Ataka Kenichi, Stripp Sven Timo, Heberle Joachim
Freie Universität Berlin, Experimental Molecular Biophysics, Arnimallee 14, 14195 Berlin, Germany.
Biochim Biophys Acta. 2013 Oct;1828(10):2283-93. doi: 10.1016/j.bbamem.2013.04.026. Epub 2013 Jun 28.
Surface-enhanced infrared absorption spectroscopy (SEIRAS) represents a variation of conventional infrared spectroscopy and exploits the signal enhancement exerted by the plasmon resonance of nano-structured metal thin films. The surface enhancement decays in about 10nm with the distance from the surface and is, thus, perfectly suited to selectively probe monolayers of biomembranes. Peculiar to membrane proteins is their vectorial functionality, the probing of which requires proper orientation within the membrane. To this end, the metal surface used in SEIRAS is chemically modified to generate an oriented membrane protein film. Monolayers of uniformly oriented membrane proteins are formed by tethering His-tagged proteins to a nickel nitrilo-triacetic acid (Ni-NTA) modified gold surface and SEIRAS commands molecular sensitivity to probe each step of surface modification. The solid surface used as plasmonic substrate for SEIRAS, can also be employed as an electrode to investigate systems where electron transfer reactions are relevant, like e.g. cytochrome c oxidase or plant-type photosystems. Furthermore, the interaction of these membrane proteins with water-soluble proteins, like cytochrome c or hydrogenase, is studied on the molecular level by SEIRAS. The impact of the membrane potential on protein functionality is verified by monitoring light-dark difference spectra of a monolayer of sensory rhodopsin (SRII) at different applied potentials. It is demonstrated that the interpretations of all of these experiments critically depend on the orientation of the solid-supported membrane protein. Finally, future directions of SEIRAS including cellular systems are discussed. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.
表面增强红外吸收光谱（SEIRAS）是传统红外光谱的一种变体，它利用了纳米结构金属薄膜的等离子体共振所产生的信号增强效应。表面增强效应在距表面约10nm的距离内衰减，因此非常适合选择性地探测生物膜的单层结构。膜蛋白的独特之处在于其矢量功能，对其进行探测需要在膜内具有适当的取向。为此，对SEIRAS中使用的金属表面进行化学修饰，以生成取向的膜蛋白膜。通过将带有His标签的蛋白连接到镍氮三乙酸（Ni-NTA）修饰的金表面上，形成均匀取向的膜蛋白单层，并且SEIRAS具有分子敏感性，可探测表面修饰的每一步。用作SEIRAS等离子体基底的固体表面，也可以用作电极来研究电子转移反应相关的系统，例如细胞色素c氧化酶或植物型光系统。此外，通过SEIRAS在分子水平上研究了这些膜蛋白与水溶性蛋白（如细胞色素c或氢化酶）的相互作用。通过监测不同施加电位下感觉视紫红质（SRII）单层的明暗差异光谱，验证了膜电位对蛋白功能的影响。结果表明，所有这些实验的解释都严重依赖于固体支持的膜蛋白的取向。最后，讨论了包括细胞系统在内的SEIRAS的未来发展方向。本文是名为“膜蛋白和肽研究中的傅里叶变换红外光谱”的特刊的一部分。
