Photoaffinity labeling

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A photoaffinity label is a biochemical method for the molecular marker by a UV light-activated label transfer or cross-linking of a molecule after binding of another molecule in vivo or in vitro .

principle

Diazirine

Proteins and other molecules bind to other molecules with a specific affinity . Photoaffinity labeling uses photoreactive molecules (e.g. aryl azides , diazirines ) as one of the reactive groups of a crosslinker in proteins in order to better control the time of crosslinking, since crosslinking is only triggered with UV radiation. Arylazides produce reactive nitrenes on UV radiation , and diazirines reactive carbenes . Due to the lower selectivity of the free radical crosslinker, the functionality of the protein is often reduced by reaction of the free radical crosslinker with important functions (e.g. an active center or a binding site ). For this reason, photoreactive crosslinkers are mostly used when no or only one amine or sulfhydryl group is available for selective crosslinking, or when subsequent functionality is insignificant. The crosslinker can then be selectively cleavable or contain other signal-carrying parts (reporter molecules) to facilitate identification, e.g. B. radioactive isotopes or fluorophores . The affinity of the crosslinker can be checked by adding radical scavengers .

There are also photoreactive diazirine-containing analogs of the amino acids leucine ( photo-leucine ), methionine and p-benzoyl-phenylalanine , which can already be incorporated into the protein during translation in vivo . The amino acid derivatives are generated by metabolic labeling by feeding with the photoreactive amino acid derivatives and reduced concentrations of the respective unmodified amino acid. The protein characterization is mostly done by mass spectrometry , western blot or protein sequencing .

When developing a photoaffinity label, the chemical stability of the reporter molecules when coupling to the photoreactive group is a development goal.

history

Photoaffinity labeling was developed by Frank Westheimer in 1962 . From 1969 onwards, arylazides were used to mark antibodies .

literature

  • Vila-Perello, M., et al. (2007). Covalent capture of phospho-dependent protein oligomerization by site-specific incorporation of a diazirine photo-cross-linker . In: J. Am. Chem. Soc. 129 (26): 8068-69.
  • Bomgarden, R. (2008). Studying Protein Interactions in Living Cells . In: Gen. Closely. News. , Volume 28, Number 7.
  • P.-O. Hétu, et al. (2008) Photo-crosslinking of proteins in intact cells reveals a dimeric structure of cyclooxygenase-2 and an inhibitor-sensitive oligomeric structure of microsomal prostaglandin E2 synthase-1 . In: Arch Biochem Biophys . doi : 10.1016 / j.abb.2008.04.038
  • Weaver, MS, et al. (2008) The copper-binding domain of sparc mediates cell survival in vitro via interaction with integrin beta 1 and activation of integrin-linked kinase . In: J Biol Chem . doi : 10.1074 / jbc.M706563200

Individual evidence

  1. ↑ J. Sumranjit, SJ Chung: Recent advances in target characterization and identification by photoaffinity probes. In: Molecules . Volume 18, Number 9, 2013, pp. 10425-10451. doi : 10.3390 / molecules180910425 . PMID 23994969 .
  2. ↑ JB Denny, Günter Blobel : 125I-labeled crosslinking reagent that is hydrophilic, photoactivatable, and cleavable through an azo linkage. In: Proceedings of the National Academy of Sciences . Volume 81, Number 17, September 1984, pp. 5286-5290. PMID 6433347 . PMC 391688 (free full text).
  3. ↑ AE Ruoho, H. Kiefer, PE Roeder, SJ Singer: The mechanism of photoaffinity labeling. In: Proceedings of the National Academy of Sciences . Volume 70, Number 9, September 1973, pp. 2567-2571, ISSN  0027-8424 . PMID 4517671 . PMC 427057 (free full text).
  4. ^ M. Suchanek, A. Radzikowska, C. Thiele: Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells . In: Nature Methods . 2, No. 4, 2005, pp. 261-268. doi : 10.1038 / nmeth752 . PMID 15782218 .
  5. ↑ D. Robinette, N. Neamati, KB Tomer, CH Borchers: photoaffinity labeling combined with mass spectrometric Approaches as a tool for structural proteomics. In: Expert review of proteomics. Volume 3, Number 4, August 2006, pp. 399-408. doi : 10.1586 / 14789450.3.4.399 . PMID 16901199 . PMC 2266983 (free full text).
  6. ^ SJ Evans, FL Moore: Nonradioactive photoaffinity labeling of steroid receptors using western blot detection system. In: Methods in molecular biology (Clifton, NJ). Volume 176, 2001, pp. 261-272. doi : 10.1385 / 1-59259-115-9: 261 . PMID 11554327 .
  7. ↑ M. Dong, LJ Miller: Direct demonstration of unique mode of natural peptide binding to the type 2 cholecystokinin receptor using photoaffinity labeling. In: Peptides . Volume 46, August 2013, pp. 143-149. doi : 10.1016 / j.peptides.2013.06.007 . PMID 23770253 . PMC 3739435 (free full text).
  8. ↑ Haijun Guo, Zhengqiu Li: Developments of bioorthogonal handle-containing photo-crosslinkers for photoaffinity labeling. In: Med. Chem. Commun. 2017, doi : 10.1039 / C7MD00217C .
  9. ↑ A. Singh, ER Thornton, FH Westheimer: The photolysis of diazoacetylchymotrypsin. In: The Journal of biological chemistry. Volume 237, September 1962, pp. 3006-3008. PMID 13913310 . PDF .
  10. ↑ RJ Vaughan, FH Westheimer: A method for marking the hydrophobic binding sites of enzymes. An insertion into the methyl group of an alanine residue of trypsin. In: Journal of the American Chemical Society. Volume 91, Number 1, January 1969, pp. 217-218. PMID 5782459 .
  11. ↑ GWJ Fleet, RR Porter, JR Knowles: Affinity Labeling of Antibodies with aryl nitrenes as Reactive Group. In: Nature. 224, 1969, pp. 511-512, doi : 10.1038 / 224511a0 .
  12. ^ CA Converse, FF Richards: Two-stage photosensitive label for antibody combining sites. In: Biochemistry. Volume 8, Number 11, November 1969, pp. 4431-4436. PMID 4187591 .