Poster abstracts
Poster number 16 submitted by David Bowles
Modifying the Stability of Tumor Suppressor p53 Through S7-S8 Loop Mutagenesis as Suggested by Comparison of Human and Worm Structures.
David Bowles (OSBP), David Rabinovich (Undergraduate Researcher)
Abstract:
The tumor suppressor p53 is a critical 54 kDa protein responsible for the regulation of cellular differentiation during times of cellular stress. This protein is so critical is to be found to be mutated in approximately 50% of all human cancer cases. The majority of these mutations are found in the central, ordered DNA-binding domain of the protein. One way in which these mutations can disrupt DNA binding is to lower the overall stability of the protein, making it unable to properly interact with DNA. Consequently, any knowledge that can be gained in stabilizing the p53 DNA-binding domain could potentially be very valuable. It has been observed that the p53 homolog in C. elegans is considerably more stable than its human counterpart. By comparing the sequences of human p53 and worm p53, it was proposed that shortening the S7S8 turn is an important factor in this stability.(1) This initial work was done in silico, and the Magliery lab is constructing mutants based on this hypothesis to be expressed and screened in vitro. Compared to human p53, worm p53 has four fewer residues in the S7S8 loop. By expressing and screening various four- and five- residue deletion mutants, insight was gained into which residues are important in the loop. These results will be used to design a loop mutant library that will probe sequence space for mutants that are more stable than the control construct. There is also evidence that the final few residues of the disordered N-terminal domain of p53 has a profound effect on p53 solubility and a moderate effect on stability.(2) Potential effects of this change to the DNA binding domain of p53 have been explored. In addition to the worm p53 comparison strategy, algorithmic improvement of p53 has been attempted using methodology developing in the Magliery lab.(3) The resulting construct is algorithm derived p53, or Algo p53, has been expressed and characterized.
References:
1. Pan, Y.; Ma, B.; Levine, A. J.; Nussinov, R., Comparison of the human and worm p53 structures suggests a way for enhancing stability. Biochemistry 2006, 45 (12), 3925-33.
2. Wells, M.; Tidow, H.; Rutherford, T. J.; Markwick, P.; Jensen, M. R.; Mylonas, E.; Svergun, D. I.; Blackledge, M.; Fersht, A. R., Structure of tumor suppressor p53 and its intrinsically disordered N-terminal transactivation domain. Proc Natl Acad Sci U S A 2008, 105 (15), 5762-7.
3. Durani, V.; Magliery, T. J., Protein engineering and stabilization from sequence statistics: variation and covariation analysis. Methods Enzymol 2013, 523, 237-56.
Keywords: Protein Engineering, Protein Dynamics, Cancer-related protein