How arginine derivatives alter the stability of lipid membranes: dissecting the roles of side chains, backbone and termini.

Arginine (R)-rich peptides constitute the most relevant class of cell-penetrating peptides and other membrane-active peptides that can translocate across the cell membrane or generate defects in lipid bilayers such as water-filled pores. The mode of action of R-rich peptides remains a topic of controversy, mainly because a quantitative and energetic understanding of arginine effects on membrane stability is lacking. Here, we explore the ability of several oligo-arginines R[Formula: see text] and of an arginine side chain mimic R[Formula: see text] to induce pore formation in lipid bilayers employing MD simulations, free-energy calculations, breakthrough force spectroscopy and leakage assays. Our experiments reveal that R[Formula: see text] but not R[Formula: see text] reduces the line tension of a membrane with anionic lipids. While R[Formula: see text] peptides form a layer on top of a partly negatively charged lipid bilayer, R[Formula: see text] leads to its disintegration. Complementary, our simulations show R[Formula: see text] causes membrane thinning and area per lipid increase beside lowering the pore nucleation free energy. Model polyarginine R[Formula: see text] similarly promoted pore formation in simulations, but without overall bilayer destabilization. We conclude that while the guanidine moiety is intrinsically membrane-disruptive, poly-arginines favor pore formation in negatively charged membranes via a different mechanism. Pore formation by R-rich peptides seems to be counteracted by lipids with PC headgroups. We found that long R[Formula: see text] and R[Formula: see text] but not short R[Formula: see text] reduce the free energy of nucleating a pore. In short R[Formula: see text], the substantial effect of the charged termini prevent their membrane activity, rationalizing why only longer [Formula: see text] are membrane-active.

Permeabilization assay for antimicrobial peptides based on pore-spanning lipid membranes on nanoporous alumina.

Screening tools to study antimicrobial peptides (AMPs) with the aim to optimize therapeutic delivery vectors require automated and parallelized sampling based on chip technology. Here, we present the development of a chip-based assay that allows for the investigation of the action of AMPs on planar lipid membranes in a time-resolved manner by fluorescence readout. Anodic aluminum oxide (AAO) composed of cylindrical pores with a diameter of 70 nm and a thickness of up to 10 µm was used as a support to generate pore-spanning lipid bilayers from giant unilamellar vesicle spreading, which resulted in large continuous membrane patches sealing the pores. Because AAO is optically transparent, fluid single lipid bilayers and the underlying pore cavities can be readily observed by three-dimensional confocal laser scanning microscopy (CLSM). To assay the membrane permeabilizing activity of the AMPs, the translocation of the water-soluble dyes into the AAO cavities and the fluorescence of the sulforhodamine 101 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanol-l-amine triethylammonium salt (Texas Red DHPE)-labeled lipid membrane were observed by CLSM in a time-resolved manner as a function of the AMP concentration. The effect of two different AMPs, magainin-2 and melittin, was investigated, showing that the concentrations required for membrane permeabilization and the kinetics of the dye entrance differ significantly. Our results are discussed in light of the proposed permeabilization models of the two AMPs. The presented data demonstrate the potential of this setup for the development of an on-chip screening platform for AMPs.

Combining reflectometry and fluorescence microscopy: an assay for the investigation of leakage processes across lipid membranes.

The passage of solutes across a lipid membrane plays a central role in many cellular processes. However, the investigation of transport processes remains a serious challenge in pharmaceutical research, particularly the transport of uncharged cargo. While translocation reactions of ions across cell membranes is commonly measured with the patch-clamp, an equally powerful screening method for the transport of uncharged compounds is still lacking. A combined setup for reflectometric interference spectroscopy (RIfS) and fluorescence microscopy measurements is presented that allows one to investigate the passive exchange of uncharged compounds across a free-standing membrane. Pore-spanning lipid membranes were prepared by spreading giant 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles on porous anodic aluminum oxide (AAO) membranes, creating sealed attoliter-sized compartments. The time-resolved leakage of different dye molecules (pyranine and crystal violet) as well as avidin through melittin induced membrane pores and defects was investigated.

Benefits and limitations of porous substrates as biosensors for protein adsorption.

Porous substrates have gained widespread interest for biosensor applications based on molecular recognition. Thus, there is a great demand to systematically investigate the parameters that limit the transport of molecules toward and within the porous matrix as a function of pore geometry. Finite element simulations (FES) and time-resolved optical waveguide spectroscopy (OWS) experiments were used to systematically study the transport of molecules and their binding on the inner surface of a porous material. OWS allowed us to measure the kinetics of protein adsorption within porous anodic aluminum oxide membranes composed of parallel-aligned, cylindrical pores with pore radii of 10-40 nm and pore depths of 0.8-9.6 µm. FES showed that protein adsorption on the inner surface of a porous matrix is almost exclusively governed by the flux into the pores. The pore-interior surface nearly acts as a perfect sink for the macromolecules. Neither diffusion within the pores nor adsorption on the surface are rate limiting steps, except for very low rate constants of adsorption. While adsorption on the pore walls is mainly governed by the stationary flux into the pores, desorption from the inner pore walls involves the rate constants of desorption and adsorption, essentially representing the protein-surface interaction potential. FES captured the essential features of the OWS experiments such as the initial linear slopes of the adsorption kinetics, which are inversely proportional to the pore depth and linearly proportional to protein concentration. We show that protein adsorption kinetics allows for an accurate determination of protein concentration, while desorption kinetics could be used to capture the interaction potential of the macromolecules with the pore walls.