Vinogradova O, Badola P, Czerski L, Sönnichsen F D, Sanders C R
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Biophys J. 1997 Jun;72(6):2688-701. doi: 10.1016/S0006-3495(97)78912-4.
Diacylglycerol kinase (DAGK) is a 13-kDa integral membrane protein that spans the lipid bilayer three times and which is active in some micellar systems. In this work DAGK was purified using metal ion chelate chromatography, and its structural properties in micelles and organic solvent mixtures studies were examined, primarily to address the question of whether the structure of DAGK can be determined using solution NMR methods. Cross-linking studies established that DAGK is homotrimeric in decyl maltoside (DM) micelles and mixed micelles. The aggregate detergent-protein molecular mass of DAGK in both octyl glucoside and DM micelles was determined to be in the range of 100-110 kDa-much larger than the sum of the molecular weights of the DAGK trimers and the protein-free micelles. In acidic organic solvent mixtures, DAGK-DM complexes were highly soluble and yielded relatively well-resolved NMR spectra. NMR and circular dichroism studies indicated that in these mixtures the enzyme adopts a kinetically trapped monomeric structure in which it irreversibly binds several detergent molecules and is primarily alpha-helical, but in which its tertiary structure is largely disordered. Although these results provide new information regarding the native oligomeric state of DAGK and the structural properties of complex membrane proteins in micelles and organic solvent mixtures, the results discourage the notion that the structure of DAGK can be readily determined at high resolution with solution NMR methods.
二酰甘油激酶（DAGK）是一种13 kDa的整合膜蛋白，它三次跨越脂质双层，并且在某些胶束系统中具有活性。在这项工作中，使用金属离子螯合色谱法纯化了DAGK，并研究了其在胶束和有机溶剂混合物中的结构性质，主要是为了解决是否可以使用溶液核磁共振方法确定DAGK结构的问题。交联研究表明，DAGK在癸基麦芽糖苷（DM）胶束和混合胶束中是同三聚体。测定了DAGK在辛基葡萄糖苷和DM胶束中的总去污剂 - 蛋白质分子量在100 - 110 kDa范围内，远大于DAGK三聚体和无蛋白质胶束分子量之和。在酸性有机溶剂混合物中，DAGK - DM复合物高度可溶，并产生分辨率相对较好的核磁共振谱。核磁共振和圆二色性研究表明，在这些混合物中，该酶采用动力学捕获的单体结构，其中它不可逆地结合几个去污剂分子，主要是α螺旋结构，但其三聚体结构在很大程度上是无序的。尽管这些结果提供了关于DAGK天然寡聚状态以及复合膜蛋白在胶束和有机溶剂混合物中结构性质的新信息，但这些结果不支持可以用溶液核磁共振方法轻易确定DAGK高分辨率结构的观点。
