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

Mayer S, Rüdiger S, Ang HC, Joerger AC, Fersht AR

Cambridge University Chemical Laboratory and Cambridge Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge CB2 0QH, UK.

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.

Published 16 August 2007 in J Mol Biol, 372(1): 268-76.
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