title: Scope, limitations and mechanistic aspects of the photo-induced cross-linking of proteins by water-soluble metal complexes.
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.
authors.affiliation: Department of Internal Medicine, Center for Biomedical Inventions, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8573, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Fancy
authors.forename: C
authors.initials: C
authors.lastname: Denison
authors.forename: K
authors.initials: K
authors.lastname: Kim
authors.forename: Y
authors.initials: Y
authors.lastname: Xie
authors.forename: T
authors.initials: T
authors.lastname: Holdeman
authors.forename: F
authors.initials: F
authors.lastname: Amini
authors.forename: T
authors.initials: T
authors.lastname: Kodadek
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry; D009942:Organometallic Compounds; D010084:Oxidation-Reduction; D033903:Periplasmic Proteins; D028061:Phosphate Transport Proteins; D010782:Photolysis; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012428:Ruthenium; D029701:Saccharomyces cerevisiae Proteins; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012995:Solubility; D014157:Transcription Factors / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry*; D014867:Water
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009942:Organometallic Compounds; D033903:Periplasmic Proteins; D028061:Phosphate Transport Proteins; D011506:Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C092864:UvsY protein, Enterobacteria phage T4; D014764:Viral Proteins; C415768:tris(2,2'-bipyridyl)ruthenium(II); D014867:Water; D012710:Serum Albumin, Bovine; C055831:phosphate permease; D012428:Ruthenium; D004247:DNA