EFFECTS OF SUBSTITUTION OF TYROSINE-57 WITH ASPARAGINE AND PHENYLALANINE ON THE PROPERTIES OF BACTERIORHODOPSIN
EFFECTS OF SUBSTITUTION OF TYROSINE-57 WITH ASPARAGINE AND PHENYLALANINE ON THE PROPERTIES OF BACTERIORHODOPSIN
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DOI：
10.1021/bi00014a040
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发表时间：
1995-04-11
期刊：
影响因子：
2.9
通讯作者：
EBREY, TG
中科院分区：
文献类型：
--
作者：
GOVINDJEE, R;KONO, M;EBREY, TG
酪氨酸57是存在于视网膜结合袋中的残基之一，在所有的嗜盐性视网膜蛋白中都是保守的。我们研究了盐盐杆菌中表达的细菌视紫红质突变体，其中酪氨酸57被天冬酰胺（Y57N）或苯丙氨酸（Y57F）取代。在Y57N中，光循环只进行到L中间体；L中间体的寿命极长，约为500 ms。两个突变体的质子释放都受到严重影响，这表明Y57与质子释放途径有关。通过比较Y57N和Y57F与野生型（WT） ph诱导的紫外吸收变化，我们确定Y57的pK(a)为10.2。在显示M形成的Y57F中，L—> M转变的速率常数依赖于pH (pK(a) 8.7)，这表明Y57可能不是通常控制向碱性光循环转变的残基。Y57要么是反离子复合物的一部分，要么靠近D85，因为它的突变会影响Asp85的pK(a)。在Y57F中，D85的pK(a)近似于4.9（而在WT中近似于2.9）。Y57N突变体在紫色到蓝色的转变中显示了两个pK(a)，类似于3.8和
Tyrosine 57 is one of the residues present in the retinal binding pocket and is conserved in all the halophilic retinal proteins. We have studied mutants of bacteriorhodopsin, expressed in Halobacterium salinarium, in which tyrosine 57 is replaced by an asparagine (Y57N) or phenylalanine (Y57F). In Y57N the photocycle proceeds only up to the L intermediate; no M is formed at neutral pH. The lifetime of L intermediate is extremely long, ca. 500 ms. Proton release is severely affected in both the mutants which suggests that Y57 is associated with the proton release pathway. By comparing the pH-induced absorption changes in the UV in Y57N and Y57F with those in the wild-type (WT), we determined that the pK(a) of Y57 is 10.2. In Y57F, which shows M formation, the rate constant of the L --> M transition is pH dependent (pK(a) 8.7) suggesting that Y57 is probably not the residue,that normally controls the transition into the alkaline photocycle. Y57 is either part of the counterion complex or in close proximity to D85 since its mutation influences the pK(a) of Asp85. In Y57F the pK(a) of D85 is similar to 4.9 (compared to similar to 2.9 in the WT). The Y57N mutant shows two pK(a)'s in the purple to blue transition, similar to 3.8 and