Trieschmann M D, Pattus F, Tadros M H
Institut für Biologie II/Mikrobiologie, Universität, Freiburg, Germany.
Mol Gen Genet. 1996 Nov 27;253(1-2):253-8. doi: 10.1007/s004380050320.
The pore-forming outer-membrane protein from Rhodobacter (R.) capsulatus (wild-type B10 strain) was isolated and purified under non-denaturing conditions. The monomer unit of the isolated porin has a molecular mass of about 28 kDa, as judged by SDS-PAGE, whereas the native protein migrates at 75 kDa. This suggests that the native porin from R. capsulatus B10 exists in a trimeric form. The N-terminal amino acid sequence was used to design an oligonucleotide which was utilised to screen a pBluescript library containing EcoRI fragments of R. capsulatus B10 DNA. A 5.3-kb DNA fragment, which included the entire structural porin gene (named porCa) and its flanking regions, was identified. A 945-bp open reading frame, coding for a mature protein of 295 amino acid residues (molecular mass 30,586 Da) plus a presequence of 20 amino acids, was found. The directly determined sequence of the amino-terminus and of four tryptic peptides of the purified porin matched perfectly with the deduced amino acid sequence. Northern blot analysis showed that the porin gene encodes an RNA transcript of 1050 nucleotides. In addition, there is no differential response in terms of either the size or abundance of the mRNA under different environmental conditions. Primer extension experiments confirmed a putative promoter upstream of the porin gene; and localised the RNA transcription start site 73 bp upstream of the ATG start codon, which is close to the putative promoter (-10/-35). As shown using a plasmid-borne porin-lacZ gene fusion, [in R. capsulatus] expression of the lacZ gene under the control of the porCa promoter is not regulated under the two different environmental conditions tested. The promoter of the porin gene was localised within 305 bp upstream of the ATG start codon. A model of the 3-D structure of porin B10 was deduced by comparative modelling with the R. capsulatus 37b4 and Rhodopseudomonas blastica porin crystallographic structures using the ProMod program on the Swiss-Model protein modelling e-mail Server. Analysis of the B10 sequence and comparison of a model of the B10 porin structure with the crystallographic structure of porin from the capsuleless strain 37B4 has revealed some important differences at the level of the protein surface, the pore and the putative ligand binding site.
来自荚膜红细菌（R. capsulatus，野生型B10菌株）的形成孔道的外膜蛋白在非变性条件下被分离和纯化。通过SDS-PAGE判断，分离得到的孔蛋白单体单元的分子量约为28 kDa，而天然蛋白在75 kDa处迁移。这表明来自荚膜红细菌B10的天然孔蛋白以三聚体形式存在。利用N端氨基酸序列设计了一种寡核苷酸，用于筛选包含荚膜红细菌B10 DNA的EcoRI片段的pBluescript文库。鉴定出一个5.3 kb的DNA片段，其中包括整个孔蛋白结构基因（命名为porCa）及其侧翼区域。发现了一个945 bp的开放阅读框，编码一个由295个氨基酸残基组成的成熟蛋白（分子量30,586 Da）加上一个20个氨基酸的前导序列。纯化的孔蛋白的N端直接测定序列和四个胰蛋白酶肽段与推导的氨基酸序列完全匹配。Northern印迹分析表明，孔蛋白基因编码一个1050个核苷酸的RNA转录本。此外，在不同环境条件下，mRNA的大小或丰度没有差异反应。引物延伸实验证实了孔蛋白基因上游的一个推定启动子；并将RNA转录起始位点定位在ATG起始密码子上游73 bp处，该位点靠近推定启动子（-10/-35）。如使用质粒携带的孔蛋白-lacZ基因融合所显示的，[在荚膜红细菌中]在porCa启动子控制下的lacZ基因表达在测试的两种不同环境条件下不受调控。孔蛋白基因的启动子定位在ATG起始密码子上游305 bp范围内。使用瑞士模型蛋白质建模电子邮件服务器上的ProMod程序，通过与荚膜红细菌37b4和 Blastica红假单胞菌孔蛋白晶体结构进行比较建模，推导了孔蛋白B10的三维结构模型。对B10序列的分析以及将B10孔蛋白结构模型与无荚膜菌株37B4的孔蛋白晶体结构进行比较，揭示了在蛋白质表面、孔道和推定的配体结合位点水平上的一些重要差异。
