Protonation state of Asp (Glu)-85 regulates the purple-to-blue transition in bacteriorhodopsin mutants Arg-82----Ala and Asp-85----Glu: the blue form is inactive in proton translocation.
Protonation state of Asp (Glu)-85 regulates the purple-to-blue transition in bacteriorhodopsin mutants Arg-82----Ala and Asp-85----Glu: the blue form is inactive in proton translocation.
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Asp (Glu)-85 的质子化状态调节细菌视紫红质突变体 Arg-82----Ala 和 Asp-85----Glu 中的紫色到蓝色转变：蓝色形式在质子易位中不活跃。
DOI：
10.1073/pnas.87.3.1013
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发表时间：
1990
影响因子：
11.1
通讯作者：
Khorana,HG
中科院分区：
文献类型：
--
作者：
Subramaniam,S;Marti,T;Khorana,HG
以前对细菌视紫红质定点突变体的研究表明，Asp-85或Arg-82的替换会影响吸收光谱。在pH 5.5和7之间，Asp-85-Glu和Arg-82-Ala突变体在紫色(最大波长约550/540 nm)和蓝色(最大波长约600/590 nm)之间存在pH依赖的平衡。对重组囊泡中质子传输随波长变化的测量表明，上述突变体的质子泵活性仅存在于它们各自的紫色物种中。对于这两个突变体，随着pH的下降，蓝色形式的形成伴随着质子转运活性的丧失。Asp-85-Asn突变体显示蓝色发色团(最大波长约588 nm)，在pH 5至7.5的范围内质子转运不活跃，也没有向紫色过渡。相反，Asp-212-Asn突变体是紫色的(最大波长约为555 nm)，并且没有随着pH的降低而转变为蓝色发色团。实验表明：(1)紫蓝转变的pKA直接受85位羧酸的pKA影响；(2)质子化Schiff碱、Asp-85、Asp-212和Arg-82之间相互作用的相对强度对细菌视紫红质的颜色和功能的调节有重要贡献。
Previous studies with site-specific mutants of bacteriorhodopsin have demonstrated that replacement of Asp-85 or Arg-82 affects the absorption spectrum. Between pH 5.5 and 7, the Asp-85----Glu and Arg-82----Ala mutants exist in a pH-dependent equilibrium between purple (lambda max approximately 550/540 nm) and blue (lambda max approximately 600/590 nm) forms of the pigment. Measurement of proton transport as a function of wavelength in reconstituted vesicles shows that proton-pumping activities for the above mutants reside exclusively in their respective purple species. For both mutants, formation of the blue form with decreasing pH is accompanied by loss of proton transport activity. The Asp-85----Asn mutant displays a blue chromophore (lambda max approximately 588 nm), is inactive in proton translocation from pH 5 to 7.5, and shows no transition to the purple form. In contrast, the Asp-212----Asn mutant is purple (lambda max approximately 555 nm) and shows no transition to a blue chromophore with decreasing pH. The experiments suggest that (i) the pKa of the purple-to-blue transition is directly influenced by the pKa of the carboxylate at residue 85 and (ii) the relative strengths of interaction between the protonated Schiff base, Asp-85, Asp-212, and Arg-82 make a major contribution to the regulation of color and function of bacteriorhodopsin.