Structure/function studies of humanp53 protein
Nichols, Nicole Magnasco
Matthews, Kathleen S.
Doctor of Philosophy
Cellular p53 response, including apoptosis and cell cycle arrest, is reportedly due to "activation" of otherwise "latent" protein, a process that has been mimicked in vitro by the use of agents that interact with the p53 C-terminal domain. We have therefore undertaken a biochemical investigation of the wild-type protein and an "activated" C-terminal mutant (S392E) to characterize the in vitro structure and function of the "latent" and "activated" forms of human p53 expressed in Escherichia coli. Differential reactivity was observed for mutant and wild-type p53 to the DNA binding domain antibody, Ab1620. Reactivity with Ab1620 is not directly correlated with p53 functional activity as previously thought, since loss of this epitope occurs at temperatures at which specific DNA binding can still be measured. Fluorescence studies established that tryptophans in wild-type and S392E p53 are quenched but DNA binding and potential conformational changes due to Ab1620 binding result in increased fluorescence signal. Structure prediction algorithms indicate that the great majority of beta-sheet structure occurs in the p53 core DNA binding domain. Circular dichroism spectra demonstrate significant stability for the wild-type protein, with a transition midpoint of ∼73°C. The "activated" mutant, S392E, displays increased stability with no detectable loss of beta-sheet structure even at 100°C. Increased alpha-helical structure is also observed for the mutant protein. The persistent beta-sheet CD signal of both proteins correlates with significant DNA binding (Kd∼nM range) to temperatures as high as 50°C. Surprisingly, the S392E mutant showed no increase in affinity for specific DNA compared to wild-type p53, although binding was maintained to a higher degree over the temperature range investigated. Interestingly, in comparison with wild-type p53, the mutant protein displays increased affinity for nonspecific DNA and for a DNA target containing both specific and nonspecific sequences. These data confirm the thermostability of both structure and specific DNA binding for both the "latent" and "activated" proteins and suggest that activation of p53 may derive from alterations in nonspecific binding by the C-terminal domain.
Cell biology; Biochemistry