Escherichia coli diacylglycerol kinase is an alpha-helical polytopic membrane protein and can spontaneously insert into preformed lipid vesicles.
Escherichia coli diacylglycerol kinase (DAGK) is a 13.2 kDa enzyme which spans the cytoplasmic membrane three times. Functional DAGK was purified to homogeneity using a polyhistidine tag and Ni(II)-chelate chromatography. Transmission Fourier transform infrared spectroscopy (FT-IR) of DAGK in phosphatidylcholine multilayers led to the conclusion that > or = 90 of DAGK's native 121 residues are alpha-helical, consistent with a model in which DAGK consists of two amphipathic alpha-helices and three transmembrane helices. Polarized attenuated total reflection FT-IR studies of DAGK in oriented multilamellae yielded data consistent with a topological arrangement in which the three transmembrane helices are well-aligned with the bilayer normal while the two amphipathic helices are approximately parallel with the membrane plane. The ability of DAGK to spontaneously insert into preformed lipid vesicles was examined using a novel assay system involving DAGK-catalyzed phosphorylation of a fluorescently tagged diacylglycerol. When micellar DAGK is diluted into L alpha-phase vesicles spontaneous insertion of the enzyme is fairly efficient (ca. 30%). DAGK refolding and insertion from delipidated urea-solubilized DAGK into lipid vesicles is also modestly efficient (3.8 +/- 2.1%) above the gel to liquid crystalline phase transition temperature. The insertion studies indicate that the difference in energy barriers (delta delta G++) between pathways leading to catalytically productive folding and insertion of DAGK relative to unproductive pathways is < 4 kcal/mol. However, additional studies carried out with mutant forms of DAGK indicated that the differences between refolding/insertion pathways for DAGK in vivo and in vitro can be significant.
Sanders
C R
CR
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA. crs4@po.cwru.edu
Czerski
L
L
Vinogradova
O
O
Badola
P
P
Song
D
D
Smith
S O
SO
D000595:Amino Acid Sequence; D019852:Diacylglycerol Kinase; D004926:Escherichia coli / Q000201:enzymology*; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared
D008051:Lipid Bilayers; D008565:Membrane Proteins; D008823:Micelles; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase