A novel photoactivatable cross‐linker for the functionally‐directed region‐specific fluorescent labeling of proteins

A cleavable cross‐linking reagent, sulfosuccinimidyl‐2(7‐azido‐4‐methycoumarin‐3‐acetamido)‐ethyl‐1,3′‐dithiopropionate (SAED), was synthesized for the selective transfer of a coumarin fluorophore from a ‘donor’ protein to a position near the binding site of an interacting ‘target’ protein. SAED contains a terminal N‐sulfoscuccinimidyl ester for conjugation to the donor, a terminal photoactivatable azido‐coumarin species for cross‐linking with the interacting target, and a central disculfide spacer for the release of the labeled target after cleavage. To evaluate the effectiveness of this labeling reagent, soynean trypsin inhibitor (STI) was derivatized (∼ 0.5 mol/mol) with SAED and then photolyzed in the presence of trypsin. A single fluorescent cross–linked species (6–7 mol% of total STI) was observed by SDS/PAGE and, after refuctive cleavage, was shown to be a 1: 1 STI‐trypsin complex. This complex was not detected without photolysis or with an inactivated cross‐linker. Importantly, complex formation was inhibited by an excess of unmodified STI and prevented by substitution of a non‐interacting protein for trypsin. Cleavage of the cross‐linked complex revealed that the trypsin, but not the STI, was fluorescent; the uncomplexed trypsin fraction remained unlabeled. These results demonstrated the specificty of the labeling of trypsin by fluorescent‐transfer cross‐linking with SAED. An efficiency of about 15% for this cross‐linking mediated labeling of trypsin was calculated. The short cross‐linking span of SAED (≤1.8nm) strictly limited the labeling to the vicinity of the contact region of trypsin with STI. thus, this novel cross‐linker permits the region‐specific targeting of a fluorophore near a functionally important binding site.