Friedrich Marcel G, Kirste Vinzenz U, Zhu Jiapeng, Gennis Robert B, Knoll Wolfgang, Naumann Renate L C
Max Planck Institute for Polymer Research, Ackermannweg 10, Mainz, Germany.
J Phys Chem B. 2008 Mar 13;112(10):3193-201. doi: 10.1021/jp709717k. Epub 2008 Feb 19.
A systematic study of the influence of the packing density of proteins on their activity is performed with cytochrome c oxidase (CcO) from R. sphaeroides as an example. The protein was incorporated into a protein-tethered bilayer lipid membrane and CcO was genetically engineered with a histidine-tag, attached to Subunit II, and then tethered by an interaction with functionalized thiol compounds bound to a gold electrode. The packing density was varied by diluting the functionalized thiol with a nonfunctionalized thiol that does not bind to the enzyme. After attaching the CcO to the gold surface, a lipid bilayer was formed to incorporate the tethered proteins. The reconstituted protein-lipid bilayer was characterized by surface enhanced infrared reflection absorption spectroscopy (SEIRAS), electrochemical impedance spectroscopy, surface plasmon resonance, and atomic force microscopy. The activity of the proteins within the reconstituted bilayer was probed by direct electrochemical electron injection and was shown to be very sensitive to the packing density of protein molecules. At low surface density of CcO, the bilayer did not effectively form, and protein aggregates were observed, whereas at very high surface density, very little lipid is able to intrude between the closely packed proteins. In both of these cases, redox activity, measured by the efficiency to accept electrons, is low. Redox activity of the enzyme is preserved in the biomimetic structure but only at a moderate surface coverage in which a continuous lipid bilayer is present and the proteins are not forced to aggregate. Electrostatic and other interaction forces between protein molecules are held responsible for these effects.
以球形红细菌的细胞色素c氧化酶（CcO）为例，对蛋白质堆积密度对其活性的影响进行了系统研究。该蛋白质被整合到蛋白质连接的双层脂质膜中，CcO通过基因工程在亚基II上连接了组氨酸标签，然后通过与结合在金电极上的功能化硫醇化合物相互作用而连接。通过用不与酶结合的非功能化硫醇稀释功能化硫醇来改变堆积密度。将CcO连接到金表面后，形成脂质双层以纳入连接的蛋白质。通过表面增强红外反射吸收光谱（SEIRAS）、电化学阻抗谱、表面等离子体共振和原子力显微镜对重构的蛋白质-脂质双层进行了表征。通过直接电化学电子注入探测重构双层内蛋白质的活性，结果表明其对蛋白质分子的堆积密度非常敏感。在CcO的低表面密度下，双层不能有效形成，观察到蛋白质聚集，而在非常高的表面密度下，很少有脂质能够侵入紧密堆积的蛋白质之间。在这两种情况下，通过接受电子效率测量的氧化还原活性都很低。酶的氧化还原活性在仿生结构中得以保留，但仅在中等表面覆盖率下，此时存在连续的脂质双层且蛋白质不会被迫聚集。蛋白质分子之间的静电和其他相互作用力是造成这些影响的原因。
