Sanders C R, Czerski L, Vinogradova O, Badola P, Song D, Smith S O
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Biochemistry. 1996 Jul 2;35(26):8610-8. doi: 10.1021/bi9604892.
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.
大肠杆菌二酰基甘油激酶（DAGK）是一种13.2 kDa的酶，它三次穿过细胞质膜。使用多组氨酸标签和镍（II）螯合色谱法将功能性DAGK纯化至同质。对磷脂酰胆碱多层膜中的DAGK进行透射傅里叶变换红外光谱（FT-IR）分析得出结论，DAGK的121个天然残基中≥90%为α螺旋，这与DAGK由两个两亲性α螺旋和三个跨膜螺旋组成的模型一致。对取向多层膜中的DAGK进行偏振衰减全反射FT-IR研究，得到的数据与一种拓扑排列一致，即三个跨膜螺旋与双层法线良好对齐，而两个两亲性螺旋与膜平面大致平行。使用一种涉及DAGK催化荧光标记二酰基甘油磷酸化的新型检测系统，研究了DAGK自发插入预先形成的脂质体的能力。当将胶束状DAGK稀释到Lα相脂质体中时，该酶的自发插入相当有效（约30%）。在高于凝胶到液晶相转变温度时，脱脂尿素溶解的DAGK重折叠并插入脂质体的效率也适中（3.8±2.1%）。插入研究表明，导致DAGK催化性有效折叠和插入的途径与非生产性途径之间的能垒差异（ΔΔG++）<4 kcal/mol。然而，对DAGK突变体形式进行的额外研究表明，DAGK在体内和体外重折叠/插入途径之间的差异可能很大。
