Swanson R V, Glazer A N
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
J Mol Biol. 1990 Aug 5;214(3):787-96. doi: 10.1016/0022-2836(90)90293-u.
The phycobiliproteins contain a conserved unique modified residue, gamma-N-methylasparagine at beta-72. This study examines the consequences of this methylation for the structure and function of phycocyanin and of phycobilisomes. An assay for the protein asparagine methylase activity was developed using [methyl-3H]S-adenosylmethionine and apophycocyanin purified from Escherichia coli containing the genes for the alpha and beta subunits of phycocyanin from Synechococcus sp. PCC 7002 as substrates. This assay permitted the partial purification, from Synechococcus sp. PCC 6301, of the activity that methylates phycocyanin and allophycocyanin completely at residue beta-72. Using the methylase assay, two independent nitrosoguanidine-induced mutants of Synechococcus sp. PCC 7942 were isolated that do not exhibit detectable phycobiliprotein methylase activity. These mutants, designated pcm 1 and pcm 2, produce phycocyanin and allophycocyanin unmethylated at beta-72. The phycobiliproteins in these mutants are assembled into phycobilisomes and can be methylated in vitro by the partially purified methylase from Synechococcus sp. PCC 6301. The mutants produce phycobiliproteins in amounts comparable to those of wild-type and the mutant and wild-type phycocyanins are equivalent with respect to thermal stability profiles. Monomeric phycocyanins purified from these strains show small spectral shifts that correlate with the level of methylation. Phycobilisomes from the mutant strains exhibit defects in energy transfer, both in vivo and in vitro, that are also correlated with deficiencies in methylation. Unmethylated or undermethylated phycobilisomes show greater emission from phycocyanin and allophycocyanin and lower fluorescence emission quantum yields than do fully methylated particles. The results support the conclusion that the site-specific methylation of phycobiliproteins contributes significantly to the efficiency of directional energy transfer in the phycobilisome.
藻胆蛋白含有一个保守的独特修饰残基，即β-72位的γ-N-甲基天冬酰胺。本研究考察了这种甲基化对藻蓝蛋白和藻胆体的结构与功能的影响。利用[甲基-³H]S-腺苷甲硫氨酸和从含有集胞藻属PCC 7002藻蓝蛋白α和β亚基基因的大肠杆菌中纯化得到的脱辅基藻蓝蛋白作为底物，开发了一种蛋白质天冬酰胺甲基转移酶活性测定方法。该测定方法使得能够从集胞藻属PCC 6301中部分纯化出能在β-72位将藻蓝蛋白和别藻蓝蛋白完全甲基化的活性物质。利用该甲基转移酶测定方法，分离出了集胞藻属PCC 7942的两个独立的亚硝基胍诱导突变体，它们不表现出可检测到的藻胆蛋白甲基转移酶活性。这些突变体命名为pcm 1和pcm 2，它们产生的藻蓝蛋白和别藻蓝蛋白在β-72位未被甲基化。这些突变体中的藻胆蛋白组装成藻胆体，并且在体外可被从集胞藻属PCC 6301中部分纯化得到的甲基转移酶甲基化。这些突变体产生的藻胆蛋白数量与野生型相当，并且突变型和野生型藻蓝蛋白在热稳定性方面相当。从这些菌株中纯化得到的单体藻蓝蛋白显示出与甲基化水平相关的小光谱位移。突变菌株的藻胆体在体内和体外的能量转移方面均表现出缺陷，这也与甲基化不足相关。未甲基化或甲基化不足的藻胆体比完全甲基化的颗粒显示出更高的藻蓝蛋白和别藻蓝蛋白发射以及更低的荧光发射量子产率。结果支持这样的结论，即藻胆蛋白的位点特异性甲基化对藻胆体中定向能量转移的效率有显著贡献。
