Structural Study of Channel-type Membrane Protein using ^<19>F Solid-state NMR

1998 Fiscal Year Final Research Report Summary

• Project/Area Number: 08640751
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 機能・物性・材料
• Research Institution: Suntory Institute for Bioorganic Research
• Principal Investigator: IWASHITA Takashi Suntory Institute for Bioorganic Research, Chief Chemist, 主任研究員 (00150144)
• Co-Investigator(Kenkyū-buntansha): SEKIYA Noriko Suntory Institute for Bioorganic Research, Researcher, 研究員 (20236102)
• Project Period (FY): 1996 – 1998
• Keywords: bacteriorhodopsin / fluorotryptophan / proton-pump / MALDI-TOF MS / X-ray diffraction / CD

Research Abstract

Membrane proteins like receptors which contain alpha-helices play an important role in the biological system. Bacteriorhodopsin is a kind of photoreceptors which has 7 alpha-helices and it is thought to be the model of GTP-binding protein coupled receptors. Tryptophan residues of bacteriorhodopsin were labeled by ^<19>F on their imidazole ring to obtain strucural informations using the ^<19>Fsolid-state NMR.However, the signals from ^<19>F might be overlapped, so that 4-F, 5-F and 6-F-tryptophan were tested to overcome the problem. We obtained three kind of fluorinated bacteriorhodopsin. The fluorinated bacteriorhodopsin which contains 6-F-tryptophan has different property from others on the purification step by sucrose gradient and the MALDI-TOF MS.Usually, bacteriorhodopsins form two dimensional trimer crystal on the membrane. Howerver, it is clarified that the bacteriorhodopsin which contains 6F-tryptophan makes no crystal structure from X-ray diffraction analysis and CD spectrum.
To study the distnance between two fluorines in the opsin and the chromophor, the synthetic chromophor which had an aromatic ring with a CF_3 group was incorporated to the fluorine labeled bacterioopsin with 5-F-tryptophan. The magnetization transfer due to the dipolar-dipolar coupling was inestigated. It seems that the magnetization transfer might be observed by 1 dimensional RFDR and 2 dimensional CP/EXCY experiments. Next, MELODRAMA pulse sequence was tested for the observation of magnetization transfer. The MELODRAMA spectrum showed the anti-phase cross-peaks to the diagonal peaks. This means that the observation of magnetization transfer between the fluorinated opsin and the CF3 group of the synthetic chromophore has been confirmed.