Figure 2: Generation and labeling of FGFR1 KD variants. | Scientific Reports

Figure 2: Generation and labeling of FGFR1 KD variants.

From: Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

Figure 2

(a) Schematic representation of FGFR1 KD variants (KD; grey), a tetracysteine motif (TC; green) and a C-terminal decahistidine tag (His10; black). Three tyrosine phosphorylation sites (Y653, Y654 and Y766) are also indicated. Kinase insert region and activation loop are underlined in black and magenta. The control protein is designated FGFR1KD3Y.TC (top). Two FGFR1 KD variants incorporating replacement of H589 or L662 by BCNK (red spheres) are designated H589BCNK.TC (middle) and L662BCNK.TC (bottom). Incorporation of a TC motif at the C-terminus enables fluorescent labeling with the dye FlAsH-EDT2 (black arrow). (b) Diagram depicting expression (step 1) and labeling (step 2) of FGFR1 KD via genetically encoded UAA, BCNK. In the first step, BCNKRS/tRNACUAPyl pair is required for incorporation of BCNK to positions corresponding to H689 and L662, directed by reassigned TAG codons. In the second step, a bioorthogonal inverse electron-demand Diels-Alder cycloaddition reaction takes place between the strained alkyne group in BCNK and the tetrazine moiety in Tet1-TAMRA-X. Structures of BCNK and Tet1 are shown while TAMRA-X is represented as a star with increased fluorescence (red) upon protein binding. (c) Analysis of incorporation of BCNK into H589BCNK.TC (left) and L662BCNK.TC (right) by TAMRA-X fluorescence. Cell lysates were prepared from conditions lacking one of the indicated components required for BCNK incorporation and incubated in the presence of Tet1-TAMRA-X; following SDS-PAGE, in-gel fluorescence was detected at 532 nm (bottom) and the protein visualized by Coomassie Blue staining (top). (d) Purification and labeling of H589BCNK.TC (left) and L662BCNK.TC (right) proteins. Samples obtained at different stages of purification were analyzed by SDS-PAGE; lane 1: E. coli pellet before induction, lane 2: E. coli pellet after IPTG induction, lane 3: Clarified lysate, lane 4: Immobilized-Metal Affinity Chromatography (IMAC) Ni2+ eluate, lane 5: Steptavidin Trap affinity purification eluate, lane 6: Size Exclusion Chromatography eluate (top panels).Following single or double labeling of proteins and SDS-PAGE, in gel fluorescence was detected at 532 nm and 473 nm and the protein visualized by Coomassie Blue staining (bottom panels).See also Supplementary Figs S1 and S2a.

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