Sudo Yuki, Furutani Yuji, Shimono Kazumi, Kamo Naoki, Kandori Hideki
Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.
Biochemistry. 2003 Dec 9;42(48):14166-72. doi: 10.1021/bi035678g.
Pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronobacterium pharaonis. It forms a 2:2 complex with its transducer protein, pHtrII, in membranes and transmits light signals through the change in the protein-protein interaction. We previously found that the ppR(K) minus ppR spectrum in D(2)O possesses vibrational bands of ppR at 3479 (-)/3369 (+) cm(-1) only in the presence of pHtrII [Furutani, Y., Sudo, Y., Kamo, N., and Kandori, H. (2003) Biochemistry 42, 4837-4842]. A D/H-unexchangeable X-H group appears to form a stronger hydrogen bond upon retinal photoisomerization in the ppR-pHtrII complex. This article aims to identify the group by use of various mutant proteins. According to the crystal structure, Tyr-199 of ppR forms a hydrogen bond with Asn-74 of pHtrII in the complex. Nevertheless, the 3479 (-)/3369 (+) cm(-1) bands were preserved in the Y199F mutant, excluding the possibility that the bands are O-H stretches of Tyr-199. On the other hand, Thr-204 and Tyr-174 form a hydrogen bond between the retinal chromophore pocket and the binding surface of the ppR-pHtrII complex. These FTIR measurements revealed that the bands at 3479 (-)/3369 (+) cm(-1) disappeared in the T204A mutant, while being shifted to 3498 (-) and 3474 (+) cm(-1) in the T204S mutant. They appear at 3430 (-)/3402 (+) cm(-1) in the Y174F mutant. From these results, we concluded that the bands at 3479 (-)/3369 (+) cm(-1) originate from the O-H stretch of Thr-204. A stronger hydrogen bond as shown by a large spectral downshift (110 cm(-1)) suggests that the specific hydrogen bonding alteration of Thr-204 takes place upon retinal photoisomerization, which does not occur in the absence of the transducer protein. Thr-204 has been known as an important residue for color tuning and photocycle kinetics in ppR. The results presented here point to an additional important role of Thr-204 in ppR for the interaction with pHtrII. Specific interaction in the complex that involves Thr-204 presumably affects the decay kinetics and binding affinity in the M intermediate.
法老视紫红质（ppR，也称为法老嗜盐菌感官视紫红质II，psRII）是嗜盐栖热菌中负趋光性的受体。它在膜中与其转导蛋白pHtrII形成2:2复合物，并通过蛋白质-蛋白质相互作用的变化来传递光信号。我们之前发现，仅在存在pHtrII的情况下，重水（D₂O）中的ppR(K)减去ppR光谱在3479（-）/3369（+）cm⁻¹处具有ppR的振动带[古谷洋一、须藤洋、加茂直、神鸟博（2003年）《生物化学》42卷，4837 - 4842页]。在ppR - pHtrII复合物中，一个D/H不可交换的X - H基团似乎在视黄醛光异构化时形成了更强的氢键。本文旨在通过使用各种突变蛋白来鉴定该基团。根据晶体结构，复合物中ppR的Tyr - 199与pHtrII的Asn - 74形成氢键。然而，在Y199F突变体中，3479（-）/3369（+）cm⁻¹的谱带得以保留，排除了这些谱带是Tyr - 199的O - H伸缩振动的可能性。另一方面，Thr - 204和Tyr - 174在视黄醛发色团口袋与ppR - pHtrII复合物的结合表面之间形成氢键。这些傅里叶变换红外光谱测量结果表明，在T204A突变体中，3479（-）/3369（+）cm⁻¹处的谱带消失，而在T204S突变体中，它们分别移至3498（-）和3474（+）cm⁻¹。在Y174F突变体中，它们出现在3430（-）/3402（+）cm⁻¹处。根据这些结果，我们得出结论，3479（-）/3369（+）cm⁻¹处的谱带源自Thr - 204的O - H伸缩振动。如大的光谱下移（110 cm⁻¹）所示的更强氢键表明，Thr - 204的特定氢键改变发生在视黄醛光异构化时，而在没有转导蛋白的情况下不会发生。Thr - 204已知是ppR中颜色调谐和光循环动力学的重要残基。此处呈现的结果表明Thr - 204在ppR中与pHtrII相互作用方面还有一个重要作用。复合物中涉及Thr - 204的特异性相互作用可能会影响M中间体的衰减动力学和结合亲和力。
