User:Jukakim/sandbox

My article: Photo-Induced Cross-Linking of Unmodified Proteins (PICUP)

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. 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.

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. 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.

PICUP allows for visualization of quantitative bands of metastable protein oligomer distribution prior to cross-linking when it is coupled with protein fractionation techniques. 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. It is necessary to investigate the aggregation tendencies of the respective amyloidogenic proteins into oligomers when exploring possible prevention and treatment procedures.

History section:

Fancy and Kodadek developed Photo-induced cross-linking of unmodified proteins (PICUP) to study and analyze stable proteins in 1999. It was the first time cross-linking of proteins were able to be performed in such a short period of time (1 second) and without modifying the structure of the proteins in question. Additionally, it allowed for more applications for protein cross-linking due to the fact that PICUP allowed for the reaction to take place at physiological pH. Being able to study proteins at physiological pH can be extremely beneficial in attempting to study the human body and biochemical mechanisms that proteins participate in.

In 2001, Gal Bitan, Aleksey Lomakin, and David B. Teplow applied PICUP to study protein oligomers that are often metastable and even lack a native structure. This allowed for studying amyloidogenic proteins associated with neurodegenerative diseases and opened doors for possible therapeutic mechanisms. Neurodegenerative diseases are currently suggested to be caused by neurotoxic proteins and the ability to study its oligomer distribution is effective in understanding how and under what conditions these oligomers are formed.

Mechanism section(in detail):

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. The highly unstable radicals on the amino acid side chains will proceed through reactions to reach a more stable state. 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. Higher oligomers - trimers, tetramers, pentamers - can be produced through covalent-links in the same way.

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, Ru²⁺ is oxidized to Ru³⁺. Ru³⁺ is now an extremely reactive oxidizer that only wants to accept one electron instead of the standard two. Ru³⁺ will pick up a single electron from amino acids of the neighboring proteins, specifically Tryptophan and Tyrosine. 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. 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. Hence, we are able to see the protein oligomer distribution when PICUP is coupled with SDS-PAGE. 

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, the 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. 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. 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 1:2 as well. 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.

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. The PCR tube is then placed in a glass vial to hold it still inside the bellows of the camera. Upon closing the lid that covers the camera, pressing down the camera shutter illuminates the mixture in the tube for second. This ensures that the irradiation time and the distance from the light sourced is controlled for every PICUP experiment.

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)

Suggested Edits

Peer Review by Tnzhou

For Cross-link

Pronoun agreement conflict between “cross-link” and “They can” in the first sentence.

The distinguishment between the physical use of the term and the biological use of the term is a good add.

I would suggest to remove the sentence beginning with “of course” in the second paragraph. I don’t feel like it adds much to the overall understanding of the topic, although it is true and is thoughtful insight.

The last paragraph in the introduction could fit better somewhere else; it feels very out of place in the introduction.

I believe that the “IUPAC definition” box would be better if it came first before the box about Vulcanization, since it gives the overview of the term and Vulcanization is just an example of cross-linking

I noticed that quite a few of the sentences begin with “when ___ is/are __”, so changing that sentence structure would allow the text to be read more smoothly.

For: edit to Cross-link page + Outline:

Good work on linking terms and adding references

Avoid using first person “we”, “us” in the article

Would be helpful to link terms as you did in the section that you added to the cross-link page

Some of the content in the lead section I feel could belong in the history section.

Aside from the all the edits, I believe you did a great job on creating content and your text reads nicely and sounds polished. I would have added more suggestions if I found anything I would thought needed change, but your article is well written.

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Peer Review by User:PrasannR

Instead of listing out my comments like he did above, I decided to create a Google Docs version of your article and leave my comments there, so please look over my comments on your article here: https://docs.google.com/document/d/1OKzyv92oBQh6Qd8ThtHHWmzbgRz0aKEIlpnflPMnrgE/edit?usp=sharing

Hope that makes my comments/edits easier to understand! PrasannR (talk) 20:17, 1 June 2017 (UTC)

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