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

我们已经报道了一种新的高氯酸可溶性蛋白（PSP）的分离和鉴定大鼠肝细胞溶质部分。PSP是一种同源二聚体，每个亚基由136个氨基酸残基组成，分子量为14，149 Da。它以不同于RNase A的方式抑制兔网织红细胞裂解物中的无细胞蛋白质合成。最近，从人单核细胞和小鼠肝脏中鉴定了一种14 kDa的抑制翻译的蛋白质。此外，编码14-kDa蛋白和PSP的互补DNA（cDNA）序列与编码一个新的功能未知的小蛋白家族（YER 057 c/YJGF）的cDNA序列高度相似。因此，PSP样蛋白的序列在原核生物、蓝藻、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 ...更多信息 提示这些蛋白在细胞调控中起着重要作用，尽管PSP的信息体不断增加，但目前还没有关于其三维结构的报道。本文首先构建了一个高效表达系统，用于生产高氯酸可溶性蛋白（PSP）。将编码PSP的cDNA插入到可诱导的细菌表达载体pGEX-4 T-1中。将该质粒导入大肠杆菌中，经异戊二醇（IPTG）表达后，用谷胱甘肽-琼脂糖4 B亲和层析纯化重组产物。纯化的产物显示预期的NH_2-末端序列，但该产物的翻译抑制活性比从大鼠肝中分离的真实PSP低10倍。以分子量为8000 Da的聚乙二醇为沉淀剂，采用悬滴法制备了粒径为0.5 mm的八面体晶体。这些晶体在内部X射线源上可达到2.44“A”，在ESRE Grenoble使用弯曲的Agnet光束线可达到1.8“A”。晶体属于立方空间群P213，晶胞参数a=B=c=89.90“A”，每个不对称单元有两个分子，VM值为2.12“A“3/Da，自旋转函数计算表明。对重原子衍生物的筛选确定了与蛋白质上的单个位点结合的铂化合物。对重原子衍生物的进一步筛选表明了两侧为两个α螺旋的β折叠。结构相似性给出了最佳命中ftsz（信号识别颗粒受体）、分支酸酯和热休克同源蛋白70 kDa。少

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