Sato Maki, Kikukawa Takashi, Araiso Tsunehisa, Okita Hirotaka, Shimono Kazumi, Kamo Naoki, Demura Makoto, Nitta Katsutoshi
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan.
Biophys Chem. 2003 May 1;104(1):209-16. doi: 10.1016/s0301-4622(02)00368-x.
Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump from Natronobacterium pharaonis. In order to clarify the role of Ser-130(phR) residue which corresponds to Ser-115(shR) for salinarum hR on the anion-binding affinity, the wild-type and Ser-130 mutants substituted with Thr, Cys and Ala were expressed in E. coli cells and solubilized with 0.1% n-dodecyl beta-D-maltopyranoside The absorption maximum (lambda(max)) of the S130T mutant indicated a blue shift from that of the wild type in the absence and presence of chloride. For S130A, a large red shift (12 nm) in the absence of chloride was observed. The wild-type and all mutants showed the blue-shift of lambda(max) upon Cl(-) addition, from which the dissociation constants of Cl(-) were determined. The dissociation constants were 5, 89, 153 and 159 mM for the wild-type, S130A, S130T and S130C, respectively, at pH 7.0 and 25 degrees C. Circular dichroic spectra of the wild-type and the Ser-130 mutants exhibited an oligomerization. The present study revealed that the Ser-130 of N. pharaonis halorhodopsin is important for the chloride binding.
法老盐红菌视紫红质（phR）是一种来自法老嗜盐菌的内向光驱动氯离子泵。为了阐明与盐沼盐红菌视紫红质（shR）的Ser-115相对应的Ser-130（phR）残基对阴离子结合亲和力的作用，将用苏氨酸、半胱氨酸和丙氨酸取代的野生型和Ser-130突变体在大肠杆菌细胞中表达，并用0.1%的正十二烷基-β-D-麦芽糖苷进行增溶。在不存在和存在氯离子的情况下，S130T突变体的最大吸收波长（λmax）相对于野生型表现出蓝移。对于S130A，在不存在氯离子的情况下观察到较大的红移（12 nm）。野生型和所有突变体在添加Cl-后均表现出λmax的蓝移，据此测定了Cl-的解离常数。在pH 7.0和25℃下，野生型、S130A、S130T和S130C的解离常数分别为5、89、153和159 mM。野生型和Ser-130突变体的圆二色光谱显示出寡聚化。本研究表明，法老盐红菌视紫红质的Ser-130对氯离子结合很重要。
