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.
Address (URL): http://