Arnaud Marquette

Structural Determinants of Antimicrobial and Antiplasmodial Activity and Selectivity in Histidine-rich Amphipathic Cationic Peptides

Designed histidine-rich amphipathic cationic peptides, such as LAH4, have enhanced membrane disruption and antibiotic properties when the peptide adopts an alignment parallel to the membrane surface. Although this was previously achieved by lowering the pH, here we have designed a new generation of histidine-rich peptides that adopt a surface alignment at neutral pH. In vitro, this new generation of peptides are powerful antibiotics in terms of the concentrations required for antibiotic activity; the spectrum of target bacteria, fungi, and parasites; and the speed with which they kill. Further modifications to the peptides, including the addition of more hydrophobic residues at the N terminus, the inclusion of a helix-breaking proline residue or using d-amino acids as building blocks, modulated the biophysical properties of the peptides and led to substantial changes in toxicity to human and parasite cells but had only a minimal effect on the antibacterial and antifungal activity. Using a range of biophysical methods, in particular solid-state NMR, we show that the peptides are highly efficient at disrupting the anionic lipid component of model membranes. However, we also show that effective pore formation in such model membranes may be related to, but is not essential for, high antimicrobial activity by cationic amphipathic helical peptides. The information in this study comprises a new layer of detail in the understanding of the action of cationic helical antimicrobial peptides and shows that rational design is capable of producing potentially therapeutic membrane active peptides with properties tailored to their function.

Helix orientations in membrane-associated Bcl-XL determined by 15N-solid-state NMR spectroscopy

Controlled cell death is fundamental to tissue hemostasis and apoptosis malfunctions can lead to a wide range of diseases. Bcl-xL is an anti-apoptotic protein the function of which is linked to its reversible interaction with mitochondrial outer membranes. Its interfacial and intermittent bilayer association makes prediction of its bound structure difficult without using methods able to extract data from dynamic systems. Here we investigate Bcl-xL associated with oriented lipid bilayers at physiological pH using solid-state NMR spectroscopy. The data are consistent with a C-terminal transmembrane anchoring sequence and an average alignment of the remaining helices, i.e. including helices 5 and 6, approximately parallel to the membrane surface. Data from several biophysical approaches confirm that after removal of the C-terminus from Bcl-xL its membrane interactions are weak. In the presence of membranes Bcl-xL can still interact with a Bak BH3 domain peptide suggesting a model where the hydrophobic C-terminus of the protein unfolds and inserts into the membrane. During this conformational change the Bcl-xL hydrophobic binding pocket becomes accessible for protein–protein interactions whilst the structure of the N-terminal region remains intact.

Membrane structure and interactions of human catestatin by multidimensional solution and solid-state NMR spectroscopy

Catestatin is a natural peptide of higher organisms including humans, with a wide variety of biological functions involved in catecholamine inhibition, cardiovascular regulation, control of blood pressure, inflammation, and innate immunity. It is derived from the natural processing of chromogranin A, induced in the skin after injury, and produced by chromaffin cells and neutrophils. With neutrophils, the peptide enters the cell by crossing the plasma membrane where it interacts with internal targets to induce calcium influx. Therefore, we investigated the membrane interactions and structure of several catestatin‐derived peptides. Whereas fluorescence dye release experiments are indicative of membrane permeabilization, multidimensional solution NMR and circular dichroism spectroscopies show that catestatin adopts α‐helical conformations between Ser‐6 and Tyr‐12 in the presence of dodecylphosphocholine micelles. Furthermore, proton‐decoupled 15N solid‐state NMR spectroscopy of sequences labeled with 15N and reconstituted into oriented lipid bilayers indicates that this domain is aligned in a strongly tilted to inplanar alignment. Proton‐decoupled 31P NMR spectra of the same samples are indicative of conformational and/or orientational heterogeneity at the level of the lipid bilayer head groups due to the presence of catestatin. The sequence and 3‐dimensional structure of catestatin exhibit homologies with penetratin, which is suggestive that they both enter the cells by related mechanisms to target internal structures.—Sugawara, M., Resende, J. M., Moraes, C. M., Marquette, A., Chich, J.‐F., Metz‐Boutigue, M.‐H., Bechinger, B. Membrane structure and interactions of human catestatin by multidimensional solution and solid‐state NMR spectroscopy. FASEB J. 24, 1737–1146 (2010). www.fasebj.org

