Highly synergistic antimicrobial activity of magainin 2 and PGLa peptides is rooted in the formation of supramolecular complexes with lipids

Magainin 2 and PGLa are cationic, amphipathic antimicrobial peptides which when added as equimolar mixture exhibit a pronounced synergism in both their antibacterial and pore-forming activities. Here we show for the first time that the peptides assemble into defined supramolecular structures along the membrane interface. The resulting mesophases are quantitatively described by state-of-the art fluorescence self-quenching and correlation spectroscopies. Notably, the synergistic behavior of magainin 2 and PGLa correlates with the formation of hetero-domains and an order-of-magnitude increased membrane affinity of both peptides. Enhanced membrane association of the peptide mixture is only observed in the presence of phophatidylethanolamines but not of phosphatidylcholines, lipids that dominate bacterial and eukaryotic membranes, respectively. Thereby the increased membrane-affinity of the peptide mixtures not only explains their synergistic antimicrobial activity, but at the same time provides a new concept to increase the therapeutic window of combinatorial drugs

Molecular packing of amphipathic peptides on the surface of lipid membranes

When polypeptides bind to the membrane surface, they become confined to a restricted quasi-two-dimensional space where peptide-peptide interactions become highly relevant, and the concept of a crowded medium is appropriate. Within this crowded environment interesting effects like clustering, separation of phases, cooperative alignment, and common movements occur. Here we investigated such effects by measuring distances between fluorophore-labeled peptides in the range </=1 nm by fluorescence self-quenching. For helical peptides with dimensions of approximately 1 x 3 nm such a small "ruler" is sensitive to the packing of the labeled peptides and thereby to their molecular arrangement. A novel approach to characterize peptide-peptide interactions within membranes is presented using the designer peptide LAH4. This sequence changes membrane topology in a controlled manner being transmembrane at neutral conditions but oriented parallel to the surface at low pH. Experimental measurements of the fluorescence self-quenching of close-by chromophores and the changes that occur upon dilution with unlabeled peptides are used to analyze the peptide distribution within the membrane surface. The data show a strong effect of electrostatic interactions and under some experimental conditions clustering of the peptides. Furthermore, the results suggest that at pH 4 the peptides arrange along the membrane surface in an ordered mesophase-like arrangement

Entropic Tension in Crowded Membranes

Unlike their model membrane counterparts, biological membranes are richly decorated with a heterogeneous assembly of membrane proteins. These proteins are so tightly packed that their excluded area interactions can alter the free energy landscape controlling the conformational transitions suffered by such proteins. For membrane channels, this effect can alter the critical membrane tension at which they undergo a transition from a closed to an open state, and therefore influence protein function \emph{in vivo}. Despite their obvious importance, crowding phenomena in membranes are much less well studied than in the cytoplasm. Using statistical mechanics results for hard disk liquids, we show that crowding induces an entropic tension in the membrane, which influences transitions that alter the projected area and circumference of a membrane protein. As a specific case study in this effect, we consider the impact of crowding on the gating properties of bacterial mechanosensitive membrane channels, which are thought to confer osmoprotection when these cells are subjected to osmotic shock. We find that crowding can alter the gating energies by more than $2\;k_BT$ in physiological conditions, a substantial fraction of the total gating energies in some cases. Given the ubiquity of membrane crowding, the nonspecific nature of excluded volume interactions, and the fact that the function of many membrane proteins involve significant conformational changes, this specific case study highlights a general aspect in the function of membrane proteins.Comment: 20 pages (inclduing supporting information), 4 figures, to appear in PLoS Comp. Bio

A Coiled-Coil Peptide Shaping Lipid Bilayers upon Fusion

NWOSupramolecular & Biomaterials Chemistr

Solid-State NMR/Dynamic Nuclear Polarization of Polypeptides in Planar Supported Lipid Bilayers

