Site-specific protein propargylation using tissue transglutaminase.
Transglutaminases (TGases) catalyse the transamidation of glutamine residues with primary amines. Herein we report the first FRET-based activity assay for the direct detection of the ligation (transamidation) reaction mediated by tissue TGase (TG2). This novel assay was then used in a microtiter plate-based screen of a library of 18 potential amine substrates. From this screen it was discovered that propargyl amine serves as an excellent substrate for TG2. Subsequently, propargyl amine and 2-azidoethyl amine were validated independently as TG2 substrates with K(M) values of 44 ± 4 μM, and 0.99 ± 0.06 mM, respectively. In a proof-of-principle protein labelling experiment, the protein casein was selectively functionalized with propargyl amine using TG2 and subsequently fluorescently labelled through a dipolar cycloaddition reaction with an azido-fluorescein conjugate. This application demonstrates the strong potential of using TG2 for site-specific protein modification through a combination of enzymatic and bioorthogonal chemistry.
Département de Chimie, Université de Montreal, Succursale centre-ville, C.P. 6128, Montréal, Québec H3C 3J7, Canada.
Claudio
C
Gnaccarini
Wajih
W
Ben-Tahar
Amina
A
Mulani
Isabelle
I
Roy
William D
WD
Lubell
Joelle N
JN
Pelletier
Jeffrey W
JW
Keillor
D019204:GTP-Binding Proteins / Q000378:metabolism*; D015394:Molecular Structure; D010293:Pargyline / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011437:Propylamines / Q000737:chemistry / Q000378:metabolism*; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
D010455:Peptides; D011437:Propylamines; C009054:propargylamine; D010293:Pargyline; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins