Geiss Andreas F, Khandelwal Raghav, Baurecht Dieter, Bliem Christina, Reiner-Rozman Ciril, Boersch Michael, Ullmann G Matthias, Loew Leslie M, Naumann Renate L C
Biosensor Technologies, Austrian Institute of Technology GmbH, AIT , Donau-City Street 1, 1220 Vienna, Austria.
University of Natural Resources and Life Sciences , Gregor-Mendel-Straße 33, 1180 Wien, Austria.
J Phys Chem B. 2017 Jan 12;121(1):143-152. doi: 10.1021/acs.jpcb.6b11116. Epub 2017 Jan 4.
His-tag technology is employed to bind membrane proteins, such as the bc complex and the reaction center (RC) from Rhodobacter sphaeroides, to spherical as well as planar surfaces in a strict orientation. Subsequently, the spherical and planar surfaces are subjected to in situ dialysis to form proteo-lipobeads (PLBs) and protein-tethered bilayer membranes, respectively. PLBs based on Ni-nitrileotriacetic acid-functionalized agarose beads that have diameters ranging from 50 to 150 μm are used to assess proton release and membrane potential parameters by confocal laser-scanning microscopy. The pH and potential transients are thus obtained from bc activated by the RC. To assess the turnover of bc excited by the RC in a similar setting, we used the planar surface of an attenuated total reflection crystal modified with a thin gold layer to carry out time-resolved surface-enhanced IR absorption spectroscopy triggered by flash lamp excitation. The experiments suggest that both proteins interact in a cyclic manner in both environments. The activity of the proteins seems to be preserved in the same manner as that in chromatophores or reconstituted in liposomes.
His标签技术用于将膜蛋白，如来自球形红细菌的bc复合物和反应中心（RC），以严格的方向结合到球形和平面表面上。随后，对球形和平面表面进行原位透析，分别形成蛋白脂质珠（PLB）和蛋白质 tethered 双层膜。基于直径范围为50至150μm的镍 - 次氮基三乙酸功能化琼脂糖珠的PLB用于通过共聚焦激光扫描显微镜评估质子释放和膜电位参数。由此从被RC激活的bc获得pH和电位瞬变。为了在类似的环境中评估被RC激发的bc的周转，我们使用用薄金层修饰的衰减全反射晶体的平面表面来进行由闪光灯激发触发的时间分辨表面增强红外吸收光谱。实验表明，两种蛋白质在两种环境中均以循环方式相互作用。蛋白质的活性似乎以与在载色体中或在脂质体中重构时相同的方式得以保留。
