User:MichaHalle/Protein cross-linking
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Protein cross-linking is a biochemical method to probe structures
of proteins or detrmine interaction sites between proteins. Proteins can for example be cross linked to their substrates, in the case of enzymes, or to binding partners, like nucleic acids or small molecules. Cross-linking can also occur between two proteins or even intramolecular. Cross links can be analysed for example by SDS-PAGE. The exact site of the cross link can be determined using mass spectrometry. {{cite journal|last=Sinz|first=Andrea|journal=Mass spectrometry reviews|year=2006|volume=25|issue=4|pages=663-682|doi=10.1002/mas.20082|pmid=16477643|accessdate=28 November 2013}}. This information can be used to probe the 3-dimensional structure, as identified cross links can be used as a molecular ruler giving constraints for protein structure modeling.
Chemistry
Chemical cross linking reagents generally consist of 2 reactive groups and a spacer. In addition to that there can be chemical modifications for improved detection (e.g. isotope labelling), for purification (e.g. biotin) or cleavage (e.g. disulfide). Often used reactive sites are NHS-esters that target amines in lysines or the N-terminus of proteins. Maleimide esters can react with sulfhydryl groups in cysteine. Photoreactive groups like benzophenone or diazirine react non-specific[http://www.piercenet.com/Objects/View.cfm?type=Page&ID=FE7F690D-58AE-4342-AE85-BA94DCA642F8 Crosslinking Reagents Technical Handbook, Pierce Biotechnology, Inc., 2006].
= Isotope labelling =
Cross linkers might be differentially labelled with isotopes for improved detection in mass spectrometry. In a cross-linking experiment a mixture of the light and heavy cross linker are mixed at a ratio of 1:1. This results in a distinct isotope pattern that is used to diferentiate cross linked from non-cross linked peptide species.
= Spacer Length =
Zero-length cross links up to NHS-PEG-NHS....
= Reactive Sites =
| class="wikitable" | ||
| Name | Reactivity against | Header text |
|---|---|---|
| NHS-esters | Lys and N-Terminus (+ hydroxyl groups in Ser, Thr, Tyr) | Example |
| Maleimide esters | Cys | Example |
| benzophenone | non-specific | Example |
| diazirine | non-specific | Example |
Mass spectrometry
Software analysis
References
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External links
- [http://www.example.com/ example.com]