D A Fancy1, C Denison, K Kim, Y Xie, T Holdeman, F Amini, T Kodadek. 1. Department of Internal Medicine, Center for Biomedical Inventions, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8573, USA.
Abstract
BACKGROUND: Chemical cross-linking is a valuable tool with which to study protein-protein interactions. Recently, a new kind of cross-linking reaction was developed in which the photolysis of associated proteins with visible light in the presence of ammonium persulfate and tris(2,2'-bipyridyl)ruthenium(II) dication or palladium(II) porphyrins results in rapid and efficient covalent coupling (Fancy, D.A. & Kodadek, T. (1999). Proc. Natl. Acad. Sci. USA 96, 6020-6024 and Kim, K., Fancy, D.A. & Kodadek, T. (1999). J. Am. Chem. Soc. 121, 11896-11897). Here, mechanistic and practical aspects of the reaction of importance for its application to biochemical problems are examined. RESULTS: It is shown that the photo-initiated cross-linking chemistry can be optimized for the analysis of protein-protein interactions in crude cell extracts. A number of commonly used epitope or affinity tags survive the reaction in functional form, allowing the simple visualization of the cross-linked products, or their isolation. It is shown that very little light-independent oxidation of protein residues occurs and that significant perturbation of complexes of interest prior to the brief photolysis period does not occur. Finally, evidence is presented that is consistent with a mechanistic model in which ammonium persulfate functions simply as an electron acceptor, facilitating the generation of the key high valent metal complex from the photoexcited species by electron transfer. In the absence of an electron acceptor, a much lower efficiency reaction is observed that appears to involve products resulting from reaction of the excited state metal complex with molecular oxygen. CONCLUSIONS: These results provide useful practical information for chemists and biochemists who may wish to employ this new cross-linking chemistry for the analysis of protein complexes. They also shed new light on the mechanism of this interesting reaction.
BACKGROUND: Chemical cross-linking is a valuable tool with which to study protein-protein interactions. Recently, a new kind of cross-linking reaction was developed in which the photolysis of associated proteins with visible light in the presence of ammonium persulfate and tris(2,2'-bipyridyl)ruthenium(II) dication or palladium(II) porphyrins results in rapid and efficient covalent coupling (Fancy, D.A. & Kodadek, T. (1999). Proc. Natl. Acad. Sci. USA 96, 6020-6024 and Kim, K., Fancy, D.A. & Kodadek, T. (1999). J. Am. Chem. Soc. 121, 11896-11897). Here, mechanistic and practical aspects of the reaction of importance for its application to biochemical problems are examined. RESULTS: It is shown that the photo-initiated cross-linking chemistry can be optimized for the analysis of protein-protein interactions in crude cell extracts. A number of commonly used epitope or affinity tags survive the reaction in functional form, allowing the simple visualization of the cross-linked products, or their isolation. It is shown that very little light-independent oxidation of protein residues occurs and that significant perturbation of complexes of interest prior to the brief photolysis period does not occur. Finally, evidence is presented that is consistent with a mechanistic model in which ammonium persulfate functions simply as an electron acceptor, facilitating the generation of the key high valent metal complex from the photoexcited species by electron transfer. In the absence of an electron acceptor, a much lower efficiency reaction is observed that appears to involve products resulting from reaction of the excited state metal complex with molecular oxygen. CONCLUSIONS: These results provide useful practical information for chemists and biochemists who may wish to employ this new cross-linking chemistry for the analysis of protein complexes. They also shed new light on the mechanism of this interesting reaction.
Authors: Chi Zhang; Ekaterina V Vinogradova; Alexander M Spokoyny; Stephen L Buchwald; Bradley L Pentelute Journal: Angew Chem Int Ed Engl Date: 2019-02-15 Impact factor: 15.336
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