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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