Proteins in biomimetic surface architectures

Renate L. Naumann 1Wolfgang Knoll 

1. Max Planck Institute for Polymer Research (MPIP), Ackermannweg 10, Mainz 55128, Germany

Abstract

Renate Naumann and Wolfgang Knoll

Max Planck Institute for Polymer Research

Ackermannweg 10, 55128 Mainz (Germany)

naumannr@mpip-mainz.mpg.de

Membrane proteins represent a crucial class of biological agents. They are the key factors in many vital functions of the cell, such as nerve excitation, respiration, photosynthesis and transport of ions and nutrients. Attempts have been made to design model systems for their investigation which pinpoint bilayer lipid membranes to surfaces. The proteins are then reconstituted into the lipid membrane to form a biomimetic surface architecture which can be investigated by a combination of spectroscopic and electrochemical techniques.
Thiolipid-based tethered bilayer lipid membranes (tBLMs) have been used to incorporate porines, ion carriers and channels such as hemolysin, valinomycin, melittin, gramicidin [1], MaxiK, the M2 channel of the AChR. The kinetics of ion transport through these moieties has been analysed using impedance measurements as a function of various parameters, such as ion concentration, transmembrane potential, leak resistance etc. The spectra are simulated using the computer program Spice designed to analyze bioelectrochemical processes. Larger complex proteins such as the cytochrome c oxidase from Rh. sphaeroidis, CcO, are incorporated in the so-called protein-tethered bilayer Lipid membrane (ptBLM) developed in our laboratories [2]. Recombinant CcO solubilized in detergent is immobilized in different defined orientations on a chemically modified gold surface to a nickel chelating nitrilo-triacetic acid (NTA) surface. The CcO monolayer is reconstituted into the lipid by substitution of detergent molecules with lipids using in-situ dialysis or adsorption. An overview is given of the investigations performed so far on this system in our laboratories, in particular using electrochemistry in combination with surface-enhanced ATR-FTIR spectroscop), and surface enhanced resonance Raman spectroscopy (SERRS) [3]. When the protein is immobilized with the cytochrome c binding side directed towards the electrode and reconstituted in-situ into a lipid bilayer, it is addressable by direct electron transfer to the redox centers. Electron transfer to the enzyme via the spacer, referred to as electronic wiring, shows an exceptionally high rate constant. This allows to perform a kinetic analysis of all four consecutive electron transfer steps within the enzyme. Structural changes of the peptide backbone are deduced from spectral changes of certain bands (e.g. Amide I), which are assigned to specific redox states of the redox centers. These spectral changes are monitored by Surface Enhanced ATR-FTIR Spectroscopy in the rapid scan and in the step scan mode. Stationary measurements at different fixed potentials show that redox transitions occur in all four redox centers. Time-resolved measurements, applying repeated potential pulses allow to follow the change of the redox state of the CuA, the heme a and the heme a3 center as a function of time. Time constants of the transitions are obtained by fitting three parameter exponentials to the time dependent absorption data [7].

[1] He, L. et al., Langmuir, 21, (2005), 11666-11672

[2] Giess, F. et al.., Biophys. J. 87, (2004), 3213-3220

[3] Friedrich, M.G. et al. Chem. Commun. (2004), 2376-237

[4] Kirste, V.U. et al. in preparation

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Presentation: Keynote lecture at SMCBS'2007 International Workshop, by Renate L. Naumann
See On-line Journal of SMCBS'2007 International Workshop

Submitted: 2007-07-12 12:40
Revised:   2009-06-07 00:44
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