応用研究部門, 研究副主幹 (50391248)
ADACHI Motoyasu Quantum Beam Science Directorate, Japan Atomic Energy Agency, Researcher, 量子ビーム応用研究部門, 研究員 (60293958)
|
|---|
| Project Period (FY) |
2005 – 2006
|
| Project Status |
Completed (Fiscal Year 2006)
|
| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2006: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
|
|---|
| Keywords | disulfide bond / T4 phage lysozyme / x-ray crystallography / protein engineering |
|---|
| Research Abstract |
In order to observe tertiary structures in different solvent conditions, intermolecular disulfide cross-link was designed into T4 phage lysozyme (T4L). Since the wild type T4L is crystallized into the space group of P3221, a mutant T4L(SNAT-T4L) in which the four residues, Ser44, Asn68, Ala93, and Thrll5, located at the crystal contacts of T4L were mutated to cysteines, so that every neighbor protein can be linked by disulfide bridges. The mutant SNAT-T4L was successfully prepared as a monomer protein at the reduced condition. Then the SNAT-T4L was crystallized to the same space group. The obtained crystal diffracted to 1.5 A resolution at the cryogenic condition in the same buffer system as crystallized. The crystal diffracted after transferring to several different pHs from 4 to 10. Although the crystal diffract neither in simple water nor in 30% DMSO solution, it diffracted well after replacing the solution with the 50mM sodium phosphate buffer (pH7) containing 100mM sodium chloride and 30% glycerol solution. The crystal was stable enough to exchange mother liquor to other solution containing organic solvent, suggesting the possibility of the reutilization of the protein crystal.
|
