Tsukamoto Takashi, Demura Makoto, Sudo Yuki
Division of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University , 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
J Phys Chem B. 2014 Oct 30;118(43):12383-94. doi: 10.1021/jp507374q. Epub 2014 Oct 15.
Assembly is one of the keys to understand biological molecules, and it takes place in spatial and temporal domains upon stimulation. Microbial rhodopsin (also called retinal protein) is a membrane-embedded protein that has a retinal chromophore within seven-transmembrane α-helices and shows homo-, di-, tri-, penta-, and hexameric assemblies. Those assemblies are closely related to critical physiological properties such as stabilizing the protein structure and regulating their photoreaction dynamics. Here we investigated the assembly and disassembly of thermophilic rhodopsin (TR), which is a novel proton-pumping rhodopsin derived from a thermophile living at 75 °C. TR was characterized using size-exclusion chromatography and circular dichroism spectroscopy, and formed a trimer at 25 °C, but irreversibly dissociated into monomers upon thermal stimulation. The transition temperature was estimated to be 68 °C. The irreversible nature made it possible to investigate the photochemical properties of both the trimer and the monomer independently. Compared with the trimer, the absorption maximum of the monomer is blue-shifted by 6 nm without any changes in the retinal composition, pKa value for the counterion or the sequence of the proton movement. The photocycling rate of the monomeric TR was similar to that of the trimeric TR. A similar trimer-monomer transition upon thermal stimulation was observed for another eubacterial rhodopsin GR but not for the archaeal rhodopsins AR3 and HwBR, suggesting that the transition is conserved in bacterial rhodopsins. Thus, the thermal stimulation of TR induces the irreversible disassembly of the trimer.
组装是理解生物分子的关键之一，并且它在受到刺激时在空间和时间域中发生。微生物视紫红质（也称为视网膜蛋白）是一种膜嵌入蛋白，在七个跨膜α-螺旋中有一个视黄醛发色团，并呈现同聚体、二聚体、三聚体、五聚体和六聚体组装形式。这些组装形式与关键的生理特性密切相关，如稳定蛋白质结构和调节其光反应动力学。在这里，我们研究了嗜热视紫红质（TR）的组装和解组装，TR是一种源自生活在75°C的嗜热菌的新型质子泵视紫红质。使用尺寸排阻色谱法和圆二色光谱法对TR进行了表征，它在25°C时形成三聚体，但在热刺激下不可逆地解离为单体。转变温度估计为68°C。这种不可逆的性质使得能够独立研究三聚体和单体的光化学性质。与三聚体相比，单体的最大吸收波长蓝移了6 nm，而视黄醛组成、抗衡离子的pKa值或质子移动序列没有任何变化。单体TR的光循环速率与三聚体TR的相似。对于另一种真细菌视紫红质GR，在热刺激下也观察到了类似的三聚体-单体转变，但古细菌视紫红质AR3和HwBR没有，这表明这种转变在细菌视紫红质中是保守的。因此，TR的热刺激诱导三聚体不可逆地解组装。
