Knierim Bernhard, Hofmann Klaus Peter, Ernst Oliver P, Hubbell Wayne L
Institut für Medizinische Physik und Biophysik, Charité Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20290-5. doi: 10.1073/pnas.0710393104. Epub 2007 Dec 11.
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 alpha-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.
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之间的相互关系，使用了在十二烷基麦芽糖苷溶液中的自旋标记视紫红质，以便在激活光脉冲后，可将时间分辨的TM6运动和质子交换分别作为pH和温度的函数进行监测。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不同步，在30摄氏度时其速度要慢一个数量级。然而，TM6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进程。重要的是，TM6的运动实际上与pH无关，在所使用的条件下席夫碱去质子化也是如此，而质子摄取的滴定pK为6.5。这一发现表明质子摄取是螺旋运动的结果而非原因。激活的视紫红质与同源G蛋白转导素结合并随后激活它。已经表明，源自转导素α亚基C末端的肽部分模拟完整G蛋白的结合。发现这些肽在TM6移动后与视紫红质结合，导致质子释放。总体而言，数据表明激活过程中涉及以下时间顺序的事件：（i）内部席夫碱质子转移；（ii）TM6移动；（iii）从溶液中摄取质子和转导素结合。
