Fig. 5
From: Selective N-terminal acylation of peptides and proteins with a Gly-His tag sequence
Functionalization of GH6-BIR2 with an azide, a fluorophore and biotin. a 3D structure of XIAP(124–240) with the linker region known to interact with caspase-3 and -7 depicted on the left and the BIR2 domain on the right. The reaction scheme of the inhibition assay is shown as well. The tetrapeptide DEVD is hydrolyzed by caspase-7 between the second aspartic acid and the AFC reporter group. AFC = 7-amino-4-trifluoromethylcoumarin. b Kinetic curves of the hydrolysis of Ac-DEVD-AFC by caspase-7, without inhibition (filled triangles) or when inhibited by unmodified (horizontal lines) or acylated (open circles) GH6-BIR2. The substrate only (crosses) was included as reference as well. c Two-step fluorescent labeling of GH6-BIR2 through acylation with 4-methoxyphenyl ester 18 followed by Cu(I)-catalyzed conjugation of alkyne cyanine dye 718, visualized by SDS-PAGE analysis. A fluorescence image and an image of the Coomassie-stained version of the same gel are shown. Lane 1: GH6-BIR2 treated with alkyne cyanine dye 718 and Cu(I) (negative control), lane 2: azido-functionalized GH6-BIR2 treated with alkyne cyanine dye 718 and Cu(I). d Direct biotinylation of GH6-BIR2 with 4-methoxyphenyl ester 20, visualized by Western blot analysis. Binding of streptavidin to biotinylated GH6-BIR2 (lane 2) was detected by incubating the blot with HRP-SAv, a conjugate of streptavidin and horseradish peroxidase (HRP), followed by the addition of a chemoluminescent substrate of HRP. Unmodified GH6-BIR2 was loaded in lane 1 as negative control
