Crosslinking is the process of chemically joining to or more molecules by a covalent bond. Photo-reactive crosslinking require energy from light to initiate. These groups are chemically inert compounds that become reactive when exposed to UV or visible light.

1. Dipali Simoniya,
M.Sc Biochemistry

2. Cross-linking
 Cross-linking is the process of chemically joining two or more
molecules by a covalent bond.
 Cross-linkers are molecules that contain two or more reactive ends
capable of chemically attaching to specific functional groups on
proteins or other molecules.
1) Intra molecular crosslink:- Attachment between two groups on a
single protein structure.
2) Inter molecular crosslink:- Attachment between groups on
two different proteins.

3. CROSSLINKING
INTER
MOLECULAR
INTRA
MOLECULAR

4. Fig. 1. The typical analysis of a cross-linked sample by shotgun proteomics. (A) The protein or proteins are incubated with a
residue specific cross-linking reagent. (B) Residues within the range of the cross-linking reagent are then covalently bonded and
transient interactions are stabilized. (C) The protein is then digested by a specific protease to form peptides. (D) Data-dependent
acquisition is used to identify peptides as they elute from an HPLC directly coupled to the mass spectrometer. (E) The identified
peptides are then fragmented to provide sequence specific information.

5. Formation of cross linking
 Cross-links can be formed by chemical reactions that are
initiated by heat, pressure, change in pH, or radiation.
 Cross-linking can also be induced in materials that are
normally thermoplastic through exposure to a radiation source,
such as electron beam exposure, gamma-radiation, or UV light.

6. In-vivo cross-linking
 In-vivo cross-linking of protein complexes using photo-reactive
amino acid analogs was introduced in 2005 by researchers
from the Max Planck Institute of Molecular Cell Biology and
Genetics.
 In this method, cells are grown
with photoreactive diazirine analogs to leucine and methionine,
which are incorporated into proteins.

7. Photo-reactive amino acid analogue
 Photo-reactive amino acid analogues are artificial
analogues of natural amino acids that amino acid
analogs may be incorporated into proteins and
peptides in vivo or in vitro.
 Photo-reactive amino acid analogs in common use are
photoreactive diazirin analogs to leucine and
methionine, and para-benzoylphenylalanine.

8. Fig- Structures of L-Photo-Leucine and L-Photo-Methionine and
natural analogues.

10. L-Photo-leucine and L-Photo-methione
 L-Photo-Leucine and L-Photo-Methionine are amino acid
derivatives that contain diazirine rings for ultraviolet (UV)
photocross-linking of proteins.
 These photo-active amino acids substitute for their respective
natural amino acids and are incorporated directly into proteins
using the endogenous mammalian translation machinery.
 Using L-Photo-Leucine and L-Photo-Methionine together
maximizes the probability of cross-linking protein complexes.

11.  The resulting complexes have an increased molecular weight
that is apparent by SDS-PAGE and Western blot.
Fig:- Standard Diazirine ring

12. Fig:- Photoreactive amino acid protocol
Upon exposure to ultraviolet light, they are activated and
covalently bind to interacting proteins that are within a few
angstroms of the photo-reactive amino acid analog.

13. Uses of L-Photo-Leucine
 L-Photo-Leucine is used as a substitute for leucine
 Used for studying protein-protein interactions
 Used for Photo affinity labelling
 They are used non-toxic and are used in combination with
chemical cross-linkers

14. References
1. Suchanek, M., Radzikowska, A., and Thiele, C. (2005).
“Photo-leucine and photo-methionine allow identification of
protein-protein interactions in living cells”. Nature Methods 2
:261. doi:10.1038/nmeth752.PMID 15782218
2. Bomgarden, R. (2008). Studying Protein Interactions in
Living Cells. Gen. Eng. News. Vol.28, 7._P.-O.
3. Proteomics/ protein-protein interactions/ cross-linking
4. www.thermofisher.com
5. www.sciencemag.org