Design and characterization of a photo-activatable hedgehog probe that mimics the natural lipidated form

• Sonic hedgehog was labeled on the N-terminal cysteine with maleimidobenzophenone.
• The potency of Bzm-ShhN was comparable to endogenous dual-lipidated ShhNp.
• The Bzm modified ShhN was monomeric as assessed by analytical SEC.
• UV induced crosslinking of Bzm-ShhN in presence of heparin.
• We report a photoactivatable ShhN probe that acts as a mimetic of processed ShhNp.

We have generated a photoactivatable form of sonic hedgehog protein by modifying the N-terminal cysteine with the heterobifunctional photocrosslinker 4-maleimidobenzophenone (Bzm). The Bzm modification on ShhN imparted a significant increase in activity as assessed in the C3H10T1/2 functional assay with potency comparable to that of the endogenous dual-lipidated form of ShhN (ShhNp). Reversed-phase HPLC analysis indicated that the increase in activity compared to unmodified ShhN may be due in part to the hydrophobic nature of the benzophenone group. In contrast to the fully processed ShhNp, Bzm-ShhN is monomeric as assessed by analytical SEC and does not require detergent to be soluble. Further, we demonstrated that the Bzm-ShhN was able to crosslink in vitro in the presence of a known binding partner, heparin. We suggest that Bzm-ShhN can serve as a relatively facile and preferred source of ShhNp for in vitro assays and as a probe to identify novel Hh protein interactions.