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Most proteins have not evolved for maximal thermal stability. Some are only marginally stable, as for example, the DNA-binding domains of p53 and its homologs, whose kinetic and thermodynamic stabilities are strongly correlated. Here, we applied high-throughput methods using a real-time PCR thermocycler to study the stability of several full-length orthologs and paralogs of the p53 family of transcription factors, which have diverse functions, ranging from tumour suppression to control of developmental processes. From isothermal denaturation fluorimetry and differential scanning fluorimetry, we found that full-length proteins showed the same correlation between kinetic and thermodynamic stability as their isolated DNA-binding domains. The stabilities of the full-length p53 orthologs were marginal and correlated with the temperature of their organism, paralleling the stability of the isolated DNA-binding domains. Additionally, the paralogs p63 and p73 were significantly more stable and long-lived than p53. The short half-life of p53 orthologs and the greater persistence of the paralogs may be biologically relevant.
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???displayArticle.pmcLink???PMC3480436 ???displayArticle.link???PLoS One ???displayArticle.grants???[+]
Figure 2. ITDF results for QM-Hsp53 constructs.A: Measurement at 38°C, full-length QM-Hsp53 in black and a construct missing the C-terminus (QM-Hsp53NCT, red) exhibit the second, faster unfolding event. Constructs missing the N-terminus (QM-Hsp53CTC, blue; QM-Hsp53CT, green) do not exhibit any significant unfolding. B: Measurement at 45°C using the same colour coding as in ‘A’. Fluorescence drops are only seen for constructs missing the N-terminus of p53.
Figure 3. Kinetic stabilisation of p53.A: Raw data for QM-p53 alone (black), with added heparin (red) and with added DNA (0.4 µM, blue; 5 µM, green). B: Comparison of half-lives of QM-p53 at different NaCl concentration without (black) and with 10% glycerol (red).
Figure 4. Thermodynamic stability determination.A: Examples of raw data melting curves for p53 orthologs derived from DSF experiments. Tm is defined as the maximum of the first derivative. B: DSF melting curves for p53 paralogs. C: Stacked view of DSC melting curves. D: Correlation of melting temperatures derived by DSF and DSC.
Figure 5. Overview of protein melting temperatures (small squares) within the p53 family.p53 orthologs (filled squares; Hsp53 in black, Mmp53 in red, Dmp53 in orange, Drp53 in blue and Xlp53 in green) and p53 paralogs (hollow squares) are shown. In corresponding colours body temperatures of homeothermic organisms are indicated as lines and optimal development conditions of poikilothermic organisms are illustrated as large rectangles.
Figure 6. Correlation between kinetic (T15, unfolding of DBD) and thermodynamic (Tm, DSF) stability data.Mammalian p53 proteins studied are represented by black squares, human p63 isoforms by blue triangles, human p73 constructs by green diamonds and Dmp53 by a red circle.
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