| Project/Area Number |
13680692
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Structural biochemistry
|
|---|
| Research Institution | Ehime University |
|---|
Principal Investigator |
HORI Hiroyuki Ehime University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (20256960)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
SAWASAKI Tatsuya Ehime University, Faculty of Engineering, Instructor, 工学部, 助手 (50314969)
|
|---|
| Project Period (FY) |
2001 – 2002
|
| Project Status |
Completed (Fiscal Year 2002)
|
| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
|
|---|
| Keywords | Cell-free translation / MAD / selenomethionine / X-ray crystal structure analysis / RNA modification enzymes / 翻訳 / 多波長異常分散法 / セレノメチオニン |
|---|
| Research Abstract |
We investigated the selenomethionine (SeMet) labeling of two model proteins (GFP and DHFR) by wheat germ in vitro cell-free translation system. The yields of the produced proteins were equal to those of the usual methionine (Met) proteins. We analyzed the contents of SeMet and Met in the proteins by LC/MS spectrometry. We analyzed the LC chromatograms thoroughly, however, we could not detect the Met labeled peptide fragment derived from the intrinsic Met. These results strongly suggest that the efficiency of foe labeling was near 100%. Further, we also found that SeMet residues in the model proteins were not oxidized during the store at -80℃ for 6 months.
Although the formation of the chromophore of SeMet-GFP was slow as compared to Met-GFP, the line shape of the fluorescence spectrum of the protein coincides with that of Met-GFP, suggesting that the SeMet labeling did not affect the environment around the chromophore. In the case of DHFR, Met residues are included the M20 loop, which plays a key role in the catalytic cycles. However, SeMet labeling did not change the enzymatic activity apparently.
We utilized this labeling technique to apply the X-ray crystal analysis of tRNA methyltransferase. Now, we are going on the structural refinement of the crystal at 1.5Å resolutions. Based on these experimental results, we confirmed that cell-free translation system was applicable to the structural proteomics.
|
