A major challenge for biophysical studies of membrane proteins is obtaining stable, homogenous samples. Traditional detergent solubilization and liposome-based methods of reconstitution may lead to protein inactivation, heterogeneous and polydisperse sized particles, and sample aggregation. [1] While membrane scaffold protein (MSP) stabilized nanodiscs have facilitated the formation of monodisperse protein samples, [2] a drawback is the detergent-based preparation method. Here we present a physicochemical characterization of polymer-stabilized lipid particles termed Lipodisq, a novel nanosized lipid-based platform capable of incorporating membrane proteins. [3] The polymers used in the Lipodisq technology can solubilize commonly used lipids such as dimyristoylphosphatidylcholine (DMPC) without the use of detergents. The small size of Lipodisq (diameter of around 9-10 nm at pH 7.4) renders them potentially suitable for many biophysical methodologies, including electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopies, electron microscopy (EM), and circular dichroism (CD) spectroscopy.DMPC and a polymer formed from a molar styrene to maleic acid ratio of 3:1 (termed 3:1 SMA, see Figure S1 in the Supporting Information) were used as a model system to generate Lipodisq particles. Their formation using 3:1 SMA and DMPC at a weight ratio of 1.25:1.0 was followed by dynamic light scattering (DLS) at 550 nm and 25 8C (Figure 1), resulting in solution clarification within a minute. Higher SMA to DMPC ratios of 3:1 can also be used, though the excess polymer may increase the total solution viscosity, while decreased amounts results in sub-optimal DMPC solubilization. The dynamic light scattering data indicate a monodisperse distribution of Lipodisq particles with an average diameter of 9 nm as previously reported, [3b] which is confirmed by negative stain transmission electron microscopy (TEM, Figure 1 C). Figure 1. Light scattering data and size of Lipodisq particles. Lipodisq particle formation was observed by measuring the light scattering of the resulting polymer-lipid solution at 550 nm (A; Abs = absorption).DLS data (B) suggest that the Lipodisq formed have an average diameter of 9 nm, whereas TEM images (C) reveal that the Lipodisq diameter varies between 5-15 nm.

Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix.innate host defense | de novo protein design | nanometrology | antibiotics | nanoscopy

A novel mechanism of monolayer poration leading to the rapid exfoliation and lysis of microbial membranes is reported.

Vpu from HIV-1 is an 81 amino acid type I integral membrane protein which consists of a cytoplasmic and a transmembrane (TM) domain. The TM domain is known to alter membrane permeability for ions and substrates when inserted into artificial membranes. Peptides corresponding to the TM domain of Vpu (Vpu(1-32)) and mutant peptides (Vpu(1-32)-W23L, Vpu(1-32)-R31V, Vpu(1-32)-S24L) have been synthesized and reconstituted into artificial lipid bilayers. All peptides show channel activity with a main conductance level of around 20 pS. Vpu(1-32)-W23L has a considerable flickering pattern in the recordings and longer open times than Vpu(1-32). Whilst recordings for Vpu(1-32)-R31V are almost indistinguishable from those of the WT peptide, recordings for Vpu(1-32)-S24L do not exhibit any noticeable channel activity. Recordings of WT peptide and Vpu(1-32)-W23L indicate Michaelis-Menten behavior when the salt concentration is increased. Both peptide channels follow the Eisenman series I, indicative for a weak ion channel with almost pore like characteristics.

Lipodisq‐Partikel – Polymer‐Lipid‐Komplexe – wurden mit einer detergentienfreien Methode hergestellt. Dimyristoylphosphatidylcholin(DMPC)‐haltige Lipodisq‐Partikel zeichnen sich im Vergleich mit einer DMPC‐Dispersion durch stärker geordnete Lipide aus. Die Styrol‐ und Maleinsäuregruppen des Polymers wechselwirken mit den lipiden DMPC‐Ketten in der Doppelschicht und mit den lipiden Kopfgruppen in der Liposdisc‐Peripherie (siehe Bild).

Vpu, an integral membrane protein encoded in HIV-1, is implicated in the release of new virus particles from infected cells, presumably mediated by ion channel activity of homo-oligomeric Vpu bundles. Reconstitution of both full length Vpu(1-81) and a short, the transmembrane (TM) domain comprising peptide Vpu(1-32) into bilayers under a constant electric field results in an asymmetric orientation of those channels. For both cases, channel activity with similar kinetics is observed. Channels can open and remain open within a broad series of conductance states even if a small or no electric potential is applied. The mean open time for Vpu peptide channels is voltage-independent. The rate of channel opening shows a biphasic voltage activation, implicating that the gating is influenced by the interaction of the dipole moments of the TM helices with an electric field.

Making complements: Solid‐state MAS NMR spectra of bacteriorhodopsin in its native purple membrane environment can be used to complement crystallographic studies of the protein by validating and redefining the (possibly distorted) loop structures. Backbone dihedral angles were extracted from the chemical shifts and compared to the crystal structures. Where there are conformational differences, the dihedral angles were used to recalculate the loop structure (see picture).

Nanoparticles assembled with poly(styrene-maleic acid) copolymers, identified in the literature as Lipodisq, SMALPs or Native Nanodisc, are routinely used as membrane mimetics to stabilise protein structures in their native conformation. To date, transmembrane proteins of varying complexity (up to 8 beta strands or 48 alpha helices) and of a range of molecular weights (from 27 kDa up to 500 kDa) have been incorporated into this particle system for structural and functional studies. SMA and related amphipathic polymers have become versatile components of the biochemist's tool kit for the stabilisation, extraction and structural characterization of membrane proteins by techniques including cryo-EM and X-ray crystallography. Lipodisq formation does not require the use of conventional detergents and thus avoids their associated detrimental consequences. Here the development of this technology, from its fundamental concept and design to the diverse range of experimental methodologies to which it can now be applied, will be reviewed.