Crown ether helical peptides are preferentially inserted in lipid bilayers as a transmembrane ion channels

Oriented circular dichroism was used to study the alignment crown ether-modified peptides. The influence of different N- and C-functionalities was assessed using at variable peptide:lipid ratios from 1:20 to 1:200. Neither the functionalities nor the concentration had any major effect on the orientation. The alignment of the 21-mer peptides was also examined with lipid membranes of different bilayer thickness. The use of synchrotron radiation as light source allowed the study of peptide:lipid molar ratios from 1:20 to 1:1000. For all conditions studied, the peptides were found to be predominantly incorporated as a transmembrane helix into the membrane, especially at low peptide concentration, but started to aggregate on the membrane surface at higher peptide:lipid ratios. The structural information on the preferred trans-bilayer alignment of the crown ether functional groups explains their ion conductivity and is useful for the further development of membrane-active nanochemotherapeutics

Membrane-Active Peptides and the Clustering of Anionic Lipids

AbstractThere is some overlap in the biological activities of cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs). We compared nine AMPs, seven CPPs, and a fusion peptide with regard to their ability to cluster anionic lipids in a mixture mimicking the cytoplasmic membrane of Gram-negative bacteria, as measured by differential scanning calorimetry. We also studied their bacteriostatic effect on several bacterial strains, and examined their conformational changes upon membrane binding using circular dichroism. A remarkable correlation was found between the net positive charge of the peptides and their capacity to induce anionic lipid clustering, which was independent of their secondary structure. Among the peptides studied, six AMPs and four CPPs were found to have strong anionic lipid clustering activity. These peptides also had bacteriostatic activity against several strains (particularly Gram-negative Escherichia coli) that are sensitive to lipid clustering agents. AMPs and CPPs that did not cluster anionic lipids were not toxic to E. coli. As shown previously for several types of AMPs, anionic lipid clustering likely contributes to the mechanism of antibacterial action of highly cationic CPPs. The same mechanism could explain the escape of CPPs from intracellular endosomes that are enriched with anionic lipids

Membrane interactions of latarcins: Antimicrobial peptides from spider venom

A group of seven peptides from spider venom with diverse sequences constitute the latarcin family. They have been described as membrane-active antibiotics, but their lipid interactions have not yet been addressed. Using circular dichroism and solid-state 15N-NMR, we systematically characterized and compared the conformation and helix alignment of all seven peptides in their membrane-bound state. These structural results could be correlated with activity assays (antimicrobial, hemolysis, fluorescence vesicle leakage). Functional synergy was not observed amongst any of the latarcins. In the presence of lipids, all peptides fold into amphiphilic α-helices as expected, the helices being either surface-bound or tilted in the bilayer. The most tilted peptide, Ltc2a, possesses a novel kind of amphiphilic profile with a coiled-coil-like hydrophobic strip and is the most aggressive of all. It indiscriminately permeabilizes natural membranes (antimicrobial, hemolysis) as well as artificial lipid bilayers through the segregation of anionic lipids and possibly enhanced motional averaging. Ltc1, Ltc3a, Ltc4a, and Ltc5a are efficient and selective in killing bacteria but without causing significant bilayer disturbance. They act rather slowly or may even translocate towards intracellular targets, suggesting more subtle lipid interactions. Ltc6a and Ltc7, finally, do not show much antimicrobial action but can nonetheless perturb model bilayers

Structure analysis of the protein translocating channel TatA in membranes using a multi-construct approach

AbstractThe twin-arginine-translocase (Tat) can transport proteins in their folded state across bacterial or thylakoid membranes. In Bacillus subtilis the Tat-machinery consists of only two integral (inner) membrane proteins, TatA and TatC. Multiple copies of TatA are supposed to form the transmembrane channel, but little structural data is available on this 70-residue component. We used a multi-construct approach for expressing several characteristic fragments of TatAd, to determine their individual structures and to cross-validate them comprehensively within the architecture of the full-length protein. Here, we report the design, high-yield expression, detergent-aided purification and lipid-reconstitution of five constructs of TatAd, overcoming difficulties associated with the very different hydrophobicities and sizes of these membrane protein fragments. Circular dichroism (CD) and oriented CD (OCD) were used to determine their respective conformations and alignments in suitable, negatively charged phospholipid bilayers. CD spectroscopy showed an N-terminal α-helix, a central helical stretch, and an unstructured C-terminus, thus proving the existence of these secondary structures in TatAd for the first time. The OCD spectra demonstrated a transmembrane orientation of the N-terminal α-helix and a surface alignment of the central amphiphilic helix in lipid bilayers, thus supporting the postulated topology model and function of TatA as a transmembrane channel