A directed approach for engineering conditional protein stability using biologically silent small molecules

Lystranne A. Maynard-Smith, Ling Chun Chen, Laura A. Banaszynski, A. G.Lisa Ooi, Thomas J. Wandless

Research output: Contribution to journalArticlepeer-review

54 Scopus citations


The ability to regulate the function of specific proteins using cell-permeable molecules can be a powerful method for interrogating biological systems. To bring this type of "chemical genetic" control to a wide range of proteins, we recently developed an experimental system in which the stability of a small protein domain expressed in mammalian cells depends on the presence of a high affinity ligand. This ligand-dependent stability is conferred to any fused partner protein. The FK506- and rapamycin-binding protein (FKBP12) has been the subject of extensive biophysical analyses, including both kinetic and thermodynamic studies of the wild-type protein as well as dozens of mutants. The goal of this study was to determine if the thermodynamic stabilities (ΔΔGU-F) of various amino acid substitutions within a given protein are predictive for engineering additional ligand-dependent destabilizing domains. We used FKBP12 as a model system and found that in vitro thermodynamic stability correlates weakly with intracellular degradation rates of the mutants and that the ability of a given mutation to destabilize the protein is context-dependent. We evaluated several new FKBP12 ligands for their ability to stabilize these mutants and found that a cell-permeable molecule called Shield-1 is the most effective stabilizing ligand. We then performed an unbiased microarray analysis of NIH3T3 cells treated with various concentrations of Shield-1. These studies show that Shield-1 does not elicit appreciable cellular responses.

Original languageEnglish (US)
Pages (from-to)24866-24872
Number of pages7
JournalJournal of Biological Chemistry
Issue number34
StatePublished - Aug 24 2007

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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