Dietrich C, Boscheinen O, Scharf K D, Schmitt L, Tampé R
Physik Department, Technische Universität München, Garching, Germany.
Biochemistry. 1996 Jan 30;35(4):1100-5. doi: 10.1021/bi952305+.
The coupling of a DNA-binding protein to self-organized lipid monolayers is examined at the air-water interface by means of film balance techniques and epifluorescence microscopy. We used two recombinant species of the heat shock factor HSF24 which differ only in a carboxy-terminal histidine tag that interacts specifically with the nickel-chelating head group of a synthetic chelator lipid. As key function, HSF24 binds to DNA that contains heat-shock responsible promoter elements. In solution, DNA-protein complex formation is demonstrated for the wild type and fusion protein. Substantial questions of these studies are whether protein function is affected after adsorption to lipid layers and whether a specific docking via histidine tag to the chelator lipid leads to functional immobilization. Using lipid mixtures that allow a lateral organization of chelator lipids within the lipid film, specific binding and unspecific adsorption can be distinguished by pattern formation of DNA-protein complexes. At the lipid interface, functional DNA-protein complexes are only detected, when the histidine-tagged protein was immobilized specifically to a chelator lipid containing monolayer. These results demonstrate that the immobilization of histidine-tagged biomolecules to membranes via chelator lipids is a promising approach to achieve a highly defined deposition of these molecules at an interface maintaining their function.
利用膜天平技术和落射荧光显微镜，在气-水界面研究了DNA结合蛋白与自组装脂质单层的偶联。我们使用了热休克因子HSF24的两种重组变体，它们仅在羧基末端的组氨酸标签上有所不同，该组氨酸标签与合成螯合剂脂质的镍螯合头部基团特异性相互作用。作为关键功能，HSF24与含有热休克反应启动子元件的DNA结合。在溶液中，野生型和融合蛋白均证明可形成DNA-蛋白质复合物。这些研究的关键问题是蛋白质吸附到脂质层后其功能是否受到影响，以及通过组氨酸标签与螯合剂脂质的特异性对接是否会导致功能固定。使用能够使螯合剂脂质在脂质膜内横向组织的脂质混合物，DNA-蛋白质复合物的图案形成可区分特异性结合和非特异性吸附。在脂质界面，只有当组氨酸标记的蛋白特异性固定到含螯合剂脂质的单层时，才能检测到功能性DNA-蛋白质复合物。这些结果表明，通过螯合剂脂质将组氨酸标记的生物分子固定到膜上是一种很有前景的方法，可在保持其功能的界面上实现这些分子的高度精确沉积。
