Resolving the functional mechanism of the lipid regulator diacylglycerolkinase by solid-state NMR

• Applicant: Professor Dr. Clemens Glaubitz
• Subject Area: Structural Biology
• Term: from 2013 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 237774529

E. coli diacylglycerolkinase (DAGK) is a homotrimeric (40 kDa, 123 residue subunits), integral membrane protein, which transfers the gamma-phosphate of ATP to the lipid diacylglycerol (DAG) converting it into phosphatidic acid (PA). Lipid substrate as well as lipid product play important roles in different signaling pathways. DAGK belongs to an important class of lipid modifying enzymes, which catalyze reactions taking place in both the aqueous and the membrane phase. DAGK is distinct from other kinases with respect to sequence and structure. It does not show sequence motifs typically found in other enzymes catalyzing phosphoryl transfer reactions and there is no structural homology model available as this protein family is very diverse in structure. The specific aim of this proposal is therefore to resolve the functional mechanism of DAGK at molecular level directly within the lipid bilayer using multinuclear steady-state as well as time-resolved MAS-NMR spectroscopy complemented by cwDNP and EPR spectroscopy. Solid-state NMR offers great potential to resolve enzymatic mechanisms at the membrane interface. The methodological toolkit for probing structure, dynamics and kinetics resulting from this project will be also applicable to other lipid regulators in the future, which is an important precondition to understand the molecular basis of lipid signaling.

• DFG Programme: Research Grants
• Major Instrumentation: MAS-NMR Probenkopf
• Instrumentation Group: 1741 Festkörper-NMR-Spektrometer