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

Abstract

A cleavable cross-linking reagent, sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-acetamido)-ethyl-l,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-sulfosuccinimidyl ester for conjugation to the donor, a terminal photoactivatable azido-coumarin species for cross-linking with the interacting target, and a central disulfide spacer for the release of the labeled target after cleavage. To evaluate the effectiveness of this labeling reagent, soybean 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 SDSjPAGE and, after reductive cleavage, was shown to be a 1 : 1 STItrypsin complex. This complex was not detected without photolysis or with an inactivated crosslinker. 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 specificity 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 ( 5 1.8 nm) 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.Chemical cross-linking reagents have been used for the determination of nearest-neighbor analysis, molecular interactions and orientations, as well as three-dimensional protein structures (Wang and Richards, 1974;Peters and Richards, 1977;Das and Fox, 1979). For these applications, the efficiency and the specificity of the coupling reaction has been increased by the development of photoaffinity heterobifunctional cross-linkers (Huang and Richards, 1977; Ji, 1979; Jaffe et al., 1980). Such reagents are first coupled to one of the components to be cross-linked while their other functional group remains chemically inert until photoactivation is initiated in the presence of the interacting component. With this controlled and sequential coupling process, self-conjugation and polymerization are kept to a minimum. More importantly, photoaffinity reagents such as aryl azides are activated into nitrenes which react extremely rapidly with numerous neighboring functional groups (Bayley and Staros, 1984).

Photochemical cross‐linking of the skeletal myosin head heavy chain to actin subdomain‐1 at Arg95 and Arg28

“…On the basis of the measured amount of residual uncross-linked 95-kDa heavy chain, we estimated the overall yield of the acto-S1 photocross-linking process to be in the range 13-25%. Although this low yield could be partly explained by an incomplete modification of the Arg residues involved in the photocross-linking, this is a comparatively high value for a photochemical coupling process (Das and Fos, 1979;Thevenin et al, 1992). Moreover, Fig.…”

Rapid labeling of intracellular His-tagged proteins in living cells

“…S3). The low quantum yield of NTA-AC is attributed to the presence of an azide at the seventh position of the coumarin moiety, which quenches the fluorescence of coumarin as reported previously (25,26). The probe Ni-NTA-AC was then generated by subsequent incubation of NTA-AC with Ni 2+ (as NiSO 4 ) in buffered aqueous solution.…”

Studying the Protein Organization of the Postsynaptic Density by a Novel Solid Phase- and Chemical Cross-linking-based Technology

“…In addition, SAED contains a hydrolyzable disulfide bond in the middle. This compound has been used to study a variety of molecular interactions (39). In this study, SAED was conjugated to DADPA-agarose beads (average diameter, ϳ80 m) via covalent links formed between its sulfo-N-hydroxysuccinimidyl ester and free amino groups on the beads (Fig.…”