Topology and Secondary Structure of the N-Terminal Domain of Diacylglycerol Kinase
- 28 September 2002
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 41 (42), 12876-12882
- https://doi.org/10.1021/bi020335o
Abstract
Prokaryotic diacylglycerol kinase (DAGK) functions as a homotrimer of 13 kDa subunits, each of which has three transmembrane segments. This enzyme is conditionally essential to some bacteria and serves as a model system for studies of membrane protein biocatalysis, stability, folding, and misfolding. In this work, the detailed topology and secondary structure of DAGK's N-terminus up through the loop following the first transmembrane domain were probed by NMR spectroscopy. Secondary structure was mapped by measuring 13C NMR chemical shifts. Residue-to-residue topology was probed by measuring 19F NMR relaxation rates for site-specifically labeled samples in the presence and absence of polar and hydrophobic paramagnetic probes. Most of DAGK's N-terminal cytoplasmic and first transmembrane segments are α-helical. The first and second transmembrane helices are separated by a short loop from residues 48 to 52. The first transmembrane segment extends from residues 32 to 48. Most of the N-terminal cytoplasmic domain lies near the interface but does not extend deeply into the membrane. Finally, catalytic activities measured for the single cysteine mutants before and after chemical labeling suggest that the N-terminal cytoplasmic domain likely contains a number of critical active site residues. The results, therefore, suggest that DAGK's active site lies very near to the water/bilayer interface.Keywords
This publication has 9 references indexed in Scilit:
- Oxygen as a Paramagnetic Probe of Membrane Protein Structure by Cysteine Mutagenesis and19F NMR SpectroscopyJournal of the American Chemical Society, 2002
- Thiol modification of diacylglycerol kinase: dependence upon site membrane disposition and reagent hydrophobicity.FEBS Letters, 2000
- Different Micellar Packing and Hydrophobicity of the Membrane Probes TEMPO and TEMPOL Influence Their Partition Between Aqueous and Micellar Phases Rather than Location in the Micelle InteriorJournal of Colloid and Interface Science, 1998
- Escherichia coli Diacylglycerol Kinase Is an Evolutionarily Optimized Membrane Enzyme and Catalyzes Direct Phosphoryl TransferJournal of Biological Chemistry, 1997
- Escherichia coli diacylglycerol kinase: a case study in the application of solution NMR methods to an integral membrane proteinBiophysical Journal, 1997
- Use of 19F NMR to probe protein structure and conformational changes.Annual Review of Biophysics and Biophysical Chemistry, 1996
- NMR View: A computer program for the visualization and analysis of NMR dataJournal of Biomolecular NMR, 1994
- Molecular characterization of a STreptococcus mutans mutant altered in environmental stress responsesJournal of Bacteriology, 1993
- Empirical correlation between protein backbone conformation and C.alpha. and C.beta. 13C nuclear magnetic resonance chemical shiftsJournal of the American Chemical Society, 1991