Elizabeth Petro - (1) Photoaffinity labeling the lipid binding site of mammalian diacylglycerol kinase

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      Publication Details (including relevant citation   information):

      Elizabeth J. Petro, Becky Tu-Sekine, Meng M. Rowland, Sammy Eni   Eni, Michael D. Best, Lauren DeVine, Robert N. Cole, Daniel M.   Raben. "Photoaffinity labeling the lipid binding site of   mammalian diacylglycerol kinase". (poster)

    • The American Society for Biochemistry and Molecular Biology   Annual Meeting, Boston, MA (April 2013)  
    • Sigma Xi Mid-Atlantic Regional Meeting, Fairfax, VA (April   2013)  
    • Graduate Student Association Annual Poster Session, The Johns   Hopkins University School of Medicine, Baltimore, MD (May 2013)  


      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. This   research is supported by Grant GM059251 from the National   Institutes of Health.

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