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Correlation of levels of folded recombinant p53 in Escherichia coli with thermodynamic stability in vitro

机译：大肠杆菌中重组p53折叠水平与体外热力学稳定性的相关性

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The amount of folded functional protein in a cell is controlled by a number of factors, including the relative rates of its biosynthetic and specific degradation processes, and its intrinsic thermodynamic stability. Mutation-induced loss of stability is a common cause of disease. Many oncogenic mutants of the tumour suppressor p53, for example, reduce the intrinsic thermodynamic stability of the protein in vitro. We have analysed the level of recombinant folded human p53 core domain (p53C) and its mutants in Escherichia coli spanning a stability range of 6 kcal/mol to assess the effects of intrinsic thermodynamic stability in vivo in the absence of specific ubiquitin-mediated pathways in human cells. The levels of folded protein were measured fluorimetrically in living cells by fusing the gene of p53C upstream to that of green fluorescent protein and measuring the fluorescence relative to a control at various temperatures. At a fixed temperature, the amount of fluorescence is correlated with the thermodynamic stability of the mutant. The level of each protein varied with temperature according to a sigmoid curve that paralleled the melting in vitro, but the apparent T-m,, was lower in vivo, because steady-state levels are observed rather than true thermodynamic equilibria. Our results show clearly that changes in the intrinsic thermodynamic stability of p53 reduce the level of folded and hence functional p53 substantially in E. coli, and provide insights into the correlation between protein instability and disease at the cellular level. (c) 2007 Elsevier Ltd. All rights reserved.
机译：细胞中功能蛋白折叠的数量受多种因素控制，包括其生物合成和特定降解过程的相对速率及其固有的热力学稳定性。突变引起的稳定性丧失是疾病的常见原因。例如，抑癌基因p53的许多致癌突变体会降低蛋白质在体外的固有热力学稳定性。我们已经分析了大肠杆菌中重组折叠的人p53核心结构域（p53C）及其突变体的水平，其稳定范围为6 kcal / mol，以评估在缺乏特定泛素介导的途径的情况下体内固有的热力学稳定性的影响。人类细胞。通过将上游的p53C基因融合到绿色荧光蛋白的基因上，并在各种温度下相对于对照测量荧光，对活细胞中的折叠蛋白水平进行了荧光测定。在固定温度下，荧光量与突变体的热力学稳定性相关。每种蛋白质的水平根据与体外融化平行的S形曲线随温度变化，但由于体内观察到稳态水平而非真正的热力学平衡，因此在体内表观T-m较低。我们的结果清楚地表明，p53固有热力学稳定性的变化可大大降低大肠杆菌中折叠后的功能p53的水平，从而在细胞水平上提供蛋白质不稳定与疾病之间相关性的见识。 （c）2007 Elsevier Ltd.保留所有权利。

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