Hackett N R, Stern L J, Chao B H, Kronis K A, Khorana H G
J Biol Chem. 1987 Jul 5;262(19):9277-84.
To test structural and mechanistic proposals about bacteriorhodopsin, a series of analogues with single amino acid substitutions has been studied. Mutants in the proposed helix F of bacteriorhodopsin were chosen for investigation because of the probable interaction of this part of the protein with the retinal chromophore. Seven mutants of the bacteriorhodopsin gene were constructed by site-directed mutagenesis, and the gene products were expressed in Escherichia coli. The resulting mutant proteins were purified and assayed for their ability to interact with retinal in phospholipid/detergent micelles to form a bacteriorhodopsin-like chromophore. Four mutants, Ser-183----Ala, Tyr-185----Phe, Ser-193----Ala, and Glu-194----Gln, bound retinal to give pigments with absorption maxima approximately the same as the wild type. Three mutant opsins bound retinal to give chromophores that were blue-shifted relative to the wild type. Two Trp----Phe substitutions at positions 182 and 189 gave absorption maxima of 480 and 524 nm, respectively, and the mutant Pro-186----Leu gave a pigment with an absorption maximum of 470 nm. However, none of the amino acid substitutions eliminated the ability of the mutant bacteriorhodopsin to pump protons in response to illumination.
为了检验关于细菌视紫红质的结构和机制假说，对一系列具有单个氨基酸取代的类似物进行了研究。选择细菌视紫红质中提议的F螺旋中的突变体进行研究，因为该蛋白质的这一部分可能与视黄醛发色团相互作用。通过定点诱变构建了七个细菌视紫红质基因的突变体，并在大肠杆菌中表达了基因产物。对所得的突变蛋白进行纯化，并测定它们在磷脂/去污剂胶束中与视黄醛相互作用以形成类似细菌视紫红质发色团的能力。四个突变体，即Ser-183→Ala、Tyr-185→Phe、Ser-193→Ala和Glu-194→Gln，结合视黄醛后产生的色素的吸收最大值与野生型大致相同。三个突变视蛋白结合视黄醛后产生的发色团相对于野生型发生了蓝移。在第182和189位的两个Trp→Phe取代分别产生了480和524nm的吸收最大值，而突变体Pro-186→Leu产生了一个吸收最大值为470nm的色素。然而，没有一个氨基酸取代消除突变型细菌视紫红质在光照下泵浦质子的能力。
