Austrian Institute of Technology GmbH , AIT, Donau-City Str. 1, 1220 Vienna, Austria.
Langmuir. 2015 Mar 3;31(8):2511-6. doi: 10.1021/la504417j. Epub 2015 Feb 20.
An artificial bilayer lipid membrane system is presented, featuring the oriented encapsulation of membrane proteins in a functionally active form. Nickel nitrilo-triacetic acid-functionalized silica nanoparticles, of a diameter of around 25 nm, are used to attach the proteins via a genetically engineered histidine tag in a uniform orientation. Subsequently, the proteins are reconstituted within a phospholipid bilayer, formed around the particles by in situ dialysis to form so-called proteo-lipobeads (PLBs). With a final size of about 50 nm, the PLBs can be employed for UV/vis spectroscopy studies, particularly of multiredox center proteins, because the effects of light scattering are negligible. As a proof of concept, we use cytochrome c oxidase (CcO) from P. denitrificans with the his tag genetically engineered to subunit I. In this orientation, the P side of CcO is directed to the outside and hence electron transfer can be initiated by reduced cytochrome c (cc). UV/vis measurements are used in order to determine the occupancy by CcO molecules encapsulated in the lipid bilayer as well as the kinetics of electron transfer between CcO and cc. The kinetic data are analyzed in terms of the Michaelis-Menten kinetics showing that the turnover rate of CcO is significantly decreased compared to that of solubilized protein, whereas the binding characteristics are improved. The data demonstrate the suitability of PLBs for functional cell-free bioassays of membrane proteins.
本文提出了一种人工双层脂质膜系统，其特点是能够以功能活性形式定向包封膜蛋白。直径约为 25nm 的镍氮三乙酸功能化硅纳米颗粒通过基因工程组氨酸标签用于以均匀的取向附着蛋白质。随后，通过原位透析在颗粒周围重新形成磷脂双层，从而形成所谓的蛋白脂球（PLB）。PLB 的最终尺寸约为 50nm，可用于 UV/vis 光谱研究，特别是多氧化还原中心蛋白，因为光散射的影响可以忽略不计。作为概念验证，我们使用来自 P. denitrificans 的细胞色素 c 氧化酶（CcO），其带有基因工程组氨酸标签，位于亚基 I。在此取向中，CcO 的 P 侧朝向外部，因此可以通过还原型细胞色素 c（cc）启动电子转移。使用 UV/vis 测量来确定脂质双层中包封的 CcO 分子的占有率以及 CcO 和 cc 之间电子转移的动力学。动力学数据根据米氏动力学进行分析，表明与溶解蛋白相比，CcO 的周转率显著降低，而结合特性得到改善。数据表明 PLB 适用于膜蛋白的功能性无细胞生物测定。
