Target Identification by Diazirine Photo-Cross-linking and Click Chemistry

Abstract

Target identification of biologically active small-molecules is often the rate-determining step in forward chemical genetics. Photo-affinity labeling (PAL) represents a useful biochemical strategy for target identification in complex protein mixtures. This unit describes the use of alkyl diazirine-based photo-affinity probes and Cu(I)-catalyzed click chemistry to covalently label and visualize the targets of biologically active small-molecules. A general method for affinity purification of probe-modified proteins, useful for identification of protein targets, is also described.

Keywords: affinity purification; click chemistry; diazirine; photo-affinity labeling; target identification.

Figure 1

(A) Generalized scheme for photo-affinity labeling and detection using a diazirine and alkyne-containing photo-affinity probe (I). UV irradiation of the diazirine generates a carbene intermediate (II) that covalently crosslinks to the protein target (III). The adduct is then detected by conjugation with an azide-containing reporter group under click chemistry conditions (IV). (B) Structures of the natural product HUN7293 (1), photo-affinity probe (2), and the photo-stable control compound (3).

Figure 2

Structures of TAMRA-azide and Biotin-azide used in the protocol.

Figure 3

Structures of compounds 6, 7, and 8 which have a fluorescent reporter group (TAMRA) directly incorporated into the natural product scaffold.

Figure 4

(A) Photo-crosslinking in ER microsomes with 2 followed by click chemistry with TAMRA-azide (4) as described in the Basic Protocol. Sec61α is marked with an asterisk (figure adapted with permission). (B) Photo-crosslinking in ER microsomes with 2 followed by click chemistry with Biotin-azide (5) and affinity purification using monomeric avidin as described in the Support Protocol. Samples representing the click reaction (Lane 1), the re-solubilized protein pellet following acetone precipitation (Lane 2), the post-monomeric avidin supernatant (Lane 3), and the eluent (Lane 4), were resolved by SDS-PAGE, transferred to nitrocellulose, and probed for biotinylated proteins with streptavidin-HRP (Strep-HRP). Percentages indicate the fraction of the total sample that was loaded in each lane. The position of Sec61α is marked with an asterisk. The biotinylated protein at ~21 kDa is a background band.