A general approach for chemical labeling and rapid, spatially controlled protein inactivation

Abstract

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.

Fig. 1.

Fluorescein conjugate of a synthetic ligand for FKBP12(F36V) (SLF′). (A) Structure of the FL-SLF′ conjugate. (B) Normalized excitation and emission spectra for fluorescein (gray lines) and FL-SLF′ (black lines).

Fig. 2.

FL-SLF′ mediates FALI in vitro when targeted to an FKBP12(F36V) fusion protein. (A) Mechanism of FALI with FL-SLF′. Irradiation of fluorescein drives formation of chemically active ROS, which tend to react with and inactivate the β-gal fusion partner because of proximity. (B) β-gal fusions were incubated with compound and then irradiated for 6 min. (C) FKBP12(F36V)-β-gal was incubated for various times with PBS (open triangles), fluorescein (open circles), or FL-SLF′ (filled circles). (D) FKBP12(F36V)-β-gal was incubated with FL-SLF′, treated with sodium azide (NaN₃), and irradiated as indicated.

Fig. 3.

FL-SLF′ specifically labels FKBP12(F36V) fusion proteins in vivo. (A) NIH 3T3 cells expressing membrane-targeted FKBP12 or FKBP12(F36V) were treated with the indicated fluorescent compounds and analyzed by flow cytometry. (B–G) Control NIH3T3s (B–D) and NIH 3T3 cells expressing a membrane-targeted FKBP12(F36V) (E–G) were treated with PBS (B and E), fluorescein (C and F), or FL-SLF′ (D and G) and imaged by confocal microscope.

Fig. 4.

FL-SLF′ specifically labels proteins expressed at varied levels in multiple cell lines. (A) Western blot with anti-FKBP12 antibody reveals low expression of Lyn-FKBP12 constructs relative to endogenous FKBP12. (B) Cell lines expressing increasing amounts of a Rac1-FKBP12(F36V) fusion. (C and D) Flow cytometry analysis of FL-SLF′-stained cells expressing increasing amounts of Rac1-FKBP12(F36V) fusion. (E) 293T cells transfected with an α1S-calcium channel fused to wild-type or F36V FKBP12 were treated with FL-SLF′ and analyzed by flow cytometry.

Fig. 5.

FL-SLF′ mediates light-driven inactivation of target proteins in living cells. (A) NIH 3T3 cell lines expressing β-gal fusion proteins were treated with fluorescein or FL-SLF′, irradiated for 5 min, then lysed and assayed for β-gal activity. (B) FKBP12(F36V)-β-gal-expressing cells were treated with PBS (open triangles), fluorescein (open circles), or FL-SLF′ (filled circles). (C–H) FKBP12(F36V)-β-gal-expressing cells (C–E) and β-gal expressing cells (F–H) were treated with FL-SLF′, then a portion of the field of view was irradiated for 15 s (C and E). Irradiation area is indicated by the red outline. Irradiation was confirmed by imaging FL-SLF′ (D and G), and then β-gal activity was assayed by addition of cell permeable dimethylacridinone (DDAO)–GAL substrate and imaging accumulation of cleaved DDAO (E and H).