Diacylglycerol kinase (DAGK) of E. coli is an integral membrane protein that functions as a 40 kDa homotrimer catalyzing the ATP-dependent phosphorylation of diacylglycerol. DAGK is a part of the membrane-derived oligosaccharide biosynthesis machinery and may be an antimicrobial drug target. In this work nuclear magnetic resonance spectroscopy (NMR) was used to characterize DAGK's structure. For NMR studies DAGK was solubilized in detergent micelles yielding a complex with the molecular weight of about 100 kDa.; As a first step towards the three dimensional structure of DAGK, backbone NMR assignment has been completed for the majority of residues by performing triple resonance experiments on a perdeuterated 15N and 13C labeled enhanced stability mutant. In addition, the membrane topology of the N-terminal region of DAGK was examined by measuring the effect of paramagnetic probes on the 19F relaxation rate of a fluorine label attached to the cysteine of single cysteine DAGK mutants. Carbon chemical shifts, 19F relaxation and nuclear Overhauser enhancement data allowed the secondary structure map of DAGK to be experimentally refined. Each monomer of 121 residues contains a micelle surface associated N-terminal domain which is composed of two helices (residues 6–15 and 18–25). Following this domain are three transmembrane (TM) helices: a relatively short TM1 (29–47) and a relatively long TM2 (52–82) and TM3 (88–118). The secondary structure elements in the DAGK monomer are connected to each other by short loops.; DAGK has also served as a model in studies of membrane protein misfolding. In this work NMR was used to demonstrate that the enhanced stability mutant misfolds to adopt a specific kinetically-trapped conformation. Misfolded DAGK is enzymatically inactive, but retains its homotrimeric form. Using guanidine hydrochloride misfolding can be reproduced in vitro, which will greatly facilitate future efforts to elucidate the nature of the misfolded conformation and the mechanism of misfolding.
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