Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light.
Chemical cross-linking is a potentially useful technique for probing the architecture of multiprotein complexes. However, analyses using typical bifunctional cross-linkers often suffer from poor yields, and large-scale modification of nucleophilic side chains can result in artifactual results attributable to structural destabilization. We report here the de novo design and development of a type of protein cross-linking reaction that uses a photogenerated oxidant to mediate rapid and efficient cross-linking of associated proteins. The process involves brief photolysis of tris-bipyridylruthenium(II) dication with visible light in the presence of the electron acceptor ammonium persulfate and the proteins of interest. Very high yields of cross-linked products can be obtained with irradiation times of <1 second. This chemistry obviates many of the problems associated with standard cross-linking reagents.
Fancy
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Departments of Internal Medicine and Biochemistry, Center for Biomedical Inventions, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-8573, USA.
Kodadek
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D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives; D001665:Binding Sites; D056831:Coordination Complexes; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D005982:Glutathione Transferase / Q000737:chemistry; D007202:Indicators and Reagents; D007700:Kinetics; D008027:Light; D008565:Membrane Proteins / Q000737:chemistry; D016877:Oxidants; D010782:Photolysis; D011506:Proteins / Q000737:chemistry* / Q000528:radiation effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D016385:TATA Box; D035181:TATA-Box Binding Protein; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry; D017975:Ruthenium Compounds
D015082:2,2'-Dipyridyl; D056831:Coordination Complexes; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D005982:Glutathione Transferase; D007202:Indicators and Reagents; D008565:Membrane Proteins; D016877:Oxidants; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D014764:Viral Proteins; C052130:GAL4 protein, S cerevisiae; C092864:UvsY protein, Enterobacteria phage T4; C547232:tris(2,2-bipyridine)-ruthenium(II); D017975:Ruthenium Compounds