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

使用^<19>F固态NMR研究通道型膜蛋白的结构

• 批准号: 08640751
• 负责人: IWASHITA Takashi
• 金额: $ 1.47万
• 依托单位: Suntory Institute for Bioorganic Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08640751/
• 关键词: bacteriorhodopsin; fluorotryptophan; proton-pump; MALDI-TOF MS; X-ray diffraction; CD; 固体NMR; レチナ-ルアナログ

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.

膜蛋白类受体含有α -螺旋，在生物系统中起着重要作用。细菌视紫红质是一种具有7个α -螺旋的光感受器，被认为是gtp结合蛋白偶联受体的模型。细菌视紫红质色氨酸残基在其咪唑环上用^<19>F标记，利用^<19>Fsolid-state NMR获得结构信息。然而，来自^<19>F的信号可能重叠，因此测试4-F， 5-F和6-F色氨酸来克服这个问题。我们得到了三种氟化细菌视紫红质。含6- f -色氨酸的氟化菌紫红质在蔗糖梯度和MALDI-TOF ms的纯化步骤中具有不同于其他方法的性质。通常，菌紫红质在膜上形成二维三聚体晶体。然而，通过x射线衍射分析和CD谱分析表明，含有6f -色氨酸的细菌视紫红质没有晶体结构。为了研究视蛋白中两个氟与发色体之间的距离，用5- f -色氨酸将合成的含CF_3基团芳环的发色体与氟标记的细菌视蛋白结合。研究了偶极-偶极耦合引起的磁化转移。通过一维RFDR和二维CP/ exy实验可以观察到磁化转移。然后，对MELODRAMA脉冲序列进行磁化转移观测。MELODRAMA谱表现为反相交叉峰和对角峰。这意味着氟化视蛋白与合成的发色团的CF3基团之间的磁化转移的观察得到了证实。