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首页> 外文期刊>Journal of Molecular Biology >Equilibrium folding properties of the yeast prion protein determinant Ure2.
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Equilibrium folding properties of the yeast prion protein determinant Ure2.

机译：酵母病毒决定因子Ure2的平衡折叠特性。

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The yeast non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The [URE3] phenotype is equivalent to loss of function of Ure2, a protein involved in regulation of nitrogen metabolism. The prion-like behaviour of Ure2 in vivo is dependent on the first 65 amino acid residues of its N-terminal region which contains a highly repetitive sequence rich in asparagine. This region has been termed the prion-determining domain (PrD). Removal of as little as residues 2-20 of the protein is sufficient to prevent occurrence of the [URE3] phenotype. Removal of the PrD does not affect the regulatory activity of Ure2. The C-terminal portion of the protein has homology to glutathione S -transferases, which are dimeric proteins. We have produced the Ure2 protein to high yield in Escherichia coli from a synthetic gene. The recombinant purified protein is shown to be a dimer. The stability, folding and oligomeric state of Ure2 and a series of N-terminally truncated or deleted variants were studied and compared. The stability of Ure2, DeltaGD-N, H2O, determined by chemical denaturation and monitored by fluorescence, is 12.1(+/-0.4) kcal mol-1at 25 degrees C and pH 8.4. A range of structural probes show a single, coincident unfolding transition, which is invariant over a 550-fold change in protein concentration. The stability is the same within error for Ure2 variants lacking all or part of the prion-determining domain. The data indicate that in the folded protein the PrD is in an unstructured conformation and does not form specific intra- or intermolecular interactions at micromolar protein concentrations. This suggests that the C-terminal domain may stabilise the PrD against prion formation by steric means, and implies that the PrD does not induce prion formation by altering the thermodynamic stability of the folded protein. Copyright 1999 Academic Press.
机译：酵母非孟德尔因子[URE3]通过a病毒样机制传播，涉及染色体编码的蛋白Ure2的聚集。 [URE3]表型等同于Ure2的功能丧失，Ure2是一种参与氮代谢调节的蛋白质。 Ure2在体内的病毒样行为取决于其N端区域的前65个氨基酸残基，该残基包含富含天冬酰胺的高度重复序列。该区域被称为the病毒决定结构域（PrD）。除去少至2-20个残基足以防止[URE3]表型的出现。去除PrD不会影响Ure2的调节活性。蛋白质的C末端部分与谷胱甘肽S-转移酶具有同源性，后者是二聚体蛋白质。我们已经通过合成基因在大肠杆菌中生产了高产量的Ure2蛋白。重组纯化的蛋白质显示为二聚体。研究和比较了Ure2和一系列N末端截短或缺失的变体的稳定性，折叠状态和寡聚状态。通过化学变性确定并通过荧光监测的Ure2，DeltaGD-N，H2O的稳定性在25摄氏度和pH 8.4下为12.1（+/- 0.4）kcal mol-1。一系列结构探针显示出一个单一的，同时发生的未折叠转变，在蛋白质浓度变化550倍时不变。对于缺少全部或部分the病毒决定域的Ure2变体，稳定性在误差范围内是相同的。数据表明，在折叠的蛋白质中，PrD处于无结构构象，并且在微摩尔蛋白质浓度下不形成特定的分子内或分子间相互作用。这表明C-末端结构域可以通过空间手段使PrD稳定以防止病毒形成，并且暗示PrD不会通过改变折叠蛋白的热力学稳定性来诱导病毒形成。版权所有1999，学术出版社。

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