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

Role of Met80 and Tyr67 in the Low-pH Conformational Equilibria of Cytochrome <i>c</i>

The low-pH conformational equilibria of ferric yeast iso-1 cytochrome <i>c</i> (ycc) and its M80A, M80A/Y67H, and M80A/Y67A variants were studied from pH 7 to 2 at low ionic strength through electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies. For wild-type ycc, the protein structure, axial heme ligands, and spin state of the iron atom convert from the native folded His/Met low-spin (LS) form to a molten globule His/H₂O high-spin (HS) form and a totally unfolded bis-aquo HS state, in a single cooperative transition with an apparent p<i>K</i>_a of ∼3.0. An analogous cooperative transition occurs for the M80A and M80A/Y67H variants. This is preceded by protonation of heme propionate-7, with a p<i>K</i>_a of ∼4.2, and by an equilibrium between a His/OH^–-ligated LS and a His/H₂O-ligated HS conformer, with a p<i>K</i>_a of ∼5.9. In the M80A/Y67A variant, the cooperative low-pH transition is split into two distinct processes because of an increased stability of the molten globule state that is formed at higher pH values than the other species. These data show that removal of the axial methionine ligand does not significantly alter the mechanism of acidic unfolding and the ranges of stability of low-pH conformers. Instead, removal of a hydrogen bonding partner at position 67 increases the stability of the molten globule and renders cytochrome <i>c</i> more susceptible to acid unfolding. This underlines the key role played by Tyr67 in stabilizing the three-dimensional structure of cytochrome <i>c</i> by means of the hydrogen bonding network connecting the Ω loops formed by residues 71–85 and 40–57

Substrate–Protein Interactions of Type II NADH:Quinone Oxidoreductase from <i>Escherichia coli</i>

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and responsible for the maintenance of NADH/NAD⁺ balance in cells. NDH-2s are the only enzymes with NADH dehydrogenase activity present in the respiratory chain of many pathogens, and thus, they were proposed as suitable targets for antimicrobial therapies. In addition, NDH-2s were also considered key players for the treatment of complex I-related neurodegenerative disorders. In this work, we explored substrate–protein interaction in NDH-2 from <i>Escherichia coli</i> (<i>Ec</i>NDH-2) combining surface-enhanced infrared absorption spectroscopic studies with electrochemical experiments, fluorescence spectroscopy assays, and quantum chemical calculations. Because of the specific stabilization of substrate complexes of <i>Ec</i>NDH-2 immobilized on electrodes, it was possible to demonstrate the presence of two distinct substrate binding sites for NADH and the quinone and to identify a bound semiprotonated quinol as a catalytic intermediate

Catalytic Activity and Proton Translocation of Reconstituted Respiratory Complex I Monitored by Surface-Enhanced Infrared Absorption Spectroscopy

Respiratory complex I (CpI) is a key player in the way organisms obtain energy, being an energy transducer, which couples nicotinamide adenine dinucleotide (NADH)/quinone oxidoreduction with proton translocation by a mechanism that remains elusive so far. In this work, we monitored the function of CpI in a biomimetic, supported lipid membrane system assembled on a 4-aminothiophenol (4-ATP) self-assembled monolayer by surface-enhanced infrared absorption spectroscopy. 4-ATP serves not only as a linker molecule to a nanostructured gold surface but also as pH sensor, as indicated by concomitant density functional theory calculations. In this way, we were able to monitor NADH/quinone oxidoreduction-induced transmembrane proton translocation via the protonation state of 4-ATP, depending on the net orientation of CpI molecules induced by two complementary approaches. An associated change of the amide I/amide II band intensity ratio indicates conformational modifications upon catalysis which may involve movements of transmembrane helices or other secondary structural elements, as suggested in the literature [Di Luca , Proc. Natl. Acad. Sci. U.S.A., 2017, 114, E6314−E6321]