Cross linking pp
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Cross-linking is a technique used to study protein structure and interactions. It involves using bifunctional reagents containing two reactive groups to form covalent bonds between amino acid residues, either within or between proteins. This captures transient or conditional interactions and provides structural data at higher resolution than other methods. The most common cross-linking reagents react with amino acids like cysteine, tyrosine, and lysine. Cross-linking has provided important insights into protein structure-function relationships and molecular interactions.
![Oxidative Cross-Linking
Introduced by Brown et al .,
Highly specific.
The process is mediated by a Ni(II)-peptide reagent [Ni(II)-
NH2-Gly-Gly-His-COOH] and
The presence of a peracid such as oxone
The initiation of the mechanism involves the oxidation of
the Ni(II) to Ni(III) by the peracid
Creates a high valent oxo species.
In turn, the species can then strip an electron from aromatic
amino acid side chains, like a tyrosine residue.](https://image.slidesharecdn.com/crosslinkingpp-120106011336-phpapp02/85/Cross-linking-pp-10-320.jpg)
Cross linking pp
- 2. ‼ Cross-links - ionic or covalent bonds that form between two polymer chains. ‼ In biological science, cross-linking - a molecular reaction - joins two molecular units - study their interactions. ‼ The technique of cross-linking - very important - information - structure and function of proteins. ‼ Cross-linking also helps - vital information - receptors, signaling cascades, and multiprotein complexes ‼The information - cross-linking - powerful - higher resolution - structural data
- 3. ‼ Mapping protein-protein interactions to specific amino acids or domains ‼ The most important advantage - allows for non-covalent protein-protein interactions ‼ That are transient or dependent on specific physiological conditions ‼ To be captured in long term covalent complexes ‼ That maintain the information even through further processing, including purification, enrichment, and analysis
- 4. Bifunctional cross-linking ‼ Reagents contain two reactive groups, thus creating a way to covalently link two target groups. ‼ These target groups can be on the same protein, or on different proteins. ‼ The usual target for these reagents are nucleophilic side chains like tyrosine or cysteine. Homobifunctional Cross-Linking ‼ Both reactive groups are the same. ‼ The majority of homobifunctional cross-linking reagents create intramolecular crosslinks.
- 5. Heterobifunctional Cross-Linking ‼ In heterobifunctional cross-linking reagents the reactive groups are different. ‼ This allows for intermolecular crosslinks. ‼ Possessing different reactive groups allows the crosslinker to make conjugations between specific functional groups on different molecules and help minimize self-conjugation.
- 6. Crosslinker Reactivities 1. Imidoesters: Imidoester crosslinkers react with primary amines to form amidine bonds. The resulting amidine is protonated and therefore has a positive charge at physiological pH. Imidoester homobifunctional crosslinkers can move through cell membranes. Crosslinking proteins within the membrane and With intramolecular crosslinking to studying protein subunit structure.
- 7. 2. NHS-Esters: React with α-amine groups on the N-termini of proteins And ε-amines on lysine residues to form amide bonds. A covalent amide bond is formed during the reaction, releasing N-hydroxysuccinimide. Performed in phosphate, bicarbonate/ carbonate, or borate buffers. Reaction with primary amines on the molecule’s surface could result in conformational change of the protein, which may result in loss of biological activity.
- 8. 3. Maleimides: Maleimide groups react with sulfhydryl groups. pH of the reaction is between pH 6.5 and 7.5. The thioether linkage that forms is stable and is not reversible.
- 9. 4. Haloacetyls : The most commonly used -haloacetyl crosslinkers contain the iodoacetyl group React with sulfhydryl groups at physiological pH. Nucleophilic substitution of iodine with a thiol producing a stable thioether linkage
- 10. Oxidative Cross-Linking Introduced by Brown et al ., Highly specific. The process is mediated by a Ni(II)-peptide reagent [Ni(II)- NH2-Gly-Gly-His-COOH] and The presence of a peracid such as oxone The initiation of the mechanism involves the oxidation of the Ni(II) to Ni(III) by the peracid Creates a high valent oxo species. In turn, the species can then strip an electron from aromatic amino acid side chains, like a tyrosine residue.
- 11. This radical cation can then, through electrophilic attack, covalently couple to a nearby tyrosine or cysteine. Finally, a hydrogen atom is abstracted by an unknown species. The final product is a cross-linked protein complex. This cross-linking method is advantageous in that through an affinity cross-linking strategy The Ni(II)-peptide can be selectively delivered to a protein And activated at a desired time by the addition of the peracid. In turn, this reaction may be highly localized and Avoids modifications to unrelated proteins Making data interpretation less complicated.
- 12. Photoreactive crosslinking The nature of most cross-linking methods prevents their use in live cells. Recently, photo-reactive amino acid analogs to create cross- links used. PCL-Between interacting proteins has allowed scientists to study protein complexes in-vivo. Analogs of leucine and methionine, both featuring photosensitive diazirine rings, are fed to growing cells. These analogs are incorporated into proteins and create cross-links between interacting proteins when exposed to ultraviolet light