Elizabeth Petro - (1) Seeking the lipid substrate binding site: structural studies of eukaryotic diacylglycerol kinases (DGKs)

Document created by Elizabeth Petro on Aug 22, 2014
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  Publication Details (including relevant citation   information):

  "Seeking the lipid substrate binding site: structural studies of   eukaryotic diacylglycerol kinases (DGKs)." Biological Chemistry   Doctoral Dissertation, The Johns Hopkins University School of   Medicine, Baltimore, MD (December 2013).


  Very little   is known about the structure of the catalytic domain of   eukaryotic diacylglycerol kinases (DGKs), a family of interfacial   enzymes implicated in a number of physiological roles and human   diseases. Constructs of the catalytic domain could be expressed   in Escherichia coli and extracted, refolded, and   purified from inclusion bodies, but when subjected to analytical   gel filtration chromatography, these constructs eluted in the   void volume in what appeared to be microscopic aggregates   unsuitable for x-ray crystallography. Adding glutathione   S-transferase, thioredoxin, or maltose binding protein as   N-terminal fusion tags did not improve the constructs’   solubility. Coexpressing with bacterial chaperones increased the   yield in the supernatant after high-speed centrifugation, but the   protein still eluted in the void upon analytical gel filtration   chromatography. DGK constructs expressed in insect cells were   likewise insoluble and unsuitable for x-ray crystallography. Loss   of enzymatic activity of purified DGK could be mitigated by   including 50% (v/v) glycerol and storing at -80°C, and by   including detergent, lipid, and protein activators following   thawing. Certain polybasic proteins and substrate analogs   increase the in vitro enzymatic activity of purified   DGK, whereas hirudin decreases its activity under certain   conditions. DGK is able to use certain diglyceride analogs as   substrates. Bimolecular fluorescence complementation was unable   to show whether DGK-theta dimerized or interacted with tau when   overexpressed in mammalian cells. Thrombin was   able to digest DGK-theta after folding, but none of the washing   conditions tested were able to separate the digested fragments,   so while digestion with thrombin appeared to activate DGK-theta,   whether the activation was due to relief from auto-inhibition or   some other effect from the thrombin could not be resolved.  Photoaffinity   labels were able to enzymatically inactivate purified DGK-theta   in a probe-, ultraviolet-, and time-dependent way, but only when   the linker between the substrate analog moiety and the   photoactivatable moiety was sufficiently short. Mass spectrometry   studies were unable to detect peptides shifted by the predicted   molecular weight of the photoaffinity labels or their fragments.   This work should guide future studies of the structure of   eukaryotic DGKs.

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