Elizabeth Petro - (1) Photoaffinity labeling of diacylglycerol kinase (DGK): seeking the lipid substrate binding site

Document created by Elizabeth Petro on Aug 26, 2014
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  Publication Details (including relevant citation   information):

  "Photoaffinity   labeling of diacylglycerol kinase (DGK): seeking the lipid   substrate binding site." Third Annual Biological Chemistry   Welcome Party & Lab Olympics Poster Session, Department of   Biological Chemistry, The Johns Hopkins University School of   Medicine, Baltimore, MD (August 2012)


  In order to identify the lipid substrate binding-site on   mammalian diacylglycerol kinase (DGK), the enzyme was expressed   in bacteria in a number of constructs for the purpose of   producing a large amount of purified protein for x-ray   crystallography. While coexpressing certain constructs with   bacterial chaperones improved the apparent solubility of DGK   after ultracentrifugation, this apparently soluble DGK elutes in   microaggregates in the void volume during analytical gel   filtration. A new photoaffinity labeling strategy was therefore   pursued, for the purpose of identifying residues near the   covalently labeled residue(s). Both probes used are substrates   for DGK, and therefore must be getting into the active site, but   do not decrease DGK’s enzymatic activity (by occluding the active   site) after exposure to UV. Preliminary studies using Triton   X-114 partitioning as an alternate means to measure covalent   labeling of DGK show that even before crosslinking, DGK   partitions into the detergent-rich phase. A new probe with a   shorter linker has recently arrived: studies are currently   underway to determine whether this new probe will properly orient   the photoactivateable part of the molecule near enough to the   enzyme to covalently crosslink to it.

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