Structural studies of perchloric acid soluble protein (PSP) from rat liver.

大鼠肝脏高氯酸可溶性蛋白（PSP）的结构研究。

基本信息

批准号：
09680594
负责人：
OKA Tatsuzo
金额：
$ 0.51万
依托单位：
Kagoshima University (1998)The University of Tokushima (1997)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680594/
关键词：
Rat Perchloric acid-soluble protein Escherichia coli Recombinant Crystallization Higher structure 蛋白質合成阻害

项目摘要

We have reported isolation and characterization of a novel perchloric acid-soluble protein (PSP) in the cytosolic fraction of rat liver. The PSP is a homodimer, each subunit consisting of 136 amino acid residues with a molecular mass of 14,149 Da. It inhibits cell-free protein synthesis in the lysate of rabbit reticulocyte in a different manner than RNase A.Recently, a 14-kDa protein which inhibit translation was characterized from human monocyte and mouse liver. The 14-kDa proteins and their mRNAs were reported to be preferentially expressed in the liver and kidney as in the case of PSP.Furthermore, the sequences of complementary DNA (cDNA) encoding the 14-kDa protein and PSP are highly similar to those of cDNA encoding a new hypothetical family (YER057c/YJGF) of small proteins with presently unknown function. Thus the sequences of PSP-like proteins are highly conserved in prokaryotic, cyanobacteria, fungi and eukaryotes. The high degree of evolutionary conservation of these proteins … More suggests that these proteins play an important role in cellular regulation.Despite the growing body of information gor PSPs, no three-dimensional structure has benn reported. In the following contribution was present the crystallization and preliminary crystallographic characterization of PSP.At first, an efficient E.coli expression system for the production of a perchloric acid-soluble protein (PSP) has been constructed. cDNA encoding PSP was inserted into an inducible bacterial expression vector pGEX-4T-1. After the plasmid introduced into E.coli was expressed by IPTG, the recombinant product was purified by glutathione-Sepharose 4B affinity chromatography. The purified product showed the expected NH2-terminal sequence, but the translation inhibitory activity of this product was 10 times lower compared with that of authentic PSP isolated from rat liver.PSP from rat liver has been crystallized in a form suitable for high resolution X-ray diffraction studies. Octahedral crystals reaching 0.5 mm in size were produced by hangin drop method using polyethylene glycol (Mr=8000 Da) as precipitant. These crystals diffract to 2.44 "A" on an in-house X-ray source and to l.8"A" using a bending, agnet beamline at ESRE Grenoble. The crystals belong to the cubic space group P213 with unit cell parameters a=b=c=89.90"A" and two molecules per asymmetric unit, as indicated from VM value of 2.12"A"3/Da and self-rotation function computation. Screening for heavy-atom derivatives identified a platinum compound that binds to a single site on the protein. Furhter screening for heavy-atom derivatives indeicated a beta sheet flanled by two alpha helice. The structural similarity gave the best hit ftsz (signal recognition particle receptor), chorismate mutase and heat-shock cognate protein 70 kDa. Less

我们报道了大鼠肝脏的胞质分数中新型高氯酸化蛋白（PSP）的分离和表征。 PSP是同型二聚体，每个亚基由136个氨基酸组成，分子质量为14,149 Da。它以与RNase不同的方式抑制兔网状细胞裂解物中无细胞的蛋白质合成。据报道，14 kDa蛋白及其mRNA在肝脏和肾脏中优选表达，如psp.furthermore而言，编码14-kDa蛋白和PSP的互补DNA（cDNA）序列与编码新的假设家族（yer057c/yjgf）的cDNA高度相似。 PSP样蛋白的序列在原核，蓝细菌，真菌和真核生物中高度保守。这些蛋白质的高度进化保守性……更多表明这些蛋白质在细胞调节中起着重要作用。尽管信息不断增长，但没有三维结构的信息。在以下贡献中存在PSP.的结晶和初步的晶体表征，首先是一种有效的大肠杆菌表达系统，用于生产高氯酸溶解蛋白（PSP）。将编码PSP的cDNA插入诱导型细菌表达载体PGEX-4T-1中。在引入大肠杆菌的质粒通过IPTG表达后，重组产物通过谷胱甘肽-Sepharose 4B亲和色谱纯化。纯化的产物显示了预期的NH2末端序列，但是与从大鼠肝脏中分离出的正宗PSP相比，该产品的翻译抑制活性降低了10倍。从大鼠肝脏中分离出的真实PSP已以适用于高分辨率X射线衍射研究的形式结晶。使用聚乙烯乙二醇（MR = 8000 DA）作为沉淀剂，通过Hangin Drop方法产生达到0.5 mm的八面体晶体。这些晶体在内部X射线源上衍射到2.44“ A”，并使用ESRE GRENOBLE的弯曲的Agnet Beamine在eSREBLEBLE上脱落。晶体属于具有单位单元参数的立方空间群P213 a = b = c = 89.90“ a”和每个非对称单位的两个分子，如VM值2.12“ A” 3/DA和自移位功能计算。对重原子衍生物的筛选确定了与蛋白质上单个位点结合的铂化合物。对重原子衍生物的筛选倍增了一个由两个alpha helice斑驳的β板。结构相似性给出了最佳的命中FTSZ（信号识别）颗粒受体），基质酸突变酶和热震的同源蛋白70 kDa。较少的