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

含有α-螺旋的膜蛋白如受体在生物系统中起着重要作用。细菌视紫红质（Bacteriorhodopsin）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。用~（13）F标记菌紫质的色氨酸残<19>基咪唑环，利用~（13）F固体核磁共振技术获得其结构信息<19>，但~（13）F标记的信号<19>可能重叠，因此用4-F、5-F和6-F-色氨酸进行了验证。我们得到了三种氟代细菌视紫红质。含6-F-色氨酸的氟代菌视紫红质在蔗糖梯度分离和MALDI-TOF MS分离纯化过程中表现出与其它氟代菌视紫红质不同的性质。为了研究视蛋白和发色团中两个氟原子的区别，将合成的发色团引入到含5-F-色氨酸的氟标记的细菌视蛋白中。研究了偶极-偶极耦合引起的磁化转移。用一维RFDR和二维CP/EXCY实验似乎可以观察到磁化转移。接下来，测试MELODRAMA脉冲序列以观察磁化转移。MELODRAMA光谱显示对角峰的反相交叉峰。这意味着已经证实了氟化视蛋白和合成发色团的CF 3基团之间的磁化转移的观察。

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.