Protein-DNA binding correlates with structural thermostability for the full-length human p53 protein

Full-length p53 protein purified from Escherichia coli in the unmodified, " latent" form was examined by several methods to correlate thermal stability of structure with functional DNA binding. Structure prediction algorithms indicate that the majority of beta -sheet structure occurs in the p53 core DNA binding domain. Circular dichroism spectra demonstrate that the intact protein is surprisingly stable with a midpoint for the irreversible unfoldi ng transition at similar to 73 degreesC. Significant beta -sheet structural signal remains even to 100 degreesC. The persistent beta -sheet CD signal correlates with significant DNA binding (K-d similar to nM range) to temper atures as high as 50 degreesC. These data confirm the ability of the DNA bi nding domain in the full-length "latent" protein to bind consensus dsDNA ta rgets effectively in the absence of activators over a broad temperature ran ge. In addition, we demonstrate that Ab1620 reactivity is not directly corr elated with the functional activity of the full-length protein since loss o f this epitope occurs at temperatures at which significant specific DNA bin ding can still be measured.