Biophysical Society Thematic Meeting - October 13-15, 2015

Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling

Thursday Speaker Abstracts

Germanium Catches Proteins in Action Jonas Schartner , Jörn Güldenhaupt, Konstantin Gavriljuk, Andreas Nabers, Klaus Gerwert, Carsten Kötting. Biology & Biotechnology, Bochum, Germany. The attenuated total reflection fourier transform infrared (ATR-FTIR) spectroscopy allows a detailed analysis of surface attached molecules, including their secondary structure, reaction mechanism, orientation, and interaction with small molecules.[1] This technique reveals vibrational changes in the attached molecules. We recently developed a universal immobilization technique for the specific immobilization of N-Ras and Photosystem I on a silane modified germanium surface.[1] We now present a new approach employing thiol chemistry on germanium.[2] 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 advantages of using thiols in comparison with silanes are that a huge variety of thiols with functional groups is commercially available and the monolayer stability is very high. 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 atomic level.[3] To further improve the S/N ratio the establishment of a 3D-surface was achieved by tethering dextran-polymers to germanium. Proteins were immobilized in multilayers with a distance of about 9 nm as shown for mCherry and GFP.[4] The difference signal was increased by more than factor three when tris-ANTA was employed for catching proteins in action. References 1: Schartner J. et al., JACS, 2013, 135, 4079-4087 2: Han, S. et al. JACS, 2001, 123, 2422–2425 3: Schartner J., et al., ChemBioChem, 2014, 2529-2534 4: Schartner J, et al., under review, 2015

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