Peripheral and Integral Membrane Binding of Peptides Characterized by Time-Dependent Fluorescence Shifts: Focus on Antimicrobial Peptide LAH4

Positioning of peptides with respect to membranes is an important parameter for biological and biophysical studies using model systems. Our experiments using five different membrane peptides suggest that the time-dependent fluorescence shift (TDFS) of Laurdan can help when distinguishing between peripheral and integral membrane binding and can be a useful, novel tool for studying the impact of transmembrane peptides (TMP) on membrane organization under near-physiological conditions. This article focuses on LAH4, a model α-helical peptide with high antimicrobial and nucleic acid transfection efficiencies. The predominantly helical peptide has been shown to orient in supported model membranes parallel to the membrane surface at acidic and, in a transmembrane manner, at basic pH. Here we investigate its interaction with fully hydrated large unilamellar vesicles (LUVs) by TDFS and fluorescence correlation spectroscopy (FCS). TDFS shows that at acidic pH LAH4 does not influence the glycerol region while at basic pH it makes acyl groups at the glycerol level of the membrane less mobile. TDFS experiments with antimicrobial peptides alamethicin and magainin 2, which are known to assume transmembrane and peripheral orientations, respectively, prove that changes in acyl group mobility at the glycerol level correlate with the orientation of membrane-associated peptide molecules. Analogous experiments with the TMPs LW21 and LAT show similar effects on the mobility of those acyl groups as alamethicin and LAH4 at basic pH. FCS, on the same neutral lipid bilayer vesicles, shows that the peripheral binding mode of LAH4 is more efficient in bilayer permeation than the transmembrane mode. In both cases, the addition of LAH4 does not lead to vesicle disintegration. The influence of negatively charged lipids on the bilayer permeation is also addressed.

Reversible Liposome Association Induced by LAH4: A Peptide with Potent Antimicrobial and Nucleic Acid Transfection Activities

We report on the reversible association of anionic liposomes induced by an antimicrobial peptide (LAH4). The process has been characterized for mixed membranes of POPC and POPS at molar ratios of 1:1, 3:1, and 9:1. Although the vesicles remain in suspension in the presence of excess amounts of peptide, the addition of more lipids results in surface charge neutralization, aggregation of the liposomes, and formation of micrometer-sized structures that coexist in equilibrium with vesicles in suspension. At low ratios of anionic lipids, vesicle aggregation is a reversible process, and vesicle disassembly is observed upon inversion of the surface charge by further supplementation with anionic vesicles. In contrast, a different process, membrane fusion, occurs in the presence of high phosphatidylserine concentrations. Upon binding to membranes containing low POPS concentrations, the peptide adopts an in-plane alpha-helical structure, a secondary structure that is conserved during vesicle association and dissociation. Our finding that peptides are essential for vesicle aggregation contributes to a better understanding of the activity of antimicrobial peptides, and suggests an additional layer of complexity in membrane-protein lipid interactions.

Magainin 2-PGLa Interactions in Membranes - Two Peptides that Exhibit Synergistic Enhancement of Antimicrobial Activity.

The structural requirements for the synergistic enhancement of antimicrobial activities of the cationic linear peptides PGLa and magainin 2 were investigated. In a first step the antimicrobial activities were evaluated for a number of modifications of the sequences and equimolar mixtures thereof. In particular fluorophore labelled peptides maintain a high degree of antimicrobial activity and considerable synergism when tested conjointly. Thereafter, circular dichroism spectroscopy indicated that these extended sequences adopt helical conformations in the presence of model membranes similar to the unmodified sequences. Energy transfer between the fluorophores suggested that the peptides reside in close proximity to each other when bound to the membrane surface at high concentrations. The fluorophore interactions quickly diminish at lower peptide-to-lipid ratios indicating that the peptide-peptide interactions are weak. Furthermore, (15)N solid-state NMR measurements of the membrane topology of [(15)N-Ala14]-PGLa and of its fluorophorecarrying analogue reconstituted into supported 1, 2-didecanoyl-sn-glycero-3-phosphocholine bilayers were performed. These experiments revealed no correlation between the topological state of PGLa and the observed synergistic enhancement of antimicrobial activities due to the presence of magainins. These results suggest that lipid mediated interactions rather than the formation of tight peptide-peptide complexes in the membrane are responsible for synergistic activities of the mixtures.

