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Biophysical analysis of Wt and mutant p53 core domain.

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posted on 20.02.2013, 12:21 by Xavier Bernard, Philip Robinson, Yves Nominé, Murielle Masson, Sebastian Charbonnier, Juan Ramon Ramirez-Ramos, Francois Deryckere, Gilles Travé, Georges Orfanoudakis

(A) Comparison of different p53 core domain proteins (WT, L265A, Y103G) with respect to secondary structure content by CD. The spectra were recorded in 20 mM sodium phosphate (pH 6.8), 20 mM NaCl, 2 mM DTT at 10°C. The far-UV spectrum of the p53wt and p53Y103G are similar and show characteristics of folded proteins with a minimum at 201 nm while the spectrum of the L265A p53 core domain suggests a large proportion of unfolded protein as indicated by the shift of the minimum towards smaller wavelength and a negative signal at 200 nm. (B) Thermal denaturation of the p53 core domain proteins monitored by far-UV CD spectroscopy at 210 nm. The spectra were recorded in 20 mM sodium phosphate (pH 6.8), 50 mM NaCl, 2 mM DTT. For clarity, spectra have been offset by 10 mdeg between each curve. (C) 1H-15N correlation spectra of p53 core domain (residues 94–312) acquired at 10°C on a Bruker DRX600 spectrometer equipped with a z-gradient triple resonance cryoprobe. The p53WT core domain is represented in black (left panel), the p53L265A core domain in red (middle panel) and the p53Y103G core domain in blue (right panel).

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