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[Display omitted] The interactions of viral fusion peptides from influenza (E4K and Ac-E4K) and human immunodeficiency virus (gp41 and Ac-gp41) with planar lipid bilayers and monolayers was investigated herein. A combination of surface-sensitive techniques, including quartz crystal microbalance with dissipation (QCM-D), Langmuir-Blodgett area-pressure isotherms with Micro-Brewster angle microscopy, and neutron reflectometry, was employed. Differences in the interactions of the viral fusion peptides with lipid bilayers featuring ordered and disordered phases, as well as lipid rafts, were revealed. The HIV fusion peptide (gp41) exhibited strong binding to the DOPC/DOPS bilayer, comprising a liquid disordered phase, with neutron reflectometry (NR) showing interaction with the bilayer's headgroup area. Conversely, negligible binding was observed with lipid bilayers in a liquid ordered phase. Notably, the influenza peptide (E4K) demonstrated slower binding kinetics with DOPC/DOPS bilayers and distinct interactions compared to gp41, as observed through QCM-D. This suggests different mechanisms of interaction with the lipid bilayers: one peptide interacts more within the headgroup region, while the other is more involved in transmembrane interactions. These findings hold implications for understanding viral fusion mechanisms and developing antimicrobials and antivirals targeting membrane interactions. The differential binding behaviours of the viral fusion peptides underscore the importance of considering membrane composition and properties in therapeutic strategy design. [ABSTRACT FROM AUTHOR]

The Spatz neutron beam instrument is the second time‐of‐flight neutron reflectometer to be installed at the OPAL research reactor. The instrument was formerly the V18 BioRef reflectometer at the BER‐II reactor in Berlin and was transferred to Australia in 2016. Subsequently the instrument was re‐installed in the neutron guide hall of the OPAL reactor at the end position of the CG2B cold‐neutron guide and recommissioned. The instrument performance has not been compromised by the move, with reflectivity achieved down to 10−7 and good counting statistics within a reasonable time frame using a wavelength range of 2–20 Å. Several different samples at the solid–air interface and the solid–liquid interface have been measured to demonstrate the instrument's capabilities. [ABSTRACT FROM AUTHOR]

A depleted antimicrobial drug pipeline combined with an increasing prevalence of Gram-negative 'superbugs' has increased interest in nano therapies to treat antibiotic resistance. As cubosomes and polymyxins disrupt the outer membrane of Gram-negative bacteria via different mechanisms, we herein examine the antimicrobial activity of polymyxin-loaded cubosomes and explore an alternative strategy via the polytherapy treatment of pathogens with cubosomes in combination with polymyxin. The polytherapy treatment substantially increases antimicrobial activity compared to polymyxin B-loaded cubosomes or polymyxin and cubosomes alone. Confocal microscopy and neutron reflectometry suggest the superior polytherapy activity is achieved via a two-step process. Firstly, electrostatic interactions between polymyxin and lipid A initially destabilize the outer membrane. Subsequently, an influx of cubosomes results in further membrane disruption via a lipid exchange process. These findings demonstrate that nanoparticle-based polytherapy treatments may potentially serve as improved alternatives to the conventional use of drug-loaded lipid nanoparticles for the treatment of "superbugs". An increasing prevalence of Gram-negative bacteria increases the interest in nanotherapies to treat antibiotic resistance. Here, the authors examine the antimicrobial activity of polymyxin-loaded cubosomes and explore a polytherapy treatment of pathogens with cubosomes in combination with polymyxin. [ABSTRACT FROM AUTHOR]

Abstract: The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug–membrane interactions may contribute to the apparent affinity of TSPO ligands. [Copyright &y& Elsevier]

