Structure-function relationships of membrane proteins - spectroelectrochemical investigation of artificial membranes

Membrane proteins exert a variety of fundamental functions, such as proton and electron transfer, voltage-gated ion translocation and enzymatic reactions. Since most of these processes can be monitored and controlled by electrochemical techniques, substantial efforts have been made to engineer membrane models on electrodes, providing a biocompatible environment for membrane proteins, e.g. tethered bilayer lipid membranes (tBLMs). To date, electrochemical impedance spectroscopy (EIS) has been extensively used to study integral proteins in such devices. However, this methodology cannot provide structural insights, which are required to understand the molecular basis of the proteins’ functions. To overcome this limitation, in this work, surface-enhanced infrared absorption (SEIRA) spectroscopy, combined with EIS, was applied. Here a nanostructured Au film, the supporting material for the tBLM, served as the IR signal amplifier of adsorbed molecules and as the working electrode in the same setup. In this work, for the first time, the assembly of stable tBLMs on nanostructured Au films is demonstrated, allowing for the structural and functional spectroelectrochemical characterization of a membrane-embedded protein. The tBLM, composed of (cholesterylpolyethylenoxy)thiol (CPEO3), 6-mercaptohexanol (6MH), and 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC), showed a capacitance of CtBLM = 0.6 0.7 μF cm−2 and, due to the presence of inevitable defects, a moderate resistance of RBilayer = (21 ± 2) kΩ cm2. The combined spectroscopic and electrochemical analysis provided novel insight into molecular details of the formation process of the tBLM, revealing a two-step process of vesicle adsorption onto 6MH fragments and the spreading to cover the CPEO3 SAM. As a proof-of-concept, the cation-channel forming peptide gramicidin A (gA) was integrated into the tBLMs and analyzed in terms of specific cation-peptide and cation-lipid interactions that are essential for the function of the channel-forming gA and the membrane structure, respectively. On the basis of EIS and SEIRA spectroscopy, the functionality of gA, acting as an ion channel for monovalent cations, was analyzed and correlated to the structural alterations. The combined approach was further extended to the human voltage-gated anion channel (hVDAC) which, besides its central key role in apoptosis, is proposed to be involved in the voltage-controlled ion flux across the membrane. This protein was used to study the direct proteo-tBLM formation from hVDAC-containing vesicles, the suitability of this tBLM system for potential-dependent SEIRA difference spectroscopy, and the mechanism of voltage-gating. Fusion and spreading of hVDAC-containing vesicles result in tBLMs with capacitances and resistances of CtBLM = 0.6 0.7 μF cm−2 and RBilayer = (14 ± 2) kΩ cm2, respectively, as well as in potential-dependent variations of the bilayer resistance. The results prove the presence of hVDAC within the POPC-POPC bilayer fragments of the tBLM. Potential-dependent SEIRA difference spectra demonstrate the motion of the bilayer towards or away from the Au surface for positive and negative potential differences, respectively. In addition, small potential-dependent changes of the protein structure were observed pointing at a mechanism of voltage-gating that involves a distortion of the β -barrel structure. This structural change may be caused either by elongation and compression (for positive and negative potentials) of the channel or by bending of the hVDAC-bilayer network leading to