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-methylcoumarin-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-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 (approximately 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 reductive 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 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 (less than or equal to 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.
Thevenin
B J
BJ
Department of Medicine, University of California, San Francisco 94143.
Shahrokh
Z
Z
Williard
R L
RL
Fujimoto
E K
EK
Kang
J J
JJ
Ikemoto
N
N
Shohet
S B
SB
D000818:Animals; D001386:Azides / Q000737:chemistry*; D001665:Binding Sites; D002417:Cattle; D003374:Coumarins / Q000737:chemistry; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D010179:Pancreas / Q000201:enzymology; D010777:Photochemistry; D013388:Succinimides / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry*; D014361:Trypsin Inhibitors / Q000737:chemistry
D001386:Azides; D003374:Coumarins; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D013388:Succinimides; D014361:Trypsin Inhibitors; C074882:sulfosuccinimidyl-2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate; D014357:Trypsin