Marti T, Otto H, Rösselet S J, Heyn M P, Khorana H G
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1219-23. doi: 10.1073/pnas.89.4.1219.
Six bacterioopsin mutants containing either single amino acid deletions (delta A84, delta L87), insertions (delta 85A, delta 88A), or both deletions and insertions (delta A84/delta 88A, delta 85A/delta L87) within the first two turns of transmembrane helix C, starting from the extracellular side, have been prepared. The mutant apoproteins refold in phospholipid/detergent micelles and display secondary structures similar to that of the wild type. However, the mutants delta 88A and delta A84/delta 88A do not form a chromophore with retinal. The regenerated chromophore of delta 85A displays absorption maxima and retinal isomer compositions in the dark- and light-adapted states similar to those of the wild type. In delta A84, delta L87, and delta 85A/delta L87 these chromophore properties are altered, and the structures are less stable than that of the wild type, as shown by an enhanced rate of reaction with hydroxylamine in the dark, an increased pKa of the denaturation at acidic pH, and a decreased pKa of Schiff base deprotonation. Proton translocation is abolished in the delta A84 and delta 85A/delta L87 mutants, whereas in delta 85A and delta L87 the activity is reduced to about 25% of the wild-type value at pH 6. The overall properties of the delta 85A, delta 85A/delta L87, and delta L87 mutants indicate that the deletions and/or insertions result in displacement of residues Arg-82, Asp-85, or Asp-96, respectively, which participate in proton translocation. The results are compatible with a helical structure for transmembrane segment C and emphasize the flexibility of intramolecular contacts in bacteriorhodopsin.
我们制备了六个细菌视紫红质突变体，这些突变体在跨膜螺旋C的前两圈（从细胞外侧开始）中，要么包含单个氨基酸缺失（ΔA84、ΔL87），要么包含插入（Δ85A、Δ88A），要么同时包含缺失和插入（ΔA84/Δ88A、Δ85A/ΔL87）。突变体脱辅基蛋白在磷脂/去污剂胶束中重新折叠，并呈现出与野生型相似的二级结构。然而，突变体Δ88A和ΔA84/Δ88A不能与视黄醛形成发色团。Δ85A再生的发色团在暗适应和光适应状态下的吸收最大值和视黄醛异构体组成与野生型相似。在ΔA84、ΔL87和Δ85A/ΔL87中，这些发色团特性发生了改变，并且结构稳定性低于野生型，这表现为在黑暗中与羟胺反应的速率加快、酸性pH下变性的pKa增加以及席夫碱去质子化的pKa降低。在ΔA84和Δ85A/ΔL87突变体中质子转运被消除，而在Δ85A和ΔL87中，在pH 6时活性降低至野生型值的约25%。Δ85A、Δ85A/ΔL87和ΔL87突变体的总体特性表明，缺失和/或插入分别导致参与质子转运的残基Arg-82、Asp-85或Asp-96发生位移。结果与跨膜片段C的螺旋结构相符，并强调了细菌视紫红质中分子内接触的灵活性。