Biophysical Investigations Elucidating the Mechanisms of Action of Antimicrobial Peptides and Their Synergism

Biophysical and structural investigations are presented with a focus on the membrane lipid interactions of cationic linear antibiotic peptides such as magainin, PGLa, LL37, and melittin. Observations made with these peptides are distinct as seen from data obtained with the hydrophobic peptide alamethicin. The cationic amphipathic peptides predominantly adopt membrane alignments parallel to the bilayer surface; thus the distribution of polar and non-polar side chains of the amphipathic helices mirror the environmental changes at the membrane interface. Such a membrane partitioning of an amphipathic helix has been shown to cause considerable disruptions in the lipid packing arrangements, transient openings at low peptide concentration, and membrane disintegration at higher peptide-to-lipid ratios. The manifold supramolecular arrangements adopted by lipids and peptides are represented by the ‘soft membranes adapt and respond, also transiently’ (SMART) model. Whereas molecular dynamics simulations provide atomistic views on lipid membranes in the presence of antimicrobial peptides, the biophysical investigations reveal interesting details on a molecular and supramolecular level, and recent microscopic imaging experiments delineate interesting sequences of events when bacterial cells are exposed to such peptides. Finally, biophysical studies that aim to reveal the mechanisms of synergistic interactions of magainin 2 and PGLa are presented, including unpublished isothermal titration calorimetry (ITC), circular dichroism (CD) and dynamic light scattering (DLS) measurements that suggest that the peptides are involved in liposome agglutination by mediating intermembrane interactions. A number of structural events are presented in schematic models that relate to the antimicrobial and synergistic mechanism of amphipathic peptides when they are aligned parallel to the membrane surface.

A fast and simple method to eliminate Cpn60 from functional recombinant proteins produced by E. coli Arctic Express

A frequent problem of recombinant protein production is their insolubility. To address this issue, engineered Escherichiacoli strains like Arctic Express that produce an exogenous chaperone facilitating protein folding, have been designed. A drawback is the frequent contamination of the protein by chaperones. A simple method, using urea at a sub-denaturing concentration, allows unbinding of Cpn60 from expressed protein. This method was successfully used to purify 2 proteins, an enzyme and a viral protein. The enzyme was fully active. The nature of interaction forces between enzyme and Cpn60 was investigated. The method is likely applicable to purify other proteins.

Simultaneous Analysis of Secondary Structure and Light Scattering from Circular Dichroism Titrations: Application to Vectofusin-1

Circular Dichroism data are often decomposed into their constituent spectra to quantify the secondary structure of peptides or proteins but the estimation of the secondary structure content fails when light scattering leads to spectral distortion. If peptide-induced liposome self-association occurs, subtracting control curves cannot correct for this. We show that if the cause of the light scattering is independent from the peptide structural changes, the CD spectra can be corrected using principal component analysis (PCA). The light scattering itself is analysed and found to be in good agreement with backscattering experiments. This method therefore allows to simultaneously follow structural changes related to peptide-liposome binding as well as peptide induced liposome self-association. We apply this method to study the structural changes and liposome binding of vectofusin-1, a transduction enhancing peptide used in lentivirus based gene therapy. Vectofusin-1 binds to POPC/POPS liposomes, causing a reversal of the negative liposome charge at high peptide concentrations. When the peptide charges exactly neutralise the lipid charges on both leaflets reversible liposome self-association occurs. These results are in good agreement with biological observations and provide further insight into the conditions required for efficent transduction enhancement.