Post-Graduate Department of Biosciences, Sardar Patel University, Bakrol, Anand, Gujarat, 388 315, India.
Małopolska Centre of Biotechnology, Jagiellonian University, 30-387, Kraków, Poland.
Photosynth Res. 2020 Jun;144(3):349-360. doi: 10.1007/s11120-020-00746-7. Epub 2020 Apr 17.
The crystal structure of phycocyanin (pr-PC) isolated from Phormidium rubidum A09DM (P. rubidum) is described at a resolution of 1.17 Å. Electron density maps derived from crystallographic data showed many clear differences in amino acid sequences when compared with the previously obtained gene-derived sequences. The differences were found in 57 positions (30 in α-subunit and 27 in β-subunit of pr-PC), in which all residues except one (β145Arg) are not interacting with the three phycocyanobilin chromophores. Highly purified pr-PC was then sequenced by mass spectrometry (MS) using LC-MS/MS. The MS data were analyzed using two independent proteomic search engines. As a result of this analysis, complete agreement between the polypeptide sequences and the electron density maps was obtained. We attribute the difference to multiple genes in the bacterium encoding the phycocyanin apoproteins and that the gene sequencing sequenced the wrong ones. We are not implying that protein sequencing by mass spectrometry is more accurate than that of gene sequencing. The final 1.17 Å structure of pr-PC allows the chromophore interactions with the protein to be described with high accuracy.
从红藻 Phormidium rubidum A09DM（P. rubidum）中分离得到的藻蓝蛋白（pr-PC）的晶体结构在 1.17Å 的分辨率下进行了描述。与之前获得的基因衍生序列相比，从晶体学数据得出的电子密度图显示氨基酸序列有许多明显的差异。这些差异出现在 57 个位置（pr-PC 的α亚基中有 30 个，β亚基中有 27 个），除了一个残基（β145Arg）外，所有残基都不与三个藻胆素发色团相互作用。然后使用 LC-MS/MS 通过质谱（MS）对高度纯化的 pr-PC 进行测序。使用两个独立的蛋白质组学搜索引擎分析 MS 数据。通过该分析，获得了多肽序列与电子密度图之间的完全一致。我们将差异归因于编码藻蓝蛋白脱辅基蛋白的细菌中的多个基因，并且基因测序测序了错误的基因。我们并不是说质谱法进行蛋白质测序比基因测序更准确。最终的 1.17Å pr-PC 结构允许高精度地描述发色团与蛋白质的相互作用。
