title: Immobilization of proteins in their physiological active state at functionalized thiol monolayers on ATR-germanium crystals.
abstract: 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.
authors.affiliation: Department of Biophysics, Faculty of Biology and Biotechnology, Ruhr-University Bochum, Universitätsstrasse 150, 44801 Bochum (Germany).
authors.forename: Jonas
authors.initials: J
authors.lastname: Schartner
authors.forename: Konstantin
authors.initials: K
authors.lastname: Gavriljuk
authors.forename: Andreas
authors.initials: A
authors.lastname: Nabers
authors.forename: Philipp
authors.initials: P
authors.lastname: Weide
authors.forename: Martin
authors.initials: M
authors.lastname: Muhler
authors.forename: Klaus
authors.initials: K
authors.lastname: Gerwert
authors.forename: Carsten
authors.initials: C
authors.lastname: Kötting
mesh_terms: D005857:Germanium / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D012243:Rhodopsin / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D055767:Immobilized Proteins; D013438:Sulfhydryl Compounds; D005857:Germanium; D006639:Histidine; D012243:Rhodopsin
keywords: ATR-FTIR; Channelrhodopsin 2; biosensors; germanium; protein immobilization