Sequence of late molecular events in the activation of rhodopsin
Sequence of late molecular events in the activation of rhodopsin
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DOI：
10.1073/pnas.0710393104
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发表时间：
2007-12-18
影响因子：
11.1
通讯作者：
Hubbell, Wayne L.
中科院分区：
文献类型：
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作者：
Knierim, Bernhard;Hofmann, Klaus Peter;Hubbell, Wayne L.
G 蛋白偶联受体视紫红质的激活涉及跨膜螺旋 6 (TM6) 的运动和质子交换事件。为了研究这些激活步骤如何相互关联，使用十二烷基麦芽糖苷溶液中的自旋标记视紫红质，以便在激活闪光后，可以分别监测时间分辨的 TM6 运动和质子交换作为 pH 和温度的函数。结果表明，TM6 的运动与将发色团与蛋白质结合的席夫碱的去质子化并不同步，但在 30 摄氏度时减慢了一个数量级。然而，TM6 的运动和在中性 pH 范围内从溶液中摄取质子遵循相同的时间过程。重要的是，TM6 的运动实际上与 pH 无关，正如所用条件下的席夫碱去质子化一样，而质子吸收滴定的 pK 为 6.5。这一发现表明，质子吸收是螺旋运动的结果而不是原因。激活的视紫质结合并随后激活同源 G 蛋白转导蛋白。已经表明，源自转导蛋白α亚基C末端的肽模拟了完整G蛋白的部分结合。这些肽被发现在 TM6 运动后与视紫红质结合，导致质子释放。总的来说，数据表明激活涉及以下事件的时间序列：（i）内部希夫碱质子转移； (ii) TM6 机芯； (iii)从溶液中摄取质子并结合转导蛋白。
Activation of the G protein-coupled receptor rhodopsin involves both the motion of transmembrane helix 6 (TM6) and proton exchange events. To study how these activation steps relate to each other, spin-labeled rhodopsin in solutions of dodecyl maltoside was used so that time-resolved TM6 motion and proton exchange could each be monitored as a function of pH and temperature after an activating light flash. The results reveal that the motion of TM6 is not synchronized with deprotonation of the Schiff base that binds the chromophore to the protein but is an order of magnitude slower at 30 degrees C. However, TM6 motion and the uptake of a proton from solution in the neutral pH range follow the same time course. Importantly, the motion of TM6 is virtually independent of pH, as is Schiff base deprotonation under the conditions used, whereas proton uptake titrates with a pK of 6.5. This finding shows that proton uptake is a consequence rather than a cause of helix motion. Activated rhodopsin binds to and subsequently activates the cognate G protein, transducin. It has been shown that peptides derived from the C terminus of the transducin a-subunit mimic in part binding of the intact G protein. These peptides are found to bind to rhodopsin after TM6 movement, resulting in the release of protons. Collectively, the data suggest the following temporal sequence of events involved in activation: (i) internal Schiff base proton transfer; (ii) TM6 movement; and (iii) proton uptake from solution and binding of transducin.