Temperature-dependent interactions between photoactivated Pharaonis phoborhodopsin and its transducer
Temperature-dependent interactions between photoactivated Pharaonis phoborhodopsin and its transducer
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DOI：
10.1021/bi060047i
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发表时间：
2006-04-18
期刊：
影响因子：
2.9
通讯作者：
Kandori, H
中科院分区：
文献类型：
--
作者：
Kamada, K;Furutani, Y;Kandori, H
Pharaonis phoborhodopsin （ppR，又称Pharaonis sensory rhodopsin ii， psRII）是法老钠单胞菌（Natronomonas Pharaonis）的负趋光受体。在膜中，它与其换能器蛋白pHtrII形成2:2的复合物，在M中间体（ppR(M)）中，这种结合被削弱了2个数量级。这种变化被认为与光信号向pHtrII的传递相对应。先前的傅里叶变换红外（F-FIR）研究观察到pHtrII在M状态下Asn74的氢键改变，表明从受体到换能器的光信号通路[Furutani， Y., Kamada， K., Sudo， Y., Shimono， K., Kamo， N., and Kandori， H.（2005）生物化学44,2909-2915]。在250-293 K范围内，我们测量了pHtrII存在和不存在时ppR(M) - ppR光谱的温度依赖性。只有ppR/pHtrII配合物的螺旋的酰胺振动对温度有明显的依赖性，在室温下酰胺振动的振幅减小。对ppR或pHtrII的c -13标记表明，这些螺旋的光谱变化来自ppR，而不是pHtrII。pHtrII中Asn74的氢键变化与温度无关，这意味着ppR中观察到的螺旋结构扰动发生在不同的区域。另一方面，ppR与pHtrII的G83C和G83F突变体复合物的螺旋结构变化减少了温度依赖性。Gly83被认为在pHtrII膜表面附近以一种灵活的扭结连接跨膜螺旋和细胞质连接区，而被Cys或Phe取代则取消了光感功能。本研究提供了直接的实验证据，证明Gly83在ppR/pHtrII复合物的激活过程中起着重要的结构作用。在现有FTIR结果的基础上，讨论了ppR/pHtrII复合物蛋白结构变化的分子机制。
Pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin Ill, psRII) is a receptor for negative phototaxis in Natronomonas pharaonis. In membranes, it forms a 2:2 complex with its transducer protein pHtrII, and the association is weakened by 2 orders of magnitude in the M intermediate (ppR(M)). Such a change is believed to correspond to the transfer of the light signal to pHtrII. A previous Fourier transform infrared (F-FIR) study observed hydrogen-bonding alteration of Asn74 in pHtrII in the M state, suggesting a light-signaling pathway from the receptor to the transducer [Furutani, Y., Kamada, K., Sudo, Y., Shimono, K., Kamo, N., and Kandori, H. (2005) Biochemistry 44, 2909-2915]. In this paper, we measure temperature dependence of the ppR(M) minus ppR spectra in the absence and presence of pHtrII at 250-293 K. Significant temperature dependence was observed for the amide-I vibrations of helices only for the ppR/pHtrII complex, where the amplitude of amide-I vibrations was reduced at room temperature. C-13-Labeling of ppR or pHtrII revealed that such spectral changes of helices originate from ppR and not pHtrII. The hydrogen-bonding alteration of Asn74 in pHtrII was temperature-independent, implying that the observed helical structural perturbation in ppR takes place in different region. On the other hand, temperature-dependent structural changes of helices were diminished for the complex of ppR with the G83C and G83F mutants of pHtrII. Gly83 is believed to connect the transmembrane helix and cytosolic linker region in a flexible kink near the membrane surface of pHtrII, and its replacement by Cys or Phe abolishes the photosensory function. The present study provides direct experimental evidence that Gly83 plays an important structural role in the activation processes of the ppR/pHtrII complex. A molecular mechanism of protein structural changes in the ppR/pHtrII complex is discussed on the basis of the present FTIR results.