Potential Distribution across Model Membranes.

Membrane models assembled on electrodes are widely used tools to study potential-dependent molecular processes at or in membranes. However, the relationship between the electrode potential and the potential across the membrane is not known. Here we studied lipid bilayers immobilized on mixed self-assembled monolayers (SAM) on Au electrodes. The mixed SAM was composed of thiol derivatives of different chain lengths such that between the islands of the short one, mercaptobenzonitrile (MBN), and the tethered lipid bilayer an aqueous compartment was formed. The nitrile function of MBN, which served as a reporter group for the vibrational Stark effect (VSE), was probed by surface-enhanced infrared absorption spectroscopy to determine the local electric field as a function of the electrode potential for pure MBN, mixed SAM, and the bilayer system. In parallel, we calculated electric fields at the VSE probe by molecular dynamics (MD) simulations for different charge densities on the metal, thereby mimicking electrode potential changes. The agreement with the experiments was very good for the calculations of the pure MBN SAM and only slightly worse for the mixed SAM. The comparison with the experiments also guided the design of the bilayer system in the MD setups, which were selected to calculate the electrode potential dependence of the transmembrane potential, a quantity that is not directly accessible by the experiments. The results agree very well with estimates in previous studies and thus demonstrate that the present combined experimental-theoretical approach is a promising tool for describing potential-dependent processes at biomimetic interfaces.

Monitoring the Orientational Changes of Alamethicin during Incorporation into Bilayer Lipid Membranes.

Antimicrobial peptides (AMPs) are the first line of defense after contact of an infectious invader, for example, bacterium or virus, with a host and an integral part of the innate immune system of humans. Their broad spectrum of biological functions ranges from cell membrane disruption over facilitation of chemotaxis to interaction with membrane-bound or intracellular receptors, thus providing novel strategies to overcome bacterial resistances. Especially, the clarification of the mechanisms and dynamics of AMP incorporation into bacterial membranes is of high interest, and different mechanistic models are still under discussion. In this work, we studied the incorporation of the peptaibol alamethicin (ALM) into tethered bilayer lipid membranes on electrodes in combination with surface-enhanced infrared absorption (SEIRA) spectroscopy. This approach allows monitoring the spontaneous and potential-induced ion channel formation of ALM in situ. The complex incorporation kinetics revealed a multistep mechanism that points to peptide-peptide interactions prior to penetrating the membrane and adopting the transmembrane configuration. On the basis of the anisotropy of the backbone amide I and II infrared absorptions determined by density functional theory calculations, we employed a mathematical model to evaluate ALM reorientations monitored by SEIRA spectroscopy. Accordingly, ALM was found to adopt inclination angles of ca. 69°-78° and 21° in its interfacially adsorbed and transmembrane incorporated states, respectively. These orientations can be stabilized efficiently by the dipolar interaction with lipid head groups or by the application of a potential gradient. The presented potential-controlled mechanistic study suggests an N-terminal integration of ALM into membranes as monomers or parallel oligomers to form ion channels composed of parallel-oriented helices, whereas antiparallel oligomers are barred from intrusion.

Electrochemical and Resonance Raman Spectroscopic Studies of Water-Oxidizing Ruthenium Terpyridyl-Bipyridyl Complexes.

The irreversible conversion of single-site water-oxidation catalysts (WOC) into more rugged catalysts structurally related to [(trpy)(5,5'-X2 -bpy)RuIV (µ-O)RuIV (trpy)(O)(H2 O)]4+ (X=H, 1-dn4+ ; X=F, 2-dn4+ ; bpy=2,2'-bipyridine; trpy=2,2':6',2"-terpyridine) represents a critical issue in the development of active and durable WOCs. In this work, the electrochemical and acid-base properties of 1-dn4+ and 2-dn4+ were evaluated. In situ resonance Raman spectroscopy was employed to characterize the species formed upon the stoichiometric oxidation of the single-site catalysts and demonstrated the formation of high-oxidation-state mononuclear Ru=O and RuO-O complexes. Under turnover conditions, the dinuclear intermediates, 1-dn4+ and 2-dn4+ as well as the previously proposed [RuVI (trpy)(O)2 (H2 O)]2+ complex (32+ ) are formed. Complex 32+ is a pivotal intermediate that provides access to the formation of dinuclear species. Single-crystal X-ray diffraction analysis of the isolated complex [RuIV (O)(trpy)(5,5'-F2 -bpy)]2+ reveals a clear elongation of the Ru-N bond trans to the oxido ligand that documents the weakness of this bond, which promotes the release of the bpy ligand and the subsequent formation of 32+ .

Mimicking Tyrosine Phosphorylation in Human Cytochrome c by the Evolved tRNA Synthetase Technique.

Phosphorylation of tyrosine 48 of cytochrome c is related to a wide range of human diseases due to the pleiotropic role of the heme-protein in cell life and death. However, the structural conformation and physicochemical properties of phosphorylated cytochrome c are difficult to study as its yield from cell extracts is very low and its kinase remains unknown. Herein, we report a high-yielding synthesis of a close mimic of phosphorylated cytochrome c, developed by optimization of the synthesis of the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF) and its efficient site-specific incorporation at position 48. It is noteworthy that the Y48pCMF mutation significantly destabilizes the Fe-Met bond in the ferric form of cytochrome c, thereby lowering the pKa value for the alkaline transition of the heme-protein. This finding reveals the differential ability of the phosphomimic protein to drive certain events. This modified cytochrome c might be an important tool to investigate the role of the natural protein following phosphorylation.