Rescue of Degradation-Prone Mutants of the FK506-Rapamycin Binding (FRB) Protein with Chemical Ligands
dc.contributor.author | Stankunas, Kryn | en_US |
dc.contributor.author | Bayle, J. Henri | en_US |
dc.contributor.author | Havranek, James J. | en_US |
dc.contributor.author | Wandless, Thomas J. | en_US |
dc.contributor.author | Baker, David | en_US |
dc.contributor.author | Crabtree, Gerald R. | en_US |
dc.contributor.author | Gestwicki, Jason E. | en_US |
dc.date.accessioned | 2007-09-20T18:50:52Z | |
dc.date.available | 2008-09-08T14:25:13Z | en_US |
dc.date.issued | 2007-07-09 | en_US |
dc.identifier.citation | Stankunas, Kryn; Bayle, J. Henri; Havranek, James J.; Wandless, Thomas J.; Baker, David; Crabtree, Gerald R.; Gestwicki, Jason E. (2007)."Rescue of Degradation-Prone Mutants of the FK506-Rapamycin Binding (FRB) Protein with Chemical Ligands." ChemBioChem 8(10): 1162-1169. <http://hdl.handle.net/2027.42/56088> | en_US |
dc.identifier.issn | 1439-4227 | en_US |
dc.identifier.issn | 1439-7633 | en_US |
dc.identifier.uri | https://hdl.handle.net/2027.42/56088 | |
dc.identifier.uri | http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=17525916&dopt=citation | en_US |
dc.description.abstract | We recently reported that certain mutations in the FK506-rapamycin binding (FRB) domain disrupt its stability in vitro and in vivo (Stankunas et al. Mol. Cell , 2003 , 12 , 1615). To determine the precise residues that cause instability, we calculated the folding free energy (Δ G ) of a collection of FRB mutants by measuring their intrinsic tryptophan fluorescence during reversible chaotropic denaturation. Our results implicate the T2098L point mutation as a key determinant of instability. Further, we found that some of the mutants in this collection were destabilised by up to 6 kcal mol −1 relative to the wild type. To investigate how these mutants behave in cells, we expressed firefly luciferase fused to FRB mutants in African green monkey kidney (COS) cell lines and mouse embryonic fibroblasts (MEFs). When unstable FRB mutants were used, we found that the protein levels and the luminescence intensities were low. However, addition of a chemical ligand for FRB, rapamycin, restored luciferase activity. Interestingly, we found a roughly linear relationship between the Δ G of the FRB mutants calculated in vitro and the relative chemical rescue in cells. Because rapamycin is capable of simultaneously binding both FRB and the chaperone, FK506-binding protein (FKBP), we next examined whether FKBP might contribute to the protection of FRB mutants. Using both in vitro experiments and a cell-based model, we found that FKBP stabilizes the mutants. These findings are consistent with recent models that suggest damage to intrinsic Δ G can be corrected by pharmacological chaperones. Further, these results provide a collection of conditionally stable fusion partners for use in controlling protein stability. | en_US |
dc.format.extent | 594839 bytes | |
dc.format.extent | 3118 bytes | |
dc.format.mimetype | application/pdf | |
dc.format.mimetype | text/plain | |
dc.publisher | WILEY-VCH Verlag | en_US |
dc.subject.other | Chemistry | en_US |
dc.subject.other | Biochemistry and Biotechnology | en_US |
dc.title | Rescue of Degradation-Prone Mutants of the FK506-Rapamycin Binding (FRB) Protein with Chemical Ligands | en_US |
dc.type | Article | en_US |
dc.rights.robots | IndexNoFollow | en_US |
dc.subject.hlbsecondlevel | Biological Chemistry | en_US |
dc.subject.hlbtoplevel | Science | en_US |
dc.subject.hlbtoplevel | Health Sciences | en_US |
dc.description.peerreviewed | Peer Reviewed | en_US |
dc.contributor.affiliationum | Department of Pathology and the Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216, USA, Fax: (+1) 734-764-1247 ; | en_US |
dc.contributor.affiliationother | Departments of Pathology and Developmental Biology, Stanford University, 279 Campus Drive, Beckman Building, Stanford, CA 94305, USA | en_US |
dc.contributor.affiliationother | Departments of Pathology and Developmental Biology, Stanford University, 279 Campus Drive, Beckman Building, Stanford, CA 94305, USA | en_US |
dc.contributor.affiliationother | Department of Biochemistry, University of Washington, J Wing, Health Sciences Building, Seattle, WA 98195, USA | en_US |
dc.contributor.affiliationother | Department of Chemical and Systems Biology, Stanford University, 318 Campus Drive, James H. Clark Center, Stanford, CA 94305, USA | en_US |
dc.contributor.affiliationother | Department of Biochemistry, University of Washington, J Wing, Health Sciences Building, Seattle, WA 98195, USA ; Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, MD 20815-6789, USA | en_US |
dc.contributor.affiliationother | Departments of Pathology and Developmental Biology, Stanford University, 279 Campus Drive, Beckman Building, Stanford, CA 94305, USA ; Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, MD 20815-6789, USA | en_US |
dc.identifier.pmid | 17525916 | en_US |
dc.description.bitstreamurl | http://deepblue.lib.umich.edu/bitstream/2027.42/56088/1/1162_ftp.pdf | en_US |
dc.identifier.doi | http://dx.doi.org/10.1002/cbic.200700087 | en_US |
dc.identifier.source | ChemBioChem | en_US |
dc.owningcollname | Interdisciplinary and Peer-Reviewed |
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