Escherichia coli diacylglycerol kinase is an alpha-helical polytopic membrane protein and can spontaneously insert into preformed lipid vesicles
Escherichia coli diacylglycerol kinase is an alpha-helical polytopic membrane protein and can spontaneously insert into preformed lipid vesicles
复制标题
DOI：
10.1021/bi9604892
复制
发表时间：
1996-07-02
期刊：
影响因子：
2.9
通讯作者：
Smith, SO
中科院分区：
文献类型：
--
作者：
Sanders, CR;Czerski, L;Smith, SO
大肠杆菌二酰基甘油激酶（DAGK）是一种13.2kDa的酶，其跨越细胞质膜三次。使用多组氨酸标签和Ni（II）-螯合层析将功能DAGK纯化至均一。磷脂酰胆碱多层膜中DAGK的透射傅立叶变换红外光谱（FT-IR）导致以下结论：DAGK的天然121个残基中大于或等于90个是α-螺旋，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。偏振衰减全反射FT-IR研究DAGK在定向多层膜产生的数据与拓扑结构的安排，其中三个跨膜螺旋排列良好的双层正常，而两个两亲性螺旋是近似平行的膜平面。DAGK自发插入到预先形成的脂质囊泡的能力进行了检查，使用一种新的检测系统，涉及DAGK催化磷酸化的荧光标记的甘油二酯。当胶束DAGK稀释到L（α）相囊泡自发插入的酶是相当有效的（约30%）。DAGK再折叠和从脱脂脲溶解的DAGK插入脂质囊泡中在凝胶至液晶相变温度以上也是适度有效的（3.8 +/-2.1%）。插入研究表明，相对于非生产性途径，导致DAGK催化生产性折叠和插入的途径之间的能量屏障（Δ Δ G（双匕首））的差异是：
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 greater than or equal to 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(double dagger)) between pathways leading to catalytically productive folding and insertion of DAGK relative to unproductive pathways is