Universal Method for Protein Immobilization on Chemically Functionalized Germanium Investigated by ATR-FTIR Difference Spectroscopy

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• 作者：Jonas Schartner ; J枚rn G眉ldenhaupt ; Bastian Mei ; Matthias R枚gner ; Martin Muhler ; Klaus Gerwert ; Carsten K枚tting
• 刊名：The Journal of the American Chemical Society
• 出版年：2013
• 出版时间：March 13, 2013
• 年：2013
• 卷：135
• 期：10
• 页码：4079-4087
• 全文大小：500K
• 年卷期：v.135,no.10(March 13, 2013)
• ISSN：1520-5126

文摘

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy allows a detailed analysis of surface attached molecules, including their secondary structure, orientation, and interaction with small molecules in the case of proteins. Here, we present a universal immobilization technique on germanium for all oligo-histidine-tagged proteins. For this purpose, new triethoxysilane derivates were developed: we synthesized a linker鈥搒ilane with a succinimidyl ester as amine-reactive headgroup and a matrix鈥搒ilane with an unreactive ethylene glycol group. A new methodology for the attachment of triethoxysilanes on germanium was established, and the surface was characterized by ATR-FTIR and X-ray photoelectron spectroscopy. In the next step, the succinimidyl ester was reacted with aminonitrilotriacetic acid. Subsequently, Ni²⁺ was coordinated to form Ni鈥搉itrilotriacetic acid for His-tag binding. The capability of the functionalized surface was demonstrated by experiments using the small GTPase Ras and photosystem I (PS I). The native binding of the proteins was proven by difference spectroscopy, which probes protein function. The function of Ras as molecular switch was demonstrated by a beryllium trifluoride anion titration assay, which allows observation of the 鈥渙n鈥?and 鈥渙ff鈥?switching of Ras at atomic resolution. Furthermore, the activity of immobilized PS I was proven by light-induced difference spectroscopy. Subsequent treatment with imidazole removes attached proteins, enabling repeated binding. This universal technique allows specific attachment of His-tagged proteins and a detailed study of their function at the atomic level using FTIR difference spectroscopy.