Tumor Suppressor p53: Loop Swapping and Mutagenesis to Improve Stability
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Date
2009-06
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The Ohio State University
Abstract
The tumor suppressor protein p53 is a transcription factor that responds to damaged DNA by initiating cell cycle arrest or apoptosis. However, p53 is a rather unstable protein with 95% of mutations affecting its function occurring on the DNA binding core domain. When p53 loses its function (often from an alteration in stability), it may lead to the development of cancer and, in fact, is associated with more than half of all human cancers. Regional stability of the p53 core domain has been calculated by Nussinov and coworkers and compared to that of the C. elegans homolog. This experiment revealed that turn S7S8 of human p53 (p53h) elicited high rates of mobility relative to that of the C. elegans p53 (p53w), which has a shorter S7S8 loop. It may be possible to increase the stability of p53h without perturbing DNA binding and other protein-protein interactions via alteration of this region. In the computational experiment, molecular dynamics simulations showed that by decreasing the length of turn S7S8, that not only was the stability of the peripheral motif enhanced, but also the stability of the entire protein was increased. However, our lab found that the loop deletion mutant fails to fold. Further probing of this region and this hypothesis are needed to fully understand the role of S7S8 in the folding pathway of p53c and its ability to produce stable mutants. Rational design is employed by inserting flexible residues into the shortened loop along with a chimeric mutant in which the worm’s turn sequence is directly inserted into the human turn. Mutants have been tested with a GFP based screen that tests for binding, chemical denaturation to test for stability, and anisotropy to test for the strength of binding to the target DNA. The culmination of these experiments has yielded a p53c loop deletion variant that is structured and active with KD values close to that of published data for other, stable p53c mutants.
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Keywords
p53, Mutagenesis