Real Time 31P and Steady State 19F/13C Solid State NMR on Integral Membrane Protein E.coli Diacylglycerol Kinase

Abstract

The integral membrane protein Diacylglycerol Kinase (DGK) from E.coli can be used as model system for prokaryotic kinases, playing an important role in microbial physiology. In its active conformation, DGK, consisting of three transmembrane helices, forms a homotrimer with one putative active site per monomer. DGK, as the smallest known kinase, catalyses the phosphorylation of diacylglycerol to phosphatidyl acid by utilizing MgATP at the interface membrane-cytoplasm. These features make DGK an attractive model protein for research on structure of membrane proteins in lipid bilayers, as well as on its function as a lipid regulator.Solid-state NMR is a unique tool for the investigation of membrane proteins in their native environment and for probing enzymatic reactions regardless of their compartmentalization. For such experiments, amount of protein and quality of the sample preparation are crucial. Expression, purification, reconstitution and sample preparation were optimised, so that a sample of DGK in high quality became available in amounts necessary for ssNMR experiments, while maintaining its specific activity.A 31P Real Time MAS experiment was designed and implemented, to investigate for the first time, simultaneously, ATP hydrolysis and phosphorylation of a substrate analog by a membrane protein, inside the lipid bilayer. From these data, the rate constants of enzymatic activity were determined. Furthermore, inhibition experiments with orthovanadate, (BeF2)x and AlF3 were carried out. The inhibiting species was identified by 19F MAS NMR. This method provides the opportunity to investigate the enzymatic mechanism in real time, with atomic resolution.A first fingerprint of structural details were obtained with a 2D 13C-13C MAS ssNMR PDSD type experiment on a uniformly 13C/15N labelled sample of a thermostable mutant- and WT-DGK. Further insight into structural details will be obtained by selective labelling.