Escherichia coli Diacylglycerol Kinase Is an α-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 phosph...
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Veröffentlicht in: | Biochemistry (Easton) 1996-07, Vol.35 (26), p.8610-8618 |
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Sprache: | eng |
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Zusammenfassung: | 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 ≥90 of DAGK's native 121 residues are α-helical, consistent with a model in which DAGK consists of two amphipathic α-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 Lα-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 (ΔΔG ‡) between pathways leading to catalytically productive folding and insertion of DAGK relative to unproductive pathways is |
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ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi9604892 |