Acrylodan

Although acryloyl crosslinking agents have not been common, the reactive group has found use in the design of the sulfhydryl-reactive fluorescent probe, 6-acryloyl-2-dimethylaminonaph-thalene (acrylodan Molecular Probes) (Epps et al., 1992 Yem et al., 1992). 

Yem, A.W. et al. (1992) Site-specific chemical modification of interleukin lb by Acrylodan at cysteine 8 and lysine 103./. Biol. Chem. 267, 3122. 

Another chemical variant of PRODAN is ACRYLODAN [6-acryloyl-2-(dimethyl-amino)naphthalene], which covalently binds to protein-SH groups. 

Acrylodan-labeled CaD, when excited at 375 nm, had an emission maximum at 515 2 nm. Addition of actin resulted in a blue shift in the emission maximum, which was not centered at 505 2 nm. There was no significant increase in fluorescence intensity around the emission maximum, but a significant increase in relative fluorescence intensity (nearly 70%) was observed at 470 nm. During the titration of labeled CaD with actin, the fluorescence intensity at 470 nm was monitored. Maximum increase in fluorescence intensity was achieved by the time 7 mol of actin were added per mol of CaD (Fig. 8). 

FIGURES Influence of actin on acrylodan-CaD (solid circle) and acrylodan CaD-CaT fluorescence (open circle). The initial CaD concentration was 1.0 X 10 M . Measurements were carried out in 25 mM Tris (pH 7.5), 42 mM NaCl, 2 mM MgCl2, 0.2 mM CaCl2, and 1 mM DTT at 20°C. Relative change in fluorescence intensities (AF/ at 470 nm are plotted as a function of actin concentration. 

The protein acrylodan-labeled bovine serum albumin (BSA-Ac) was solubilized into water-in-C02 microemulsions created with the PFPE surfactant mentioned in the previous section [73]. This particular protein was chosen because BSA is moderately large (67,000 Da) and the strong Ac fluorescence provides a measure of both BSA conformation and concentration in solution. Upon forming a microemulsion with PFPE, the BSA-Ac fluorescence is very strong and similar to that of native BSA-Ac in buffer at pH 7.0, as shown in Fig. 15. After recovery, the BSA-Ac is still recognized by the BSA antibody. These results show that BSA-Ac is solubilized within the aqueous microemulsion droplets in an environment similar to that of bulk buffered water and remains biologically active. 

Figure 2.3 First HTS for the detection allosteric Src inhibitors. In the absence of ligand, acrylodan-labeled cSrc shows two emission maxima at 475 and 505 nm. Type I ligands induce a robust loss of fluorescence intensity (arrows) at 475 nm, resulting in a red shift in the emission maxima to 510 nm (right panel). Type II and III inhibitors stabilize the inactive kinase conformation and elicit a different response in which the emissions at 475 and 505 nm are equally reduced. The emission signal at 445 nm is less sensitive to ligand binding and serves as an internal reference point, allowing for...

Key words FLiK, Fluorescence, Acrylodan, DFG-out, DFG-in, Kinase inhibitors. Activation loop. 

Fig. 1. The FLiK technology for the detection of allosteric Type ll/lll kinase Inhibitors. Kinases are regulated by an activation loop which can adopt active and inactive conformations, (a) The inactive conformation (DFG-out) presents an alternative binding site in which DFG-out ligands can bind and prevent the kinase from adopting and active conformation, (b) A Cys was mutated into the activation loop and (c) used for the attachment of acrylodan. (d) The DFG motif and activation loop adopt a different conformation in inactive kinases when compared to active kinase with ATP bound, resulting in a fluorescence change. Active kinases have an activation loop which is open and extended in contrast to the inactive DFG-out conformation. Reproduced from (7) with permission from Nature Publishing Group.

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