We are taking a photoaffinity labeling approach to identifying the lipid binding site on mammalian diacylglycerol kinase (DGK). The probe includes a diacylglycerol(DAG)-like moiety, except with ether linkages instead of acyl linkages to prevent chain migration, in order to target the probe to the lipid binding site on DGK.  The probe also includes a benzophenone moiety, in order to covalently crosslink to whatever amino acids happen to be near it when excited with 360 nm light. In this way, the probe is expected to covalently modify the enzyme near the DAG-binding site(s). When the probe is incorporated into liposomes, DGK's enzymatic activity is reduced in a probe- and UV-dependent way, but only when the linker connecting the benzophenone moiety and the DAG-like moiety on the probe is sufficiently short. Probes with longer linkers are able to be used as substrates by the enzyme, suggesting that they are able to access the active site but not to crosslink to the protein. Mass spectrometry studies are currently underway to map the site of crosslinking. Preliminary results show reduced amino acid coverage for in-gel trypsin-digested crosslinked enzyme as compared to control: these missing peptides are candidates for sites of crosslinking.

Photoaffinity labeling to map the lipid binding site of mammalian diacylglycerol kinase

246th National Meeting (2013)

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