Photo-induced cross-linking of unmodified proteins

APS and Ru(Bpy)₃ reaction to produce excited Ru(Bpy)₃

The mechanism of PICUP require the tris(bipyridyl)Ru(II) complex, an electron acceptor, ammonium persulfate (APS), and reactive amino acid side chains.[2] Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate, a tris(bipyridyl)Ru(II) complex, initially contains a Ru²⁺.[4] Upon visible light irradiation and in the presence of ammonium persulfate (APS), Ru²⁺ enters its excited state and is oxidized to Ru³⁺.[4] Ru³⁺ is now an extremely reactive oxidizer that only wants to accept one electron instead of the standard two.[4] 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.[5] 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.[4] This produces a radical that is highly unstable on the amino acid side chains, which proceeds through reactions to reach a more stable state.

Formation of covalent bond between amino acid side chains (Tyrosine)

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.[4] 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.[2]

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.[4] 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.[4] This allows for a distribution of variety of oligomers to be present in the mixture.

Camera apparatus used to irradiate the protein mixture

In every experiment, it is important to determine the relative concentration of the protein in question, ammonium persulfate, and Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate that will be used in the protein cross-linking experiment. Previous experiments have shown that for amyloid β-protein (Aβ), the peptide assumed to cause toxicity in Alzheimer's disease, the ratio of protein: Ru(Bpy)₃: APS is 1:2:40.[3] The ratio of Ru(Bpy)₃ and APS is suggested to be kept at this ratio, but the appropriate concentration of a given protein can vary. For many proteins that PICUP has not yet been used for, finding the appropriate concentrations can be done through trial and error. Generally, protein concentrations would fall in between 10 and 50μM, dissolved in the corresponding buffer, most likely sodium phosphate if testing for conditions at physiological pH. However, some studies of pure protein suggest that the protein to Ru(Bpy)₃ ratio should be kept at 1:2 as well.[4] This level arises from the fact that the lower amount of Ru(Bpy)₃ can lead to the protein sample appearing to have more than the actual number of higher order oligomers, and a greater amount of Ru(Bpy)₃ can allow for artificial cross-linking byproducts.

The general method for PICUP is as follows:

1. The appropriate amount of the protein is pipetted into the polymerase chain reaction tube (PCR).[6]
2. Ammonium persulfate (APS) and Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate are added to the sample and mixed briefly.[6]
3. The PCR tube is placed in a glass vial to hold it still inside the bellows of the camera that would irradiate the mixture. Upon closing the lid that covers the camera, pressing down the camera shutter illuminates the tube for however long you program the camera to irradiate for. Using the same camera for every replicate of the experiment ensures that the irradiation time and the distance from the light sourced is controlled for every PICUP experiment.[6]
4. After the sample in the PCR tube is irradiated, a calculated amount of 1M Dithiothreitol (DTT) is immediately added to the mixture to quench the reaction. If the reaction is not quenched, the oligomers will continually aggregate and lower order oligomers will not be present in the mixture. In addition, if SDS-PAGE is to be used to analyze the oligomer distribution of the proteins after PICUP, DTT would also act as a denaturing agent to the proteins before gel electrophoresis.[6]

Using PICUP on protein complexes is useful in providing catalytic and kinetic information about these proteins, as catalytic mechanisms are rapid and PICUP allows for fast and highly efficient cross-linking of proteins.[5] Some epitope and affinity tags were shown to be unaffected by the PICUP reaction, enabling visualization of the cross-linked proteins.[5]

Furthermore, PICUP allows for visualization of quantitative bands of protein oligomer distribution when it is coupled with protein fractionation techniques. This combination is especially useful when examining the oligomers of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, that result from protein aggregation.[1] PICUP is extremely important when possible prevention and treatment procedures for these diseases are explored, as it is necessary to investigate the aggregation propensity of the respective amyloidogenic proteins.

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