Label transfer

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A label transfer (engl. Label , marking ') describes the cross-linking of interacting proteins or protein subunits with a signal-bearing and cleavable cross-linker.

properties

When two proteins or two subunits of a protein bind to each other , this interaction can be fixed by covalently linking these two parts.

In a label transfer, crosslinkers with two different reactive groups (heterobifunctional crosslinkers) are used that simultaneously carry a measurable signal and a selectively cleavable bond. Biotin , a fluorophore or a radionuclide are usually used as signals . Selectively cleavable bonds are e.g. B. disulfide compounds or ethers . The protein for which interaction partners are sought is called bait protein (English for 'bait'), the binding protein as prey protein (English for 'prey').

The bait protein is first coupled with the crosslinker. Heterobifunctional crosslinkers are used so that the coupling can be controlled by different reaction conditions and, after the bait protein has been labeled, the second crosslinking function is only coupled after it has been brought together with the prey protein. This reduces a reaction of both reactive groups of the crosslinker with the bait protein (self- crosslinking , not neighboring crosslinking ). After adding the prey protein, a change in the ambient conditions (e.g. pH value , ultraviolet light ) makes the second reactive group of the crosslinker reactive and statistically binds to molecules in the immediate vicinity of the bait protein. Because the two proteins are bound to one another, the second reactive group is in the immediate vicinity of the prey protein and reacts preferentially with it. Since the signal contained in the crosslinker lies between the cleavable site and the second reactive group on the prey protein, after crosslinking and subsequent cleavage of the crosslinker, the signal is transferred from the originally marked bait protein to the prey protein. Since the crosslinking was removed by cleavage, can by appropriate methods, the labeled prey protein characterized to be such. B. by Western blot , by mass spectrometry or by Edman degradation .

literature

Individual evidence

  1. ^ DA Fancy: Elucidation of protein-protein interactions using chemical cross-linking or label transfer techniques. In: Current Opinion in Chemical Biology . Volume 4, Number 1, February 2000, pp. 28-33, PMID 10679368 .
  2. ↑ SS Andrews, ZB Hill, BG Perera, DJ Maly: Label transfer reagents to probe p38 MAPK binding partners. In: ChemBioChem Volume 14, Number 2, January 2013, pp. 209-216, doi : 10.1002 / cbic.201200673 . PMID 23319368 . PMC 3762675 (free full text).
  3. ^ B. Liu, CT Archer, L. Burdine, TG Gillette, T. Kodadek: Label transfer chemistry for the characterization of protein-protein interactions. In: Journal of the American Chemical Society . Volume 129, Number 41, October 2007, pp. 12348-12349, doi : 10.1021 / ja072904r . PMID 17894490 . PMC 2529226 (free full text).
  4. ↑ 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 of the United States of America . Volume 81, Number 17, September 1984, pp. 5286-5290, PMID 6433347 . PMC 391688 (free full text).
  5. ↑ CL Jaffe, H. Lis, N. Sharon: New clevable photoreactive heterobifunctional cross-linking reagents for studying membrane organization. In: Biochemistry . Volume 19, Number 19, September 1980, pp. 4423-4429, PMID 7190840 .