Babst M, Albrecht H, Wegmann I, Brunisholz R, Zuber H
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Zürich, Switzerland.
Eur J Biochem. 1991 Dec 5;202(2):277-84. doi: 10.1111/j.1432-1033.1991.tb16373.x.
To obtain information on the structural and functional role of highly conserved amino acid residues in the B870 alpha and beta light-harvesting polypeptides of Rhodobacter capsulatus, site-directed mutagenesis was performed. 18 mutants with single amino acid substitutions at nine different positions in the B870 antenna polypeptides were prepared in a B800-850-lacking strain. The characterization of the resulting phenotypes was based on a quantification of the core-complex elements (reaction center, light-harvesting polypeptides, bacteriochlorophyll a and carotenoid) and the core-complex spectral characteristics (absorption maximum, absorption coefficient and fluorescence intensity). These data generally showed that strong structural effects were caused by the amino acid substitutions. Thus, the three tryptophan exchanges at the position alpha 8 resulted in either the absence of a core complex (alpha Trp8----Leu), the absence of the core antenna (alpha Trp8----Ala) or a reduction in the carotenoid content (alpha Trp8----Tyr). Likewise, the mutants alpha Pro13Gly (i.e. alpha Pro13----Gly), beta Gly10Val and alpha Phe23Ala demonstrated an abnormal protein/pigment ratio in the core antenna, while a drastically reduced antenna size resulted from the amino acid exchange beta Arg45Asp. In contrast to the structural effects, the absorption maxima and the fluorescence intensities of the mutant antennae differed only slightly from the wild type. The strongest blue shift of the bacteriochlorophyll a (8-11 nm) was induced by substitutions of the Trp at position alpha 43 (alpha Trp43----Ala, Leu or Tyr). Contrary to the other spectral effects, the absorption coefficient of bacteriochlorophyll a was strongly influenced by the amino acid substitutions and varied by 1.6-times less (beta Arg45Asp) and 1.3-times greater (alpha Phe25Ala) than normal. The antenna-free mutant, alpha Trp8Ala, yielded a high rate of B800-850 revertants during phototrophic growth, indicating a direct energy transfer from the B800-850 antenna to the reaction center in these strains. Although conditions for growth were generally observed to influence phenotypic expression, the structural as well as spectral effects were demonstrated to differ to the greatest extent between chemotrophically grown and phototrophically grown cells.
为了获取有关荚膜红细菌B870α和β捕光多肽中高度保守氨基酸残基的结构和功能作用的信息，进行了定点诱变。在一个缺乏B800 - 850的菌株中制备了18个在B870天线多肽的9个不同位置有单个氨基酸替换的突变体。对所得表型的表征基于对核心复合物成分（反应中心、捕光多肽、细菌叶绿素a和类胡萝卜素）的定量以及核心复合物光谱特征（最大吸收波长、吸收系数和荧光强度）。这些数据总体上表明氨基酸替换会引起强烈的结构效应。因此，α8位置的三个色氨酸交换导致要么没有核心复合物（αTrp8→Leu），要么没有核心天线（αTrp8→Ala），要么类胡萝卜素含量降低（αTrp8→Tyr）。同样，突变体αPro13Gly（即αPro13→Gly）、βGly10Val和αPhe23Ala在核心天线中表现出异常的蛋白质/色素比，而氨基酸交换βArg45Asp导致天线尺寸大幅减小。与结构效应相反，突变天线的最大吸收波长和荧光强度与野生型仅略有不同。α43位置色氨酸的替换（αTrp43→Ala、Leu或Tyr）诱导了细菌叶绿素a最强的蓝移（8 - 11 nm）。与其他光谱效应相反，细菌叶绿素a的吸收系数受氨基酸替换的强烈影响，变化比正常情况小1.6倍（βArg45Asp）和大1.3倍（αPhe25Ala）。无天线突变体αTrp8Ala在光养生长过程中产生了高比例的B800 - 850回复突变体，表明在这些菌株中从B800 - 850天线到反应中心存在直接的能量转移。尽管通常观察到生长条件会影响表型表达，但结构和光谱效应在化能营养生长细胞和光养生长细胞之间的差异最为显著。
