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 journalArticle

38 Citations (Scopus)

Abstract

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
Volume282
Issue number34
DOIs
StatePublished - Aug 24 2007

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Protein Stability
Tacrolimus Binding Protein 1A
Ligands
Molecules
Thermodynamics
Tacrolimus Binding Proteins
Proteins
Tissue Array Analysis
Thermodynamic stability
Protein Engineering
Amino Acid Substitution
Biological systems
Mutant Proteins
Microarrays
Substitution reactions
Cells
Mutation
Amino Acids
Degradation
Kinetics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A directed approach for engineering conditional protein stability using biologically silent small molecules. / Maynard-Smith, Lystranne A.; Chen, Ling Chun; Banaszynski, Laura A.; Ooi, A. G.Lisa; Wandless, Thomas J.

In: Journal of Biological Chemistry, Vol. 282, No. 34, 24.08.2007, p. 24866-24872.

Research output: Contribution to journalArticle

Maynard-Smith, Lystranne A. ; Chen, Ling Chun ; Banaszynski, Laura A. ; Ooi, A. G.Lisa ; Wandless, Thomas J. / A directed approach for engineering conditional protein stability using biologically silent small molecules. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 34. pp. 24866-24872.
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