Diazirine Amino Acids for Photo Cross-Linkage in Living Cells

Published on 26.04.2018

Iris Biotech introduces a comprehensive set of photo-crosslinking amino acids bearing the diazirine moiety.

Diazirine Amino Acids for Photo Cross-Linkage in Living Cells

Irradiation of diazirines with UV light (ca. 350 nm - 360 nm) yields a highly reactive carbene species that can undergo insertions into C-C, C-H, O-H and X-H (X = heteroatom) bonds of neighboring molecules to irreversibly form a covalent bond. The diazirine moiety is the smallest of all photophores, so introduction of a diazirine-bearing amino acid into a peptide or protein usually does not impair its biological activity. Further advantages of diazirine crosslinkers are their stability at room temperature, as well as their relative stability to nucleophiles, and to both acidic and basic conditions.

Applications:

Probing protein–protein and protein- peptide interactions by photo-crosslinking (e.g. site-specific incorporation of Photo-Lys into glutathione S-transferase allowing covalent crosslinking of the two subunits of the dimeric protein in E. coli)
Probing protein- peptide interactions in order to identify cellular targets of peptides of interest
Studying protein−drug interactions and identifying new drug targets
By comparing results obtained from different proteomic setups (e.g. live cells and cell lysates), more putative targets can be identified.

These amino acids are available in Fmoc- as well as Boc-protected versions for their incorporation into synthetic peptides via standard coupling methods. Furthermore, the unprotected diazirine amino acids are also available for incorporation into expressed peptides and proteins by utilizing the appropriate aminoacyl-tRNA synthetase/tRNA pair. A combination of synthetic and recombinant approaches utilizing NCL has been demonstrated as well. Unnatural amino acids are frequently toxic to cells; however, these photo-amino acids are functional and nontoxic, which allows them to be a premium tool for studying mechanisms and interactions in living cells.

Identification of intracellular targets using a diazirine-bearing probe. By attaching a reporter group or tag to the probe, the target of the binding probe can be either identified using the reporter group, or isolated using the tag.

References:

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