Retinal chromophore structure and Schiff base interactions in red-shifted channelrhodopsin-1 from Chlamydomonas augustae.
Retinal chromophore structure and Schiff base interactions in red-shifted channelrhodopsin-1 from Chlamydomonas augustae.
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DOI：
10.1021/bi500445c
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发表时间：
2014-06-24
期刊：
影响因子：
2.9
通讯作者：
Rothschild KJ
中科院分区：
文献类型：
--
作者：
Ogren JI;Mamaev S;Russano D;Li H;Spudich JL;Rothschild KJ
视紫红质通道蛋白 (ChRs) 是微生物视紫红质家族的一个独特分支，控制着绿藻的趋光性。由于 ChR 可以作为光门控阳离子通道在神经元膜中表达和发挥作用，因此它们已迅速成为神经生物学中重要的光遗传学工具。虽然来自单细胞藻类莱茵衣藻 (CrChR2) 的视紫红质通道蛋白-2 是最常用和研究最广泛的光遗传学 ChR，但人们对其他不同 ChR 组的特性知之甚少。在这项研究中，利用近红外共焦共振拉曼光谱以及氢-氘交换和定点诱变来研究来自奥古斯衣藻 (CaChR1) 的红移 ChR1 的结构。这些测量表明，(i) CaChR1 具有与光驱动质子泵细菌视紫红质 (BR) 和感觉视紫红质 II 相似的全反式视网膜结构，但与 CrChR2 的混合视网膜成分不同，(ii) 将 pH 从 7 降低至 2 或用中性残基替换 Glu169 或 Asp299 不会显着使烯键拉伸频率移动超过 1-2 cm-1 与 BR 相比，其中发生 7-9 cm-1 的下移，反映了 Asp85 抗衡离子的中和，并且 (iii) CaChR1 质子化希夫碱 (SB) 具有比 BR 更强的氢键。提出了一个模型来解释这些结果，其中在 pH 7 时，SB 的主要抗衡离子是 Asp299（BR 中 Asp212 的同系物），而 Glu169（BR 中 Asp85 的同系物）以中性状态存在。我们观察到共振拉曼光谱在 pH 9 到 2 的广泛范围内异常恒定，并讨论了其含义。这些结果与 CaChR1 最近的可见光吸收和电流测量结果一致 [Sineshchekov, O. A., et al. (2013)视紫红质通道的分子内质子转移。生物物理学。 J. 104, 807–817；李，H.，等人。 (2014) 螺旋 B 赖氨酸残基在视紫红质通道光活性位点中的作用。生物物理学。 J. 106, 1607–1617]。
Channelrhodopsins (ChRs), which form a distinct branch of the microbial rhodopsin family, control phototaxis in green algae. Because ChRs can be expressed and function in neuronal membranes as light-gated cation channels, they have rapidly become an important optogenetic tool in neurobiology. While channelrhodopsin-2 from the unicellular alga Chlamydomonas reinhardtii (CrChR2) is the most commonly used and extensively studied optogenetic ChR, little is known about the properties of the diverse group of other ChRs. In this study, near-infrared confocal resonance Raman spectroscopy along with hydrogen–deuterium exchange and site-directed mutagenesis were used to study the structure of red-shifted ChR1 from Chlamydomonas augustae (CaChR1). These measurements reveal that (i) CaChR1 has an all-trans-retinal structure similar to those of the light-driven proton pump bacteriorhodopsin (BR) and sensory rhodopsin II but different from that of the mixed retinal composition of CrChR2, (ii) lowering the pH from 7 to 2 or substituting neutral residues for Glu169 or Asp299 does not significantly shift the ethylenic stretch frequency more than 1–2 cm–1 in contrast to BR in which a downshift of 7–9 cm–1 occurs reflecting neutralization of the Asp85 counterion, and (iii) the CaChR1 protonated Schiff base (SB) has stronger hydrogen bonding than BR. A model is proposed to explain these results whereby at pH 7 the predominant counterion to the SB is Asp299 (the homologue to Asp212 in BR) while Glu169 (the homologue to Asp85 in BR) exists in a neutral state. We observe an unusual constancy of the resonance Raman spectra over the broad range from pH 9 to 2 and discuss its implications. These results are in accord with recent visible absorption and current measurements of CaChR1 [Sineshchekov, O. A., et al. (2013) Intramolecular proton transfer in channelrhodopsins. Biophys. J. 104, 807–817; Li, H., et al. (2014) Role of a helix B lysine residue in the photoactive site in channelrhodopsins. Biophys. J. 106, 1607–1617].
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