Secondary structure and backbone dynamics of Escherichia coli diacylglycerol kinase, as revealed by site-directed solid-state 13C NMR

To gain insight into secondary structure and backbone dynamics, we have recorded (13)C NMR spectra of [3-(13)C]Ala-, [1-(13)C]Val-labeled Escherichia coli diacylglycerol kinase (DGK), using cross-polarization-magic angle spinning (CP-MAS) and single-pulse excitation with dipolar decoupled-magic angle spinning (DD-MAS) methods. DGK was either solubilized in n-decyl-beta-maltoside (DM) micelle or integrated into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers. Surprisingly, the (13)C NMR spectra were broadened to yield rather featureless peaks at physiological temperatures, both in DM solution or lipid bilayers at liquid crystalline phase, due to interference of motional frequencies of DGK with frequencies of magic angle spinning (MAS) or proton decoupling (10(4) or 10(5) Hz, respectively). In gel phase lipids, however, up to six distinct (13)C NMR peaks were well-resolved due to lowered fluctuation frequencies (<10(5) Hz) for the transmembrane region, the amphipathic alpha-helices and loops. While DGK can be tightly packed in gel phase lipids, DGK is less tightly packed at physiological temperatures, where it becomes more mobile. The fact that the enzymatic activity is low under conditions where motion is restricted and high when conformational fluctuations can occur suggests that acquisition of low frequency backbone motions, on the microsecond to millisecond time scale, may facilitate the efficient enzymatic activity of DGK.