Dynamic nuclear polarization has been developed to overcome the limitations of the inherently low signal intensity of NMR spectroscopy. This technique promises to be particularly useful for solid-state NMR spectroscopy where the signals are broadened over a larger frequency range and most investigations rely on recording low gamma nuclei. To extend the range of possible investigations, a triple-resonance flat-coil solid-state NMR probe is presented with microwave irradiation capacities allowing the investigation of static samples at temperatures of 100 K, including supported lipid bilayers. The probe performance allows for two-dimensional separated local field experiments with high-power Lee-Goldberg decoupling and cross-polarization under simultaneous irradiation from a gyrotron microwave generator. Efficient cooling of the sample turned out to be essential for best enhancements and line shape and necessitated the development of a dedicated cooling chamber. Furthermore, a new membrane-anchored biradical is presented, and the geometry of supported membranes was optimized not only for good membrane alignment, handling, stability, and filling factor of the coil but also for heat and microwave dissipation. Enhancement factors of 17-fold were obtained, and a two-dimensional PISEMA spectrum of a transmembrane helical peptide was obtained in less than 2 h

Structural remodeling and oligomerization of human cathelicidin on membranes suggest fibril-like structures as active species

Antimicrobial peptides as part of the mammalian innate immune system target and remove major bacterial pathogens, often through irreversible damage of their cellular membranes. To explore the mechanism by which the important cathelicidin peptide LL-37 of the human innate immune system interacts with membranes, we performed biochemical, biophysical and structural studies. The crystal structure of LL-37 displays dimers of anti-parallel helices and the formation of amphipathic surfaces. Peptide-detergent interactions introduce remodeling of this structure after occupation of defined hydrophobic sites at the dimer interface. Furthermore, hydrophobic nests are shaped between dimer structures providing another scaffold enclosing detergents. Both scaffolds underline the potential of LL-37 to form defined peptide-lipid complexes in vivo. After adopting the activated peptide conformation LL-37 can polymerize and selectively extract bacterial lipids whereby the membrane is destabilized. The supramolecular fibril-like architectures formed in crystals can be reproduced in a peptide-lipid system after nanogold-labelled LL-37 interacted with lipid vesicles as followed by electron microscopy. We suggest that these supramolecular structures represent the LL-37-membrane active state. Collectively, our study provides new insights into the fascinating plasticity of LL-37 demonstrated at atomic resolution and opens the venue for LL-37-based molecules as novel antibiotics.We would like to thank Sandra Delgado for the technical help in the preparation of the cryoEM vitrified grids and Dr. Isabel Uson and Dr. Ivan De Marino for the Arcimboldo software and valuable help. Funding was provided by the Unidad de Biofisica and the IKERBASQUE and MINECO science foundations

Retinal Vascular Occlusion after COVID-19 Vaccination : More Coincidence than Causal Relationship? Data from a Retrospective Multicentre Study

Background: To investigate whether vaccination against SARS-CoV-2 is associated with the onset of retinal vascular occlusive disease (RVOD). Methods: In this multicentre study, data from patients with central and branch retinal vein occlusion (CRVO and BRVO), central and branch retinal artery occlusion (CRAO and BRAO), and anterior ischaemic optic neuropathy (AION) were retrospectively collected during a 2-month index period (1 June–31 July 2021) according to a defined protocol. The relation to any previous vaccination was documented for the consecutive case series. Numbers of RVOD and COVID-19 vaccination were investigated in a case-by-case analysis. A case– control study using age- and sex-matched controls from the general population (study participants from the Gutenberg Health Study) and an adjusted conditional logistic regression analysis was conducted. Results: Four hundred and twenty-one subjects presenting during the index period (61 days) were enrolled: one hundred and twenty-one patients with CRVO, seventy-five with BRVO, fifty-six with CRAO, sixty-five with BRAO, and one hundred and four with AION. Three hundred and thirty-two (78.9%) patients had been vaccinated before the onset of RVOD. The vaccines given were BNT162b2/BioNTech/Pfizer (n = 221), followed by ChadOx1/AstraZeneca (n = 57), mRNA1273/Moderna (n = 21), and Ad26.COV2.S/Johnson & Johnson (n = 11; unknown n = 22). Our case–control analysis integrating population-based data from the GHS yielded no evidence of an increased risk after COVID-19 vaccination (OR = 0.93; 95% CI: 0.60–1.45, p = 0.75) in connection with a vaccination within a 4-week window. Conclusions: To date, there has been no evidence of any association between SARS-CoV-2 vaccination and a higher RVOD risk

Varicellovirus UL49.5 Proteins Differentially Affect the Function of the Transporter Associated with Antigen Processing, TAP

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I–restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL49.5 proteins block TAP as well, these data indicate that UL49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL49.5. Taken together, these results classify the UL49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms

correction to unpacking configurational dynamics sequence analysis and qualitative comparative analysis as a mixed method design

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