Little is known experimentally about the detailed orientation of membrane-bound maculatin 1.1 (Mac1), an antimicrobial peptide from the skin secretions of Australian tree frogs. In this work multiple 15N-labelled or 2H-labelled Mac1 with dodecylphosphocholine (DPC) micelles and isotropic DMPC/DHPC (q = 0.5) bicelles were investigated by solution NMR, circular dichroism (CD) spectroscopy, neutron reflectometry and molecular dynamics (MD) simulations in explicit solvent. In buffer, the 15N-1H HSQC and CD spectra were indicative of the peptide being random coiled. In the presence of micelles or isotropic bicelles, a unique and helical peptide structure that was confirmed by CD was found. The titration of the soluble paramagnetic agent gadolinium (Gd-DTPA) into the Mac1-DPC solution led to enhanced relaxation of all 15N labelled residues. The peptide N-terminus was more exposed to Gd-DTPA than the C-terminus in micelles, while only the Gly-4 and Ala-18 resonances were significantly reduced in the presence of isotropic bicelles. MD simulations of Mac1 fully inserted into a DPC micelle converged towards a solvent exposed orientation and a topology where Mac1 was wrapped around the DPC micelle with the more hydrophobic side facing inward. MD simulations of Mac1 fully inserted into a phosphatidylcholine (PC) bilayer converged towards a kinked transmembrane orientation with water molecules penetrating around Lys-8. A deuterium labelled Mac1 used in neutron reflectometry experiments suggested a preferred orientation in zwitterionic PC bilayers. These results give insight into the membrane disrupting activity of Mac1 against cell membranes. Unlabelled Image • Conformation of the antimicrobial peptide maculatin 1.1 was studied in planar bilayers and in micelles. • Isotopically labelled maculatin 1.1 was used to determine orientation in lipid bilayers. • Maculatin 1.1 adopts a preferred orientation in zwitterionic lipid bilayers. • In planar bilayers both the N-terminus and C-terminus are solvent exposed indicating pore-formation. [ABSTRACT FROM AUTHOR]

In Gram–negative bacteria, the multi-protein β-barrel assembly machine (BAM) complex is a nanomachine playing a vital role in the process of assembling β-barrel proteins into the outer membrane (OM). The core component of this multiprotein complex, BamA, is an evolutionarily conserved protein that carries five polypeptide-transport-associated (POTRA) domains that project from the outer membrane. BamA is essential for chaperoning the insertion of proteins into the OM surface of bacterial cells. In this work, we have reconstituted a membrane containing BamA on a gold substrate and characterized structure of each component and movement in different situation at the nanoscale level using quartz-crystal microbalance with dissipation and neutron reflectometry (NR). The purified BamA in n-dodecyl β-D-maltoside (DDM) was first engineered onto a nickel-NTA (Nα, Nα-bis-(carboxymethyl)- l -lysine) modified gold surface followed by DDM removal and bilayer assembly. The system was then used to monitor the binding and insertion of a substrate membrane protein. The data shows the total reach of BamA was 120 Å and the embedding of membrane had no effect on the BamA morphology. However, the addition of the substrate enabled the periplasmic POTRA domain of BamA to extend further away from the membrane surface. This dynamic behaviour of BamA POTRA domains is consistent with models invoking the gathering of transported substrates from the periplasmic space between the inner and outer membranes in bacterial cells. This study provides evidence that NR is a reliable tool for diverse investigations in the future, especially for applications in the field of membrane protein biogenesis. Unlabelled Image • Quartz crystal microbalance with dissipation monitoring (QCM-D) showed the interaction between BamA with a substrate protein. • The structures of BamA with/without a supported lipid bilayer were characterized by neutron reflectometry (NR). • The embedding of a lipid bilayer didn't affect the structure of BamA. • A substrate protein stimulates an extension of POTRA domain of BamA in the periplasmic space. [ABSTRACT FROM AUTHOR]

In Gram-negative bacteria, the β-barrel assembly machinery (BAM) complex catalyses the assembly of β-barrel proteins into the outer membrane, and is composed of five subunits: BamA, BamB, BamC, BamD and BamE. Once assembled, - β-barrel proteins can be involved in various functions including uptake of nutrients, export of toxins and mediating host-pathogen interactions, but the precise mechanism by which these ubiquitous and often essential β-barrel proteins are assembled is yet to be established. In order to determine the relative positions of BAM subunits in the membrane environment we reconstituted each subunit into a biomimetic membrane, characterizing their interaction and structural changes by Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and neutron reflectometry. Our results suggested that the binding of BamE, or a BamDE dimer, to BamA induced conformational changes in the polypeptide transported-associated (POTRA) domains of BamA, but that BamB or BamD alone did not promote any such changes. As monitored by neutron reflectometry, addition of an unfolded substrate protein extended the length of POTRA domains further away from the membrane interface as part of the mechanism whereby the substrate protein was folded into the membrane. [Display omitted] • Neutron reflectometry study of Bam subunits and their substrate • BamE can alter the conformational changes of BamA through its interaction with POTRA5 domain. • BamB and BamD cannot change the conformation of POTRA domain. • Substrate, Pet induced an extension of the POTRA domains of BamA. [ABSTRACT FROM AUTHOR]