A general approach for chemical labeling and rapid, spatially controlled protein inactivation.
Chemical labeling of proteins inside of living cells can enable studies of the location, movement, and function of proteins in vivo. Here we demonstrate an approach for chemical labeling of proteins that uses the high-affinity interaction between an FKBP12 mutant (F36V) and a synthetic, engineered ligand (SLF'). A fluorescein conjugate to the engineered ligand (FL-SLF') retained binding to FKBP12(F36V) and possessed similar fluorescence properties as parental fluorescein. FL-SLF' labeled FKBP12(F36V) fusion proteins in live mammalian cells, and was used to monitor the subcellular localization of a membrane targeted FKBP12(F36V) construct. Chemical labeling of FKBP12(F36V) fusion proteins with FL-SLF' was readily detectable at low expression levels of the FKBP12(F36V) fusion, and the level of fluorescent staining with FL-SLF' was proportional to the FKBP12(F36V) expression level. This FL-SLF'-FKBP12(F36V) labeling technique was tested in fluorophore assisted laser inactivation (FALI), a light-mediated technique to rapidly inactivate fluorophore-labeled target proteins. FL-SLF' mediated FALI of a beta-galactosidase-FKBP12(F36V) fusion protein, causing rapid inactivation of >90% of enzyme activity upon irradiation in vitro. FL-SLF' also mediated FALI of a beta-galactosidase fusion expressed in living NIH 3T3 cells, where beta-galactosidase activity was reduced in 15 s. Thus, FL-SLF' can be used to monitor proteins in vivo and to target rapid, spatially and temporally defined inactivation of target proteins in living cells in a process that we call FK-FALI.
Marks
Kevin M
KM
The Baxter Laboratory for Genetic Pharmacology, and Department of Molecular Pharmacology, Stanford University School of Medicine, CA 94305, USA.
Braun
Patrick D
PD
Nolan
Garry P
GP
D000818:Animals; D005456:Fluorescent Dyes; D051379:Mice; D041681:NIH 3T3 Cells; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism*
D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D022061:Tacrolimus Binding Protein 1A