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''Method section:''
''Method section:''


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. Previous experiments have shown that for peptide [[Amyloid beta]], peptide found to be toxic in Alzheimer's Disease, can apply PICUP with a ratio of 1:2:40 for Aβ peptide, Ru(Bpy), and APS respectively.<ref name=":2" /> The ratio between Ru(Bpy) and APS is suggested to be kept at this ratio, but the appropriate concentration of a given protein can vary.<ref name=":2" /> 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.
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. Previous experiments have shown that for peptide [[Amyloid beta]], peptide found to be toxic in Alzheimer's Disease, can apply PICUP with a ratio of 1:2:40 for Aβ peptide, Ru(Bpy), and APS respectively.<ref name=":2" /> The ratio between Ru(Bpy) and APS is suggested to be kept at this ratio, but the appropriate concentration of a given protein can vary.<ref name=":2" /> 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. However, studies of pure protein suggest that the protein to Ru(Bpy) ratio should be kept at 2:1 as well.<ref name=":2" /> This is due to the fact that the lower amount of Ru(Bpy) can lead to the protein sample appearing to have more than actual amount of higher order oligomers and higher amount of Ru(Bpy) can allow for artificial cross-linking products.<ref name=":3" />


The appropriate amount of the protein is pipetted into the polymerase chain reaction tube (PCR). Ammonium persulfate (APS) and Tris(2,2′‐bipyridyl)dichlororuthenium(II) hexahydrate are added to the sample and mixed.<ref name=":2" /> The PCR tube is then placed in a glass vial to hold it still inside the bellows of the camera.<ref name=":2" /> Upon closing the lid that covers the camera, pressing down the camera shutter illuminates the mixture in the tube for second.<ref name=":2" /> This ensures that the irradiation time and the distance from the light sourced is controlled for every PICUP experiment.<ref name=":2" />
The appropriate amount of the protein is pipetted into the polymerase chain reaction tube (PCR). Ammonium persulfate (APS) and Tris(2,2′‐bipyridyl)dichlororuthenium(II) hexahydrate are added to the sample and mixed.<ref name=":2" /> The PCR tube is then placed in a glass vial to hold it still inside the bellows of the camera.<ref name=":2" /> Upon closing the lid that covers the camera, pressing down the camera shutter illuminates the mixture in the tube for second.<ref name=":2" /> This ensures that the irradiation time and the distance from the light sourced is controlled for every PICUP experiment.<ref name=":2" />
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- ++ papers on PICUP
- ++ papers on PICUP

Limitations: (use 10)

Revision as of 01:07, 25 May 2017

Adding to cross-link page:

In-vitro rapid cross-linking method, termed PICUP (photo-induced cross-linking of unmodified proteins), was developed by David A. Fancy and Thomas Kodadek in 1999.[1] They devised a process in which ammonium persulfate (APS), which acts as an electron acceptor, and tris-bipyridylruthenium (II) cation ( [Ru(bpy)3]2+) are added to the protein of interest and irradiated with UV light.[1] PICUP is more expeditious and high yielding when compared to previous chemical cross-linking methods.[1]

Bibliography:

- Definition[2]

- History[1]

- Mechanism[3][4]

- Method[5]

- Application to the real world[6] [7]

- Some disadvantage [8]

Outline for Photo-Induced Cross-linking of Unmodified Proteins

Lead section (beginning, definition):

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) is a protein cross-linking method triggered by visible light irradiation of a photocatalyst in the presence of electron acceptor and protein of interest, generating a highly reactive radical that elicits the formation of a covalent bond between the amino acid side chains of the proteins to be linked.[2] Cross-linking methods developed prior to PICUP, including the use of physical, oxidative, and chemical cross-linkers, often required more time and resulted in synthetic protein byproducts as well as low yield of actual cross-linked proteins due to the characteristics of cross-linking reagents to be multifunctional.[1]

