1 research outputs found
Diazirine-Containing Photoactivatable Isoprenoid: Synthesis and Application in Studies with Isoprenylcysteine Carboxyl Methyltransferase
Photoaffinity
labeling is a useful technique employed to identify
protein–ligand and protein–protein noncovalent interactions.
Photolabeling experiments have been particularly informative for probing
membrane-bound proteins where structural information is difficult
to obtain. The most widely used classes of photoactive functionalities
include aryl azides, diazocarbonyls, diazirines, and benzophenones.
Diazirines are intrinsically smaller than benzophenones and generate
carbenes upon photolysis that react with a broader range of amino
acid side chains compared with the benzophenone-derived diradical;
this makes diazirines potentially more general photoaffinity-labeling
agents. In this article, we describe the development and application
of a new isoprenoid analogue containing a diazirine moiety that was
prepared in six steps and incorporated into an <b>a</b>-factor-derived
peptide produced via solid-phase synthesis. In addition to the diazirine
moiety, fluorescein and biotin groups were also incorporated into
the peptide to aid in the detection and enrichment of photo-cross-linked
products. This multifuctional diazirine-containing peptide was a substrate
for Ste14p, the yeast homologue of the potential anticancer target
Icmt, with <i>K</i><sub>m</sub> (6.6 μM) and <i>V</i><sub>max</sub> (947 pmol min<sup>–1</sup> mg<sup>–1</sup>) values comparable or better than <b>a</b>-factor
peptides functionalized with benzophenone-based isoprenoids. Photo-cross-linking
experiments demonstrated that the diazirine probe photo-cross-linked
to Ste14p with observably higher efficiency than benzophenone-containing <b>a</b>-factor peptides