Investigating γ-secretase protein interactions in live cells using active site-directed clickable dual-photoaffinity probes

Abstract

We have developed a suite of clickable γ-secretase active site-directed dual-photoaffinity probes possessing photoactivatable benzophenones that are located within the inhibitor scaffold as well as spaced away from the core by a linker. Through photoactivation of these dual photoprobes and subsequent click chemistry mediated conjugation of a reporter group, we have been able to specifically label PS1-N-terminal fragment (PS1-NTF), PS1-C-terminal fragment (PS1-CTF) and nicastrin and form a pseudo-full length PS1 through crosslinks between PS1-NTF and PS1-CTF. Probe-mediated protein–protein crosslinks were confirmed by in-gel fluorescence, western blotting and LC-MS/MS detection of tryptic peptides of the electrophoretically separated proteins.