The process was first applied in 1999 to unravel the interactions between polypeptides and decipher the structural differences a protein undergoes throughout a catalytic mechanism through efficient chemical cross-linking.[1] The new method, elicited through photo-oxidation by visible light, allowed for rapid (<1 second) and high production of covalently-crosslinked proteins in close proximity.[1]

The mechanism of PICUP require tris(bipyridyl)Ru(II) complex, an electron acceptor, ammonium persulfate (APS), and reactive amino acid side chains such as Tryptophan and Tyrosine. Ru2+ of the tris(bipyridyl)Ru(II) complex is oxidized to Ru3+ by visible light irradiation. As an effective single electron oxidizer, Ru3+ can produce a protein radical by oxidizing a protein molecule in its vicinity.[9] The highly unstable radicals on the amino acid side chains will proceed through reactions to reach a more stable state.[9] The unpaired electron can be used to form a covalent bond by interacting with a neighboring protein monomer, resulting in a dimer with a covalent bond.[9] Higher oligomers - trimers, tetramers, pentamers - can be produced through covalently-links in the same way.

PICUP allows for visualization of quantitative bands of metastable protein oligomer distribution prior to cross-linking when it is coupled with protein fractionation techniques.[1] This combination is especially useful when examining the oligomers of neurodegenerative diseases resulting from misfolding of the proteins such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, as it can track the dependence of oligomerization tendencies on the primary sequence of the polypeptide.[2] It is necessary to investigate the aggregation tendencies of the respective amyloidogenic proteins into oligomers when exploring possible prevention and treatment procedures.

History section:

  1. David A. Fancy and Thomas Kodadek
  2. David Teplow and Gal Bitan

Mechanism section(in detail):

Tris(2,2′‐bipyridyl)dichlororuthenium(II) hexahydrate, a tris(bipyridyl)Ru(II) complex, initially contains a Ru2+.[10] Upon visible light irradiation and in the presence of ammonium persulfate, Ru2+ is oxidized to Ru3+.[10] Ru3+ is now an extremely reactive oxidizer that only wants to accept one electron instead of the standard two.[10] Ru3+ will pick up a single electron from amino acids of the neighboring proteins, specifically Tryptophan and Tyrosine[10]. The protein that had just donated a single electron to Ru3+ is now a radical, and with the regeneration of Ru2+, the radicals are continuously formed.

The numerous unstable protein radicals come in contact with each other through simple diffusion and react both intramolecularly 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 and releasing a hydrogen atom.[10] This newly formed dimer is also able to react with numerous other monomers or dimers with the comparable mechanism, creating higher levels of cross-linked oligomers.[10]

Method section:

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. Previous experiments have shown that for peptide Amyloid beta, peptide found to be toxic in Alzheimer's Disease, can apply PICUP with a ratio of 1:2:40 for Aβ peptide, Ru(Bpy), and APS respectively.[9] The ratio between Ru(Bpy) and APS is suggested to be kept at this ratio, but the appropriate concentration of a given protein can vary.[9] 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. However, studies of pure protein suggest that the protein to Ru(Bpy) ratio should be kept at 2:1 as well.[9] This is due to the fact that the lower amount of Ru(Bpy) can lead to the protein sample appearing to have more than actual amount of higher order oligomers and higher amount of Ru(Bpy) can allow for artificial cross-linking products.[10]

The appropriate amount of the protein is pipetted into the polymerase chain reaction tube (PCR). Ammonium persulfate (APS) and Tris(2,2′‐bipyridyl)dichlororuthenium(II) hexahydrate are added to the sample and mixed.[9] The PCR tube is then placed in a glass vial to hold it still inside the bellows of the camera.[9] Upon closing the lid that covers the camera, pressing down the camera shutter illuminates the mixture in the tube for second.[9] This ensures that the irradiation time and the distance from the light sourced is controlled for every PICUP experiment.[9]

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. In addition, if SDS-PAGE is to be used to analyze the oligomer distribution of the proteins, DTT would also act as a denaturing agent to the proteins before gel electrophoresis.

