A split-ubiquitin-based assay detects the influence of mutations on the conformational stability of the p53 DNA binding domain in vivo

FEBS Lett. 2002 Nov 6;531(2):259-64. doi: 10.1016/s0014-5793(02)03533-0.

Abstract

Many mutations in the human tumor suppressor p53 affect the function of the protein by destabilizing the structure of its DNA binding domain. To monitor the effects of those mutations in vivo the stability of the DNA binding domain of p53 and some of its mutants was investigated with the split-ubiquitin (split-Ub) method. The split-Ub-derived in vivo data on the relative stability of the mutants roughly correlate with the quantitative data from in vitro denaturation experiments as reported in the literature. A variation of this assay allows visualizing the difference in stability between the wild-type p53 core and the mutant p53(V143A) by a simple growth assay.

MeSH terms

  • Cell Division
  • DNA / metabolism
  • Genetic Techniques*
  • Models, Biological
  • Mutation
  • Protein Conformation
  • Protein Folding
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / analysis
  • Saccharomyces cerevisiae / genetics
  • Tumor Suppressor Protein p53 / chemistry*
  • Tumor Suppressor Protein p53 / genetics*
  • Tumor Suppressor Protein p53 / metabolism
  • Ubiquitin / genetics*
  • Ubiquitin / metabolism

Substances

  • Recombinant Fusion Proteins
  • Tumor Suppressor Protein p53
  • Ubiquitin
  • DNA