title: A directed approach for engineering conditional protein stability using biologically silent small molecules.
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 (DeltaDeltaG(U-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.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California 94305, USA.
authors.forename: Lystranne A
authors.initials: LA
authors.lastname: Maynard-Smith
authors.forename: Ling-Chun
authors.initials: LC
authors.lastname: Chen
authors.forename: Laura A
authors.initials: LA
authors.lastname: Banaszynski
authors.forename: A G Lisa
authors.initials: AG
authors.lastname: Ooi
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Wandless
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001703:Biophysics / Q000379:methods; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D041681:NIH 3T3 Cells; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D022061:Tacrolimus Binding Protein 1A