Immobilization of Proteins on Chemically Modified Germanium Investigated by ATR-FTIR

Abstract

The attenuated total reflection fourier transform infrared spectroscopy (ATR-FTIR) allows a detailed analysis of surface attached molecules, including their secondary structure, reaction mechanism, orientation and interaction with small molecules or proteins.1 The aim of our study is the development of a universal immobilization technique on germanium for all kinds of proteins. We recently showed the specific immobilization of N-Ras and Photosystem I on a silane modified germanium surface.1We now present a new approach employing thiol chemistry on germanium.2,3 On one hand germanium crystals provide a great signal-to-noise ratio in ATR-FTIR. On the other hand protein immobilization via thiol chemistry is well-established because it is standard for modifications of gold surfaces e.g. in surface plasmon resonance. Here we combine the best of both worlds and report on germanium surface functionalization with different thiols which allowed for specific immobilization of histidine-tagged proteins with over 99% specific binding. The great advantage of using thiols in comparison with silanes is that a huge variety of thiols with functional groups for many kinds of protein immobilizations are readily available and the higher stability. Nativity of protein folding was confirmed by secondary structure analysis. Stimulus induced difference spectra were obtained for immobilized Channelrhodopsin 2, the small GTPase N-Ras and the phosphocholine-transferase AnkX, which demonstrated protein function at the atomic level.4 Protein activity was observed for Channelrhodopsin 2 for over several days.41: Schartner J. et al., J. Am. Chem. Soc., 2013, 135, 4079-40872: Hanrath, T. & Korgel, B. A., J. Am. Chem. Soc., 2004, 126, 15466-154723: Han, S. et al. J. Am. Chem. Soc., 2001, 123, 2422-24254: Schartner J., et al., ChemBioChem, 2014, accepted