Schartner Jonas, Gavriljuk Konstantin, Nabers Andreas, Weide Philipp, Muhler Martin, Gerwert Klaus, Kötting Carsten
Department of Biophysics, Faculty of Biology and Biotechnology, Ruhr-University Bochum, Universitätsstrasse 150, 44801 Bochum (Germany).
Chembiochem. 2014 Nov 24;15(17):2529-34. doi: 10.1002/cbic.201402478. Epub 2014 Sep 24.
Protein immobilization on solid surfaces has become a powerful tool for the investigation of protein function. Physiologically relevant molecular reaction mechanisms and interactions of proteins can be revealed with excellent signal-to-noise ratio by vibrational spectroscopy (ATR-FTIR) on germanium crystals. Protein immobilization by thiol chemistry is well-established on gold surfaces, for example, for surface plasmon resonance. Here, we combine features of both approaches: a germanium surface functionalized with different thiols to allow specific immobilization of various histidine-tagged proteins with over 99% specific binding. In addition to FTIR, the surfaces were characterized by XPS and fluorescence microscopy. Secondary-structure analysis and stimulus-induced difference spectroscopy confirmed protein activity at the atomic level, for example, physiological cation channel formation of Channelrhodopsin 2.
蛋白质固定在固体表面已成为研究蛋白质功能的有力工具。通过锗晶体上的振动光谱（衰减全反射傅里叶变换红外光谱，ATR-FTIR），可以以优异的信噪比揭示蛋白质的生理相关分子反应机制和相互作用。例如，通过硫醇化学将蛋白质固定在金表面，用于表面等离子体共振，这一方法已得到广泛应用。在此，我们结合了两种方法的特点：用不同硫醇功能化的锗表面，以实现各种组氨酸标签蛋白的特异性固定，特异性结合率超过99%。除了傅里叶变换红外光谱，还通过X射线光电子能谱和荧光显微镜对表面进行了表征。二级结构分析和刺激诱导差异光谱在原子水平上证实了蛋白质的活性，例如，通道视紫红质2的生理阳离子通道形成。