Applications of PICUP:

- analyze proteins (neurodegenerative diseases)

- ++ papers on PICUP

Limitations: (use 10)

  1. ^ a b c d e f g h Fancy, David A.; Kodadek, Thomas (1999-05-25). "Chemistry for the analysis of protein–protein interactions: Rapid and efficient cross-linking triggered by long wavelength light". Proceedings of the National Academy of Sciences. 96 (11): 6020–6024. doi:10.1073/pnas.96.11.6020. ISSN 0027-8424. PMID 10339534.
  2. ^ a b c Rahimi, Farid; Maiti, Panchanan; Bitan, Gal (2009-01-12). "Photo-induced cross-linking of unmodified proteins (PICUP) applied to amyloidogenic peptides". Journal of Visualized Experiments: JoVE (23). doi:10.3791/1071. ISSN 1940-087X. PMC 2763294. PMID 19229175.{{cite journal}}: CS1 maint: PMC format (link)
  3. ^ Bitan, Gal; Lomakin, Aleksey; Teplow, David B. (2001-09-14). "Amyloid β-Protein Oligomerization PRENUCLEATION INTERACTIONS REVEALED BY PHOTO-INDUCED CROSS-LINKING OF UNMODIFIED PROTEINS". Journal of Biological Chemistry. 276 (37): 35176–35184. doi:10.1074/jbc.M102223200. ISSN 0021-9258. PMID 11441003.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  4. ^ Weerasekera, Rasanjala; Schmitt-Ulms, Gerold (2006-12-01). "Crosslinking Strategies for the Study of Membrane Protein Complexes and Protein Interaction Interfaces". Biotechnology and Genetic Engineering Reviews. 23 (1): 41–62. doi:10.1080/02648725.2006.10648077. ISSN 0264-8725.
  5. ^ Vollers, SabrinaS.; Teplow, DavidB.; Bitan, Gal (2005-01-01). Sigurdsson, EinarM. (ed.). Amyloid Proteins. Methods in Molecular Biology™. Humana Press. pp. 11–18. doi:10.1385/1-59259-874-9:011. ISBN 9781588293374.
  6. ^ Rosu, Cornelia; Cueto, Rafael; Russo, Paul S. (2016-08-23). "Poly(colloid)s: "Polymerization" of Poly(l-tyrosine)-silica Composite Particles through the Photoinduced Cross-Linking of Unmodified Proteins Method". Langmuir: the ACS journal of surfaces and colloids. 32 (33): 8392–8402. doi:10.1021/acs.langmuir.6b01815. ISSN 1520-5827. PMID 27504929.
  7. ^ Rosu, Cornelia; Cueto, Rafael; Russo, Paul S. (2016-08-23). "Poly(colloid)s: "Polymerization" of Poly(l-tyrosine)-silica Composite Particles through the Photoinduced Cross-Linking of Unmodified Proteins Method". Langmuir. 32 (33): 8392–8402. doi:10.1021/acs.langmuir.6b01815. ISSN 0743-7463.
  8. ^ Sinz, Andrea (2006-07-01). "Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions". Mass Spectrometry Reviews. 25 (4): 663–682. doi:10.1002/mas.20082. ISSN 0277-7037. PMID 16477643.
  9. ^ a b c d e f g h i j Bitan, Gal (2006-01-01). "Structural Study of Metastable Amyloidogenic Protein Oligomers by Photo-Induced Cross-Linking of Unmodified Proteins". Methods in enzymology. 413: 217–236. doi:10.1016/S0076-6879(06)13012-8. ISSN 0076-6879. PMC 2782599. PMID 17046399.{{cite journal}}: CS1 maint: PMC format (link)
  10. ^ a b c d e f g Bitan, Gal (2006). "Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins". Methods in Enzymology. 413: 217–236. doi:10.1016/S0076-6879(06)13012-8. ISSN 0076-6879. PMC 2782599. PMID 17046399.{{cite journal}}: CS1 maint: PMC format (link)
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