Assessment of the multiphase interaction between a membrane disrupting peptide and a lipid membrane.
ABSTRACT Although modeling and experimental approaches to probe antimicrobial peptides-lipid membranes interaction have already been reported, quantitative evaluation of the whole process, including full dissolution of the lipid, is still missing. We report on the real-time assessment of the entire set of stages of melittin-membrane interaction, based on surface plasmon resonance (SPR) measurements, using supported lipid matrices on L1 sensors and long peptide injections. We advance a mathematical model which comprises a set of coupled kinetic equations and relates via the transfer matrix the evolution of lipid and peptide concentrations with the SPR sensorgram. Upon fitting the sensorgrams of melittin injections on POPC lipid matrices, in agreement with literature data, the model provides: association and dissociation rates, concentration thresholds, and evolution within each interacting layer of lipid and peptide concentrations as well as of peptide to lipid ratios. The proposed model combined with appropriate experimental protocols adds new depths to SPR investigation of peptide-lipid interaction offering a quantitative platform for research and controlled design of improved antimicrobial peptides. A wider applicability for quantitative assessment of other pore forming compounds on different lipid matrices is suggested.
- SourceAvailable from: Eugen Gheorghiu[Show abstract] [Hide abstract]
ABSTRACT: This study presents a multiparametric label-free analysis gathering surface plasmon resonance (SPR) and electrical impedance spectroscopy (EIS) for monitoring the progress of a model epithelial cell culture (Madin Darbey Canine Kidney - MDCK) exposed to a peptide with high bio-medical relevance, amyloid β (Aβ42). The approach surpasses the limitations in using the SPR angle for analyzing confluent cell monolayers and proposes a novel quantitative analysis of the SPR dip combined with advanced EIS as a tool for dynamic cell assessment. Long, up to 48h time series of EIS and SPR data reveal a biphasic cellular response upon Aβ42 exposure corresponding to changes in cell-substrate adherence, cell-cell tightening or cytoskeletal remodeling. The equivalent circuit used for fitting the EIS spectra provided substantiation of SPR analysis on the progress of cell adhesion as well as insight on dynamics of cell-cell junction. Complementary endpoint assays: western blot analysis and atomic force microscopy experiments have been performed for validation. The proposed label free sensing of nonlethal effect of model amyloid protein at cellular level provides enhanced resolution on cell-surface and cell-cell interactions modulated by membrane related protein apparatus, applicable as well to other adherent cell types and amyloid compounds.Biosensors & Bioelectronics 08/2013; 52C:89-97. · 6.45 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The potency and selectivity of many antimicrobial peptides (AMPs) are correlated with their ability to interact with and disrupt the bacterial cell membrane. In vitro experiments using model membranes have been used to determine the mechanism of membrane disruption of AMPs. Since the mechanism of action of an AMP depends on the ability of the model membrane to accurately mimic the cell membrane, it is important to understand the effect of membrane composition. Anionic lipids which are present in the outer membrane of prokaryotes but are less common in eukaryotic membranes are usually considered key for the bacterial selectivity of AMPs. We show by fluorescence measurements of peptide-induced membrane permeabilization that the presence of anionic lipids at high concentrations can actually inhibit membrane disruption by the AMP MSI-78 (pexiganan), a representative of a large class of highly cationic AMPs. Paramagnetic quenching studies suggest MSI-78 is in a surface-associated inactive mode in anionic SDS micelles, but is in a deeply buried and presumably more active mode in zwitterionic DPC micelles. Furthermore, a switch in mechanism occurs with lipid composition. Membrane fragmentation with MSI-78 is observable in mixed vesicles containing both anionic and zwitterionic lipids but not in vesicles composed of a single lipid of either type. These findings suggest membrane affinity and membrane permeabilization are not always correlated, and additional effects can be seen as the complexity of the model membranes is increased that may be more reflective of the actual cellular environment.Biochemistry 04/2013; · 3.38 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: We present novel solutions to surpass current analytic limitations of Magneto Optical Surface Plasmon Resonance (MOSPR) assays, concerning both the chip structure, and the method for data analysis. The structure of the chip is modified to contain a thin layer of Co-Au alloy instead of successive layers of homogenous metals, as currently used. This alloy presents improved plasmonic and magnetic properties, yet a structural stability similar to Au-SPR chips, allowing for bioaffinity assays in saline solutions. Analyzing the whole reflectivity curve at multiple angles of incidence instead of the reflectivity value at a single incidence angle provides a high signal to noise ratio suitable for detection of minute analyte concentrations. Based on assessment of solutions with known refractive index as well as of a model biomolecular interaction (i.e. IgG – AntiIgG) we demonstrate that the proposed structure of the MOSPR sensing chip and the procedure of data analysis allows for long time assessment in liquid media with increased sensitivity over standard SPR analyses.Biosensors and Bioelectronics. 08/2014;