Main content area

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

THEVENIN, Benard J.‐M., SHAHROKH, Zahra, WILLIARD, Renee L., FUJIMOTO, Edward K., KANG, Jaw‐Jou, IKEMOTO, Noriaki, SHOHET, Stephen B.
European journal of biochemistry 1992 v.206 no.2 pp. 471-477
binding sites, coumarin, cross-linking reagents, crosslinking, fluorescence, fluorescent labeling, photolysis, polyacrylamide gel electrophoresis, proteins, trypsin, trypsin inhibitors
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.