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 unfolding transition at approximately 73 degrees C. Significant beta-sheet structural signal remains even to 100 degrees C. The persistent beta-sheet CD signal correlates with significant DNA binding (K(d) approximately nM range) to temperatures as high as 50 degrees C. These data confirm the ability of the DNA binding domain in the full-length "latent" protein to bind consensus dsDNA targets effectively in the absence of activators over a broad temperature range. In addition, we demonstrate that Ab1620 reactivity is not directly correlated with the functional activity of the full-length protein since loss of this epitope occurs at temperatures at which significant specific DNA binding can still be measured.
Nichols
N M
NM
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, USA.
Matthews
K S
KS
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D001666:Binding Sites, Antibody; D002942:Circular Dichroism; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D006825:Hybridomas; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA