title: Universal method for protein immobilization on chemically functionalized germanium investigated by ATR-FTIR difference spectroscopy.
abstract: 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-silane with a succinimidyl ester as amine-reactive headgroup and a matrix-silane 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, Ni(2+) was coordinated to form Ni-nitrilotriacetic 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 "on" and "off" 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.
authors: Department of Biophysics, Faculty of Biology and Biotechnology, Ruhr-University Bochum, Germany.
authors: Jonas
authors: J
authors: Schartner
authors: Jörn
authors: J
authors: Güldenhaupt
authors: Bastian
authors: B
authors: Mei
authors: Matthias
authors: M
authors: Rögner
authors: Martin
authors: M
authors: Muhler
authors: Klaus
authors: K
authors: Gerwert
authors: Carsten
authors: C
authors: Kötting
mesh_terms: D005857:Germanium / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared; D013499:Surface Properties
chemical_list: D011506:Proteins; D005857:Germanium