page_title: Photo-induced cross-linking of unmodified proteins
section_title: Mechanism
hierarchical_section_title: Photo-induced cross-linking of unmodified proteins / Mechanism
caption_attribution_description: English: RuBpy reacting with APS in presence of light
context_page_description: Photo-Induced Cross-Linking of Unmodified Proteins is a protein cross-linking method by visible light irradiation of a photocatalyst in the presence of an electron acceptor and the protein of interest. Irradiation results in a highly reactive protein radical that forms a covalent bond between the amino acid side chains of the proteins to be linked. Cross-linking methods developed prior to PICUP, including the use of physical, oxidative, and chemical cross-linkers, often require more time and result in protein byproducts. In addition, the cross-linked protein yield is very low due to the multifunctionality of the cross-linking reagents.
  The process was first applied in 1999 to utilize protein cross-linking techniques to analyze the interactions between polypeptides as well as structural differences proteins undergo in a catalytic pathway. The techniques in the 20th century were not sufficient to be applied to cross-link fast and transient changes of these proteins in high yield. PICUP allowed for rapid and high production of covalently-linked proteins in close proximity with each other.
context_section_description: The mechanism of PICUP require the tris(bipyridyl)Ru(II) complex, an electron acceptor, ammonium persulfate (APS), and reactive amino acid side chains. Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate, a tris(bipyridyl)Ru(II) complex, initially contains a Ru²⁺. Upon visible light irradiation and in the presence of ammonium persulfate (APS), Ru²⁺ enters its excited state and is oxidized to Ru³⁺. Ru³⁺ is now an extremely reactive oxidizer that only wants to accept one electron instead of the standard two. The reaction can proceed in the absence of an electron acceptor, but it will have a much lower efficiency, producing byproducts resulting from the excited Ru(Bpy)₃ reacting with oxygen. As an effective single electron oxidizer, Ru³⁺ will pick up a single electron from amino acids of the neighboring proteins, most commonly Tryptophan and Tyrosine. This produces a radical that is highly unstable on the amino acid side chains, which proceeds through reactions to reach a more stable state.  
  The unpaired single electron on the side chain reacts with another amino acid side chain of a polypeptide in the vicinity, resulting in a dimer with a covalent bond. With the regeneration of Ru²⁺ when Ru³⁺ picks up an electron from the amino acids allows for continuous formation of radicals with Ru³⁺ being oxidized by APS.
  In the PCR tube that the reaction takes place in, numerous unstable protein radicals come in contact with each other through simple diffusion and react both intra- and intermolecularly to achieve a more stable state. The monomeric protein radicals are able to achieve a lower energy state through forming a covalent bond to produce a dimer and releasing a hydrogen atom. PLEASE NOTE: The image mistakenly shows the release of a proton and should not be used in this form (See also ref. 4). The creator has been contacted. This newly formed dimer is also able to react with numerous other monomers or dimers through the same mechanism, creating higher numbers of cross-linked oligomers. This allows for a distribution of variety of oligomers to be present in the mixture.
caption: APS and Ru(Bpy)3 reaction to produce excited Ru(Bpy